CORNELL UNIVERSITY.
THE
Roswell P. Flomer Library
THE GIFT OF ROSWELL P. FLOWER FOR THE USE OF THE N. Y. STATE VETERINARY COLLEGE. 1897
| Cornell University Library
' TX 543.S35
LT 3 1924 000 936 983 vet
BACTERIOLOGICAL METHODS
SCHNEIDER
BY THE SAME AUTHOR
PHARMACEUTICAL BACTERIOLOGY
80 Illustrations Octavo 246 Pages Cloth, $2.00 Post Paid
“The discussion of disinfectants and of the principles of disinfection and sterilization and of the practical application of these principles in the pharmacy would alone make the book well worth while to every phar- macist.’—Jnl, Amer. Pharmaceutical Ass'n.
Bacteriological Methods
IN
Food and Drugs Laboratories
WITH AN
Introduction to Micro-analytical Methods
BY
ALBERT SCHNEIDER, M. D., PH. D.
(CoLumpBia UNIVERSITY),
PROFESSOR OF PHARMACOGNOSY AND BACTERIOLOGY IN THE COLLEGE OF PHARMACY OF THE UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
87 ILLUSTRATIONS AND 6 FULL PAGE PLATES
PHILADELPHIA
P. BLAKISTON’S SON & CO. 1012 WALNUT STREET
No] 57
CopyriGHT, 191s, BY P. Biaxtston’s Son & Co.
et > "2° ad ~~ i at Sse see! or ; we
THE MAPLE PRESS YORK PA
PREFACE
The administration of the Federal Pure Food and Drugs Act and of the several State Pure Food and Drugs Laws has made the introduction of bacteriological methods into food and drugs laboratories a necessity. Because of the close relationship be- tween the work of the bacteriologist and that of the micro-analyst, it is advised that, wherever possible, these two laboratory branches be combined in the most effectual codperative manner. With such codperation in mind, a brief introduction to micro-analytical methods is added. Fuller details on micro-technique will be found in special works on the microscopy of fibers, foods, spices, drugs, of water supplies, of sewage, etc.
As is more fully set forth in the text, the bacteriological as well as micro-analytical methods in our food and drugs labora- tories are not yet fully worked out, and the present volume is submitted hoping that it will be instrumental in bringing about a unification of methods and that it will perhaps also serve as a guide to the working out of newer and inadequately tested older methods.
The volume is primarily intended as a guide to students who are interested in the bacteriological examination of foods and drugs. Its use as a laboratory guide presupposes a thorough knowledge of general bacteriology.
Acknowledgments are made to the following authors for the use of illustrations: E. R. Stitt (Bacteriology, Blood Work and Animal Parasitology), R. L. Pitfield (Compend on Bacteriology), _W. J. MacNeal (Pathogenic Micro-organisms), C. E. Marshall (Microbiology), John F. Anderson and Thomas B. McClintic (Method of Standardizing Disinfectants), and G. W. Hunter (Es- sentials of Biology).
vi PREFACE
Grateful acknowledgments are also made to A. E. Graham, Inspector in Charge, San Francisco Laboratory, Bureau of Animal Industry, for valuable suggestions regarding the examination of meats and meat products with special reference to the isolation and examination of animal fat crystals and the examination of sausage meats for starch fillers; to Professor Karl Frederick Meyer, of the Department of Bacteriology and Protozoology of the University of California, for the article on “The Precipitin Test for Meats,” and to Merck’s Report for permission to use those parts of the text which had been published in that journal. It is also desired to acknowledge the loan of several cuts by the Bausch and Lomb Optical Company. Additional acknowledgments are
made throughout the text. San Francisco, CALIFORNTA.
CONTENTS
I. OUTLINE OF MICRO-ANALYTICAL METHODS IN Foop AND Drucs LABORATORIES
oOo On An
PAGE
. Introduction . : . Grouping of Suhetuneea is be Beaming in Food ae Truss Pahoutatien, . The Work of the Micro-analyst in oo to that of the Chemist and
Bacteriologist .
. Equipment for Micro- analytical Work ;
. Organoleptic Testing ; . Methods Useful in the iadoulinn a Veoetable Tage, — Ete , . Methods Useful in the Examination of Vegetable Food Products
. Micro-chemical Color Reaction Tests . ........
. Making Analytical Reports .
. The More Important Histological Hlentents of Plants
II. BAcTERIOLOGICAL METHODS IN Foop AND Drucs LaBoRATORIES
: Introduction i ;
. Direct Baateriologtesl Reamidadtins ; 8
. Numerical Limits of Micro-organisms in Wands and Draes Pare
. Quantitative Estimations by the Cultural Methods 3
. Preparation of Standard Culture Media. General Suggestions
. Preparation of Required Standard Culture Media . .
. Technique for Making Quantitative and Qualitative Retinattons thy the
Plating Methods 2.005 66 2 5 @ A eee ee we eG
. Practical Application of the Ouenteatices Betinations by the Pathe
Methods. ... . sy (eh EDR ca men Als hep es vin ae ey es
. Qualitative Disternfvation’ « Bs hs a Beh acs ue) en Dn te ee Ca
. Evidence of Sewage Contamination
. Possible Contamination of Foods with the Typhoid Badlius,
. Possible Contamination of Food Substances with the Cholera Bacillus.
. Biological Water Analysis . .. . a a ee,
. Bacteriological Examination of Mineral Waters ; :
. The Microscopical and Bacteriological Examination of Milk
. The Bacteriological Examination of Shellfish . . .
. The Bacteriological and Toxicological Examination ar Meats aed Meat
PROdUEtS:s 4 <) wAE a 6 ee BR ee ww ER BOOS ee OPE Re
viii‘ CONTENTS
Pace 18. The Bacteriological Examination of Eggs and Egg Products ..... . 187 19. The Bacteriological Examination of Pharmaceutical Preparations . . . . 197 20. The Microscopical and Bacteriological Examination of Syrups. . . . 202
The Microscopical and een Examination of Fermented Foods 2 210
aq.
and Beverages aus 22. Standardization of Disinfectants, bs 2 230 23. Determining the Purity and Quality of Sera, sails andl Related Broducts 265
24. Special Biological and Toxicological Tests . .........2. 2... 268
|
OUTLINE OF MICRO-ANALYTICAL METHODS IN FOOD AND DRUGS LABORATORIES
1. Introduction
The value of the compound microscope in the examination of foods and drugs is as yet not generally recognized. *Efficiency in this line of work depends very largely upon a long and wide range of experience, in this regard differing very markedly from effi- ciency in the field of chemical analyses. All that is required of the chemist, as far as routine analytical work is concerned, is a very close adherence to the methods laid down for him. He is pronounced skilled in direct proportion to his adherence to methods and skill shown in the manipulation of apparatus and reagents. ‘The micro-analyst in order to be efficient must be very familiar with the appearance of the multitudinous forms of cells, tissues, cell-contents and with the behavior of certain micro- chemical reagents and this familiarity can be acquired only through long and careful observation.
2. Grouping of Substances to be Examined in Food and Drugs Laboratories
The analytical methods, as they apply to the critical examina- tion of foods and drugs, are chemical, microscopical and bacteriolog- ical. The substances to be analyzed may be grouped as follows:
T
2 MICRO-ANALYTICAL METHODS
1. Vegetable drugs, crude and powdered, pharmacopceial and other simple and compound medicinal powders. 2. Spices and condiments, whole, ground and powdered, Prepared spices and condiments. 3. Coffee, tea, cocoa, chocolate, confections, candies. 4. Tobacco and preparations made from tobacco, as snuff, smoking tobacco, cigars, etc. s. Chemicals, minerals, solutions of chemicals, etc. 6. Tablets, pills, powders. 7. Meats of all kinds, raw, cooked, canned, sausage meats, etc. 8. Dairying products, as milk, cream, cheese, butter, ice-cream, ice cream fillers, etc. g. Insect powders, dusting powders, cosmetics. to. Cattle and poultry powders. 11. Unknown powders, wholly or partly of vegetable origin. 12. Starches, dextrins, sausage meat binders (starches). 13. Vegetable foods, as jams and jellies; fresh, pickled, cooked, cafined and preserved. 14. Flours and meals. 1s. Breakfast foods, infant and invalid foods. 16. Breads and similar materials; biscuits, doughnuts, cakes, pies, pastries, etc. 17. Macaroni, spaghetti and similar preparations, noodles, etc. 18. Nuts and nut-like fruits and seeds, etc. 19. Beverages of all kinds, liquids generally. 20. Pharmaceuticals of all kinds. at. Patent and proprietary medicines. 22. Unknown foods and medicines.
In the examination of some of these substances the chemical method is all important, as in chemicals generally; in the examina- tion of others the microscopical method is all-important, as in meals, flours, spices; and again the bacteriological testing is all- important, as in sewage, contaminated water, contaminated milk, infected foods and drinks generally, etc. A properly equipped analytical laboratory, whether federal, state or private, should be prepared to apply all three methods. The bacteriological in- vestigations should be made by the micro-analyst rather than by the chemist, because of the closer relationship between bacteriology and microscopy.
INTRODUCTION 3
.3. The Work of the Micro-analyst in Relationship to that of the Chemist and Bacteriologist
Just what work should or should not be done by the micro- analyst is as yet not definitely determined; at least, there is no uniformity as to scope of action in the different analytical labora- tories. It is suggested that the following work be assigned to the micro-analyst:
1. Gross and net weight determination of all such samples as require it.
2. Moisture determination of substances which require it.
3. Ash and acid insoluble determinations of substances which are primarily subject to microscopical analysis, as vegetable drugs, pills, powders, vegetable compound powders, etc.
4. Use of certain special tests, as sublimation tests for benzoic acid, salicylic acid and boric acid; Grahe’s cinchona test, wheat gluten test, color reactions for boric acid, capsicum, guaiac, salicylic acid, morphine, etc., tests for cholesterol and phy- tosterol crystals, and others which may prove useful.
5. Bacteriological testing of foods and drugs generally, of sera, vaccines, galen- icals, syrups, milk, water, jams, jellies, catsups, etc., as may be required, following the method of the Society of the American Bacteriologists, and limiting the testing to determining the presence or absence of the colon bacillus and other sewage organ- isms, and the usual quantitative bacterial determinations for milk, water and other substances, of which the quality is usually based upon the quantitative bacterial content.
Substances subject to analysis in the laboratories mentioned should be grouped or classified according to the special or pre- ferred methods of examination to be applied. It is, of course, evident that in the majority of cases chemical as well as micro- scopical methods should be used. In some cases even all three must be used in order that conclusive results may be obtained. The following grouping is suggested:
1. Substances in which the chemical analysis is of first importance. Chemicals generally, and chemicals in solution, alcohol, alcoholic drinks, flavoring extracts, syrups, oils, fats, etc.
2. Substances in which the microscopical analysis is of first importance— vegetable substances and preparations which are essentially of vegetable origin. Meats of all kinds, variously prepared, cooked, spiced, etc.
3. Substances in which the chemical and microscopical examinations are of equal
4 MICRO-ANALYTICAL METHODS
importance—assayable vegetable drugs, all prepared food substances with chemicals in solution, compound powders, pills, tablets.
4. Substances to which the microscopical examination is not generally applied —chemicals, liquids in which the insoluble particles are slight in amount, as wines, brandies, comparatively pure solutions, etc. Here the centrifuge plays an im-
portant part. ; 7 s. Substances in which the bacterial testing is of prime importance—milk,
sewage or otherwise organically contaminated water supplies, and other liquids, beers, etc., contaminated foods generally. In this class of substances the micro- scopical and chemical examinations become necessary in addition to the bacterio- logical; in fact, a bacteriological test is incomplete without the use of a good com- pound microscope.
The work of the micro-analyst is, so to speak, on trial. The doubt in the minds of the critics is due, very largely, to the un- satisfactory results traceable to the efforts of those who are not sufficiently qualified. Even the most skillful analysts admit numerous defects in methods and shortcomings in results. For example, the quantitative estimates based upon optical judg- ment are approximate only, and with most workers there is a very marked tendency to make these estimates volumetric rather than gravimetric. This can in a measure be corrected by bring- ing into play the judgment of the relative weights of the several substances under comparison. For example, the amount of sand present in powdered belladonna root may be volumetrically estimated at 20 per cent. In this case the acid insoluble ash residue may show 35 to 4o per cent. of silica. An example like this also indicates why the micro-analyst should make the sand and ash determinations. The percentage estimates based upon microscopical examination may vary within 25 to 50 per cent. when the amounts of the admixtures are small or slight. For example, the actual amount of arrow-root starch in the so-called arrowroot biscuit is 2.5 per cent. The micro-analyst’s estimates may range from a trace or small amount to 5 per cent. When the quantities of admixtures are large, from 30 to go per cent., the estimations may approximate within ro or 1 5 per cent. of the actual amount present. These estimates can no doubt be
INTRODUCTION 5
made much more accurate by uniform methods of technique, aided by certain mechanical devices. For example, in the ex- amination of vegetable powders, spices, meals, flours and similar substances, the samples should be thoroughly mixed, and slide mounts should be of standard and uniform thickness and the relative amounts of the ingredients should be estimated by means of microscope slides having uniform ruled squares of definite measuring value in microns. These and other details in the methods should be more fully worked out.
Several micro-analysts have declared themselves as opposed to giving percentage estimates of the several ingredients of a compound. However, not to give the approximate percentages will cause great confusion and very materially lessen the value of the work done. For example, to report a pancake flour as com- posed of ‘‘buckwheat and wheat flour, the former predominating,” instead of ‘‘buckwheat approximately 75 per cent. and wheat approximately 25 per cent.,’’ would certainly be unsatisfactory.
The following examples will serve to explain the relative value of the chemical and microscopical analyses. Suppose the sub- stance to be examined is a baby food. The microscope may re- veal approximate percentages of oil globules, steam dextrinized wheat starch, unchanged wheat and arrowroot starch, wheat tissue and milk sugar. The chemical analysis will show a definite percentage of sugar, soluble starch, insoluble starch, fat, vege- table fiber and ash. This is a good example of a case where the two methods of analysis are of equal importance; one without the other would be unsatisfactory, incomplete and inconclusive. Again, the chemical assay may show that a sample of powdered belladonna leaf contains 0.35 per cent. of mydriatic alkaloids, and yet the microscopical examinations may prove the presence of 20 per cent. or more of some foreign leaf.
An adjunct in analytical work, much neglected by the chemist, is the organoleptic testing. This is especially important in the examination of unknown substances, fruit products, spices,
6 MICRO-ANALYTICAL METHODS
meats, etc., as it often gives a clue to the quality of the sub- stances and to the means of getting quick results.
4. Equipment for Micro-analytical Work
The equipment and apparatus required by the micro-analyst is comparatively inexpensive, and it is very earnestly advised to secure only those appliances which are useful or essential for the work in hand. The following list is submitted without entering into detail, as it may be assumed that the microscopist does not require explanations:
1. Simple lens. 2. Compound microscope. a. Ocular with micrometer scale. 6. Oculars, Nos. 2 and 4. c. Objectives, Nos. 3, 5 and 7. d. 1/12 in. oil-immersion objective for bacteriological work. 3. Slides and covers. 4. Section knife or razor, and strop. 5. Polarizer, for the study of starches, crystals and other substances. Should be convenient to use. The selenite plates are useful. 6. Thoma-Zeiss hemacytometer; for counting bacteria and yeast cells. 7. Stage mold and spore counter, as described in Part. II (Fig. 5). 8. Accurate metal or hard rubber millimeter ruler for measuring seeds (in fruit products), etc. 9. The required glassware and adjunct apparatus. 10. The required reagents. 11. Equipment for making moisture determinations. 12. Equipment for making ash determinations. 13. Equipment for the required bacteriological tests and determinations.
The laboratory in which the work is done should be roomy, well- lighted, provided with the necessary shelves, apparatus and supply cases, reference books, etc. The details need not be given here. The analyst must see to it that the necessary things are provided. A skillful and experienced worker should have the tcols of his choice, not those selected for him by some one not qualified to judge.
The skilled micro-analyst has little difficulty in determining
LABORATORY EQUIPMENT 7
the purity and comparative quality of the simple spices, as pepper, allspice, cloves, cinnamon and ginger. However matters are
Fic. 1.—Form of compound microscope suitable for bacteriological and general microscopical work in food and drugs laboratories. Note the desirable and necessary accessories as given in the text. The form of polarizing apparatus convenient to be used with the compound microscope, sets into the substage diaphragm ring with the iris diaphragm opened to the maximum. The analyzer takes the place of the ocular. —(Bausch & Lomb Co.)
quite different when it comes to the examination of powdered vegetable drugs, compound vegetable powders and vegetable products of unknown composition. A thorough knowledge of,
8 MICRO-ANALYTICAL METHODS
and a wide familiarity with, cell-forms, tissue elements and formed cell contents is an absolute essential in order that accurately re- liable and conclusive results may be obtained and serious con- fusion may be avoided. Differences in the reports of findings by micro-analysts are in part due to the personal equation, in part due to variable methods and differences of judgment in estimat- ing the quantity of tissue elements present and also in part due to a lack of extensive and intensive experience.
5. Organoleptic Testing
The organoleptic tests are indeed valuable adjuncts to the micro- scopical work. There are, however, some differences of opinion regarding the interpretation and valuation which aie to be placed on comparisons of color, odor and taste, even among those having had considerable experience and endowed with a fairly normal special sense development. Our color terminology is in great confusion, and so far as the olfactory sense is concerned, there are only comparatively few odors or flavors which admit of ready comparison such as tea flavor, coffee odor, vanilla odor, raspberry flavor, loganberry flavor, and the odor of such drugs as valerian, cubeb, fenugreek, asafetida, aloes, turpentine, camphor, the essen- tial oils, calamus, etc., and the odor of the spices. Our compara- tive judgment of tastes is more reliable. Much experience is necessary to form fairly reliable estimates of flavors (associations of tastes and odors), though pure fruit flavors are, as a rule, readily distinguishable, as that of apples, dried apples, peach, dried peach, quince and strawberry. Manufactured fruit preparations gener- ally lose much of their flavor due to many causes, as cooking, steaming, fermentative changes, presence of decayed (moldy) fruits, mixing of several kinds of fruits or fruit juices, etc., to say nothing of the wholly artificial or imitation fruit flavors and the
flavors of the imitation fruit products which have little or no fruit in their composition.
SPECIAL TESTS 9
6. Methods Useful in the Examination of Vegetable Drugs, Spices, Etc.
We shall give a few tests which have proven useful in the ex- amination of drugs and food products. It will be found that many of the test results are largely approximate, and some of them are primarily intended to serve as aids or checks to the chemical examination.
1. Mace Test.—To a pinch of the powdered mace add a few drops of ro per cent. sodium hydroxide solution. Banda or true mace changes color only slightly, whereas wild or Bombay mace turns a deep orange color.
2. Conium Test.—To the substance to be tested for the presence of conium fruits (as anise, caraway or other umbelliferous fruits), add 25 per cent. sodium or potassium hydroxide solution. In the presence of 1 per cent. or more of conium fruits a distinct mouse odor is developed in time (10 min. to 14 hr.). This test is not reliable with old umbelliferous fruits, as many of them de- velop.a more or less marked mouse odor with alkalies.
3. Lignin Test.—The classic phloroglucin-hydrochloric acid test is useful in making estimates of the amount of lignified tissue present, as in old belladonna root, aconite roots and stems, lobelia herb, fruit products, spices, etc.
4. Grahe’s Cinchona Test.—Drive the moisture from the inner surface of a small test-tube by holding it over a Bunsen burner. Into this dried test-tube place a pinch of finely powdered cinchona bark (No. 80) and heat rather carefully over an alcohol lamp or Bunsen burner. When the bark begins to char, red fumes begin to fill the tube and condense on the side of the tube as a reddish purplish liquid. The intensity of the reaction is approximately proportional (direct proportion) to the percentage of alkaloids present. Some skill and experience is necessary to perform this test well. The tube must not be heated too quickly or too much, and the powder should be uniformly fine.
Io MICRO-ANALYTICAL METHODS
5. Beaker Sand Test.—Pour a definite amount of the powdered spice or vegetable drug into a beaker, add water, stir until the sand is washed away from the vegetable particles and settles to the bottom of the beaker. Let a stream of water run into beaker so as to wash out the vegetable matter. The final washing and decanting must be done carefully so as not to lose the sand. Salt brine may be used instead of water, should the vegetable matter have a comparatively high specific gravity. Dry the sand and weigh to obtain the percentage of sand present.
6. Ash Determination.—According to the regulation method. The percentage of the acid-insoluble residue should also be de- termined. It should be borne in mind that the ash determination gives only approximate results as far as the presence of clay and dirt is concerned, since the organic matter of dirt is combustible. The ash percentage varies greatly in vegetable drugs, especially in herbs and leaves. The sand percentage is comparatively high in those herbs and leaves having abundant trichomes, especially if the drug plants (or herbaceous spices) bearing such trichomes are grown in dry sandy soil. Dirt (and sand) percentage is apt to be high in roots and rhizomes, particularly when rootlets are abundant and when the gathering, garbling and cleaning is carelessly done.
There are a number of chemical tests giving color reactions which can be done conveniently by the micro-analyst, as the boric acid reaction with curcuma, the H,SO, color reaction with some barks, capsicum, guaiac, resin, cubeb, etc.; the H»SO, plus for- maldehyde color reaction with morphine; the ferric chloride color reaction with salicylic acid, etc. These tests should be used when, in the judgment of the analyst, they may serve to give better information regarding the identity, purity and quality of the drug.
SPECIAL TESTS II
7. Methods Useful in the Examination of Vegetable Food Products
1. Sublimation Test for Benzoic Acid.—Place a drop or two of the suspected liquid or semi-liquid food substance into a deep watch crystal of 1 in. diameter. Place over it a clean dry slide. Now hold the watch crystal over a flame (alcohol lamp’) until the substance (as wine, vinegar, catsup, jam, jelly, etc.), comes to an active boil. The steam vapor, carrying with it the benzoic acid, is condensed on the slide. Remove the slide and set it aside until the condensed moisture has evaporated; very moderate heat may be used to hasten evaporation. Examine under the microscope, whereupon the benzoic acid crystals may be seen, provided any were present. The test is delicate, very reliable and very few substances interfere with it. It is very pronounced in the presence of o.o1 per cent. of benzoic acid.
2. Sublimation Test for Salicylic Acid— Made like the benzoic acid test. The crystal formation (plates) is very pronounced in dilutions of 1:1000. After having examined the crystals under the microscope, add a drop of weak solution of ferric chloride to the crystals upon the slide, whereupon a blue coloration develops. Boric acid is likewise deposited by sublimation, but the test is not as satisfactory as those for benzoic acid and for salicylic acid.
The sublimation test may also be extended to other crystalline substances which undergo sublimation on exposure to heat.
3. Curcuma Thread Test for Boric Acid.—Boil 5 grams of powdered curcuma in 10 cc. of alcohol. To the evaporated alco- holic extract add a little soda and several cc. of 50 per cent. alcohol. In this place paper (bast fiber), cotton or linen threads and bring to a brisk boil for a few moments. Remove threads and dry between blotting paper, lay them in a very weak solu- tion of sulphuric acid and rinse in-water. When dry the threads should be a golden yellow.
1 Alcohol lamp is preferable because the flame is small and yet the heating is more quickly done.
12 MICRO-ANALYTICAL METHODS
The test for the presence of boric acid (also for borax) is made as follows: Dip the end of a prepared thread in a 10 per cent. solution of hydrochloric acid and allow to dry. Lay the thread on a slide, cover with cover glass and examine. It should be of a reddish-brown color. To the edge of cover glass apply a droplet of a ro to 13 per cent. solution of sodium carbonate, followed by a droplet of the suspected solution. In the presence of boric acid, the thread is colored blue, which coloration remains for a longer or shorter period and then changes to gray and violet. The test is a very delicate one and is not hindered by the presence of sodium chloride, magnesium sulphate and aluminium sulphate. Strong solutions of phosphoric acid, silicic acid, calcium chlorite and magnesium chlorite, interfere with the reaction more or less.
4. Formaldehyde Test.—Concentrated hydrochloric acid added to weak solutions of formaldehyde (1 : 5000) or substances containing formaldehyde, forms stellate clusters having a some- what crystalline appearance. The formaldehyde can be de- posited on a slide by sublimation (as for benzoic acid) and the acid added. The stellate clusters appear upon evaporation of the hydrochloric acid. The test requires further verification to determine its value.
5. Sulphurous Acid Test.—Moisten starch paper with a very dilute solution of potassium-iodide iodine solution which colors it blue. In the presence of the merest trace of sulphurous acid the paper is decolorized. Do not use heat in this test.
6. Iodine Reaction.—The color reaction of starch with N/so iodine solution is of great importance in the examination of fruit products, such as jams, jellies, catsups, etc., as it shows whether or not ripe or green fruits and juices of unripe fruit were used and whether or not starch paste may have been added as a filler or thickening agent. As is known, green fruits generally contain more or less starch, whereas ripe fruits are quite generally free from starch. The reaction may be observed only in the fruit pulp cells, indicating the presence of unripe fruit, or it may be
SPECIAL TESTS 13
limited to the non-cellular portions of such substances as jams and jellies, indicating the use of fruit juices obtained from unripe fruits.
7. Microscopical Examination of Bacteria and Metals by Direct Sunlight.—Very minute quantities of certain minerals as iron, copper, mercury, and a few others, can be detected in liquids and semiliquids (in the form of metallic hydroxides) when examined (on slide mounts) by means of direct sunlight. All transmissible light must be cut off.
Direct sunlight can also be used in making bacterial counts in liquids, using the Thoma-Zeiss hemacytometer (Turck ruling). The bacteria are readily recognizable on the dark background, standing out far more clearly than in the usual examination by transmitted light, because of the more pronounced color contrasts.
8. Micro-gluten Test—Mount a bit of the flour in water on a slide, being careful not to use too much water. Cover with cover glass and move cover glass to and fro a few times on the mounted material. ‘The gluten separates into stringy fragments which may readily be seen under the low power of the compound microscope. The use of a weak solution of carbol-fuchsin, sofranin, or other stain, will bring out the gluten particles more clearly.
9. Hand Gluten Test.— Moisten wheat flour with water, making it into a dough. Knead constantly and carefully under a slow
1The optical principles of the ultra-microscope of Zsygmondy and Siedentopf depend upon the use of direct sunlight (or other intense light) combined with an absolutely dark field, with or without the use of a condenser, the rays of light being directed upon the object to be examined approximately at right angles to the optical axis of the compound microscope.
The limits of vision with the ultra-microscope are approximately 0.003, however, solid particles (as of metallic colloids) of not more than 0.003u in diameter show no structure, they appear rather as points of light.
The limits of vision with the ordinary microscope are, for air (white light) about 0,304, for homogeneous immersion (white light) about 0.254, and for homogeneous immersion when rays of shorter wave length than white light (as the blue spectrum)
are used, are about o.15p.
14 MICRO-ANALYTICAL METHODS
stream of water, washing out all of the starch. The gluten sepa- rates out as atenacious gummy mass. With care fairly accurate quantitative results may be obtained. Weigh the dried flour and compare with weight of the dried gluten mass. With cereal flours other than wheat, the entire dough mass is gradually washed away, leaving no gluten.
ro. Agar in Jams, Jellies and Similar Fruit Products.—The method generally recommended is to ash a sample of the jam or jelly at as low a temperature as possible, and to add weak hydro- chloric acid for the purpose of decomposing the carbonates, etc. If agar has been added to the substance the silicious skeletons of diatoms will appear in the ash residue examined under a com- pound microscope.
A far better method is to dissolve (with heat) about ro grams of the substance in 200 cc. of distilled water and centrifugalize (while still hot) for half an hour. Decant off the supernatant liquid and examine the residue microscopically. If agar has been added, characteristic agar diatoms (mostly Arachnodiscus ehren- bergit Baillon) will be found, also undissolved agar cell fragments and remnants of undissolved parasitic algal forms, which are quite universally found upon agar. The undissolved agar rem- nants and the algal parasites, which are in fact almost as character- istic as the diatoms, would be wholly destroyed by the ashing process. Furthermore, the ashing-acid process, no matter how carefully done, results in a comminution and destruction of some of the diatom shells. Finding one or more diatoms and one or more algal remnants in one slide mount (or in 5 to 20 fields of view) is conclusive evidence that agar has been added, though this does not indicate the exact amount that is present. If the characteristic structures (diatoms and algal remnants) are com- paratively abundant then it is safe to conclude that agar has been added in considerable amount (2-4 per cent.) or that an impure grade of agar was used. The purer the grade of agar the fewer are the diatoms present, but no agar has yet been found on the
SPECIAL TESTS I5
market which is wholly free from diatoms, undissolved agar cells and algal parasites.
The reason why distilled water should be used in making the solution for centrifugalizing is because ordinary hydrant water may contain diatoms, which might be confusing, especially to a beginner, although the marine diatoms are mostly quite different in form from the fresh water diatoms. With a high-speed centri- fuge less material and less time need be consumed. Also, the more complete the solution the better the results.
8. Micro-chemical Color Reaction Tests
There are certain micro-chemical color reactions, other than those already mentioned, which are of great value in determining the presence of impurities or adulterants in liquids and semi- liquids. The methods as perfected by F. Emich depend upon the use of cotton fibers treated with certain chemicals which convert the metallic compounds into the sulphides. The prepared threads can be readily transferred to the several solutions used and the color and precipitation effects can be observed under the micro- scope. The following are the more important reagents and reactions: ;
1. Cotton Threads for Metal Tests.—Dip absorbent cotton threads alternately into 15 per cent. solutions of sodium sulphide and zinc sulphate, pressing between blotting paper, and air-dry each time.
The threads thus prepared should assume a deep black color with a 1 per cent. solution of silver nitrate. They may be kept for a long time and are used to demon- strate the presence of As, Sb, Au, Pt, Cu, Hg, Pb and Bi, in various chemical compounds.
2. Ammonium Sulphide Vapor Test.—Place a few fibers of absorbent cotton into a drop of the suspected solution and allow the moisture to evaporate. Suspending the threads in the vapor of ammonium sulphide will indicate the presence of Cd, Hg, Ag, Fe, Co and Ni (dark to black coloration).
The prepared threads are used in the following tests:
a. Arsenical Test.—Dip a sodium sulphide thread into the suspected solution and allow to dry. In the presence of 0.008 per cent. arsenic there is a distinct yel- lowish coloration, due to the sulphide of arsenic formed in and upon the threads. The arsenical threads will also show the characteristic reactions with hydrochloric
16 MICRO-ANALYTICAL METHODS
acid, ammonia and ammonium sulphide by bringing a drop of the reagent in contact with the thread upon the slide. (See also Biological Test for Arsenic in Part II.)
b. Zinc Test.—Dip cotton fibers into the suspected solution, allow the moisture to evaporate, and then dip the threads into a solution of gold chloride. A violet coloration develops which remains in the presence of acids but vanishes in the presence of chlorine water, indicating the presence of zinc chlorite. The reaction is appreciable in the presence of 0.003 wg of zinc chlorite, whereas in the form of the sulphite, 0.1 wg of zinc is required to show the reaction.
c. Antimony Test.—Dip a sulphide thread into the solution, allow solution to evaporate and then expose the thread to the vapor of ammonium sulphide. If the solution to be tested contains considerable hydrochloric acid, sulphide of anti- mony is formed upon evaporation.
d. Gold Test.—Gives a brown coloration with the sulphide thread, which color disappears upon prolonged exposure to ammonium sulphide, more quickly on ex- posure to chlorine, bromine and sodium hypochlorite. The threads which have been decolorized with chlorine are colored blue to black with iron chlorite and violet to red with zinc chlorite.
e. Silver Test.—A neutral or faintly acid silver nitrate solution gives a brown to black coloration with the sulphide thread, the depth of the reaction depending upon the concentration of the solution. The fibers can be decolorized by placing in sodium hypochlorite, and the color can be restored by means of zinc chlorite or an alkaline solution of grape sugar. Sulphuric acid will again decolorize.
f. Mercuric Chloride.—Cotton threads dipped into a solution containing mer- curic chloride and exposed to the vapors of ammonium sulphide or ammonia, are colored black. The color is quite permanent in the presence of acids. A sulphide thread is colored yellow in neutral solution of mercuric chloride, changing to black in the ammonium sulphide vapor.
g. Lead Test.—Neutral lead solutions (lead nitrate) turn the sulphide threads yellow and black on prolonged exposure to ammonium sulphide. In acid solutions the color reaction with the sulphide thread is black. The yellow coloration is promptly changed to black upon exposure to ammonium sulphide, or when placed in weak sulphuric acid (1:15). The latter reaction distinguishes between lead and mercury, as the yellow coloration of the mercury is changed very slowly with dilute sulphuric acid.
h. Bismuth Test.—Solutions color the sulphide thread reddish-brown. Bromine causes the color to disappear. Potassium dichromate causes a yellow coloration, while alkaline solutions of zinc chlorite produce a black coloration, Lead solutions are not reduced by alkaline solutions of zinc chlorite.
i. Iron Test.—Ammonium sulphide vapor gives a black precipitate which is soluble in weak solutions of hydrochloric acid. Potassium ferrocyanide gives a blue coloration.
Jj. Copper Test.—Solutions of copper sulphate give a brown coloration to the sulphide thread, which color persists in 10 per cent. hydrochloric acid, but disappears on exposure to bromine vapor. The threads which have been bleached with bro-
SPECIAL TESTS 17
mine give the copper ferrocyanide reaction when placed in an acidulated solution of potassium ferrocyanide.
The following table from the work by Koenig gives the relative sensitiveness of the tests above described :1
Elements in conn - combination i Reaction Limit Comparative
valency (mg. X 108) sensitiveness
| BO patted eke | Curcuma thread................ oO. 1 in 33,000 AG ietana ne Sulphide thread... 2.22.0 ca0e0ss 10.0 ) I in 2,500 SU Garrett Sulphide thread................ 1.0 | 1 in 40,000 Sn”......... | Violet color with sulphide thread 3.0 + TI in 20,000 Au’”’........ | Sulphide thread—brown, purple. . Ro) ) rin 22,000 12) ci nee Sulphide thread................ 8.0 | 1 in 6,000 CU aes aexs Sulphide thread + ferrocyanides. 8.0 I in 4,000 Ag’......... | Sulphide thread + Ag......... 5.0 r in 22,000 FA secre a exces || ANUEL MAD OR: canctappienn cima < aes cto ac 8.0 1 in 25,000 He onan wey Sulphide thread wa. cae cea nes 5.0 I in 20,000 Pb eaeexecy | Sulphide +PbCrOmeccces geese 8.0 , TAM Tg ico Be Jawa ae Sulphide + chromate + Bi....... 8.0 | 1 in 9,000 (crcl canara een (NH;SH) vapor...............- 6.0 | 1 in 9,000 Fe ccsacce an | CNA 3S A) — DIE. oe oe essences se 8.0 | 1 in 3,500 Co’’......... | NH3;SH or nitroso—beta—naph- : EO ester ap seseoer aie. ates chat pal 3 | I in 100,000
INI eget es | INTE GS His ce g canner a asic se ee me | 0.3. ++ JT in 100,000
9. Making Analytical Reports
The methods of micro-analysts, whether in private, commercial or government laboratories, should be uniform. Much could be
1 The comparative degree of sensitiveness of the different chemical compounds concerned in the color reactions above described and tabulated is indicated by the number of cubic centimeters in which 1 gram of the substance in solution is still appreciable. The actual limit, determined experimentally, is indicated in terms of milligrams, that is 0.001 mg., represented by wg. Expressing the com- parative sensitiveness (CS) in a formula we have
pg limit molecular weights _ amount limit “* combination valency
CS =
or to give the example for boron, we have
_ 0.00001 |, 59 _ = 9.00000006 ** 3 332009
18 MICRO-ANALYTICAL METHODS
done to bring this about if the analysts were to meet for the purpose of comparing methods and results. Uniform blank re- port forms should be adopted and used in the micro-analytical laboratories, somewhat like those used by chemists. It cannot, however, be denied that the efficiency in the work done depends largely upon the ability, judgment and experience of the analyst.
The reports of the micro-analysts may be made according to the following groups:
I. Drugs and foods of vegetable origin, including dry or solid products of both animal and vegetable origin.
II. Liquid or moist products of animal and vegetable origin (canned and pre-
served products generally). III. Bacterial examinations of liquids, foods and drugs.
There should be a special blank report card for each group of substances, arranged as follows:
Form No. I
ING iuhdieetennany Salelapeineeer (I. S., laboratory or other serial number) TTT Ul ctcnivteka tenctnisotacncel ort eens Nelda path ostnciatinlteenaichietedameuat Sample received............... Sample examined............. Condition of wrappings and seals............00.000 000 c eee eee One ame eRe TOS Sic, sacs aterialnen nica aneseredogsin & aris reoreenacs drenireloybnn mecrens
WOTTSTS PETES? GEA CE] te anianiutresn aansrndncin heuponty miptrelishean cmccenay
(C10) (5) Seay ORR Nee aE Tannen re mn - Vern Set amr OEE TAN nae
DOE ses eer ih mt lO ee es ag Mgt sk ie id alg idcses
CESS asses: crore apy er at YA stain manor be ele ade sue a PNET UGH HOSES, spores ase imere © dite: insnounrensie woke unin snesansuisn tinea sevens
Sand (beaker test)............. Per cent.
ENS Be acne te hain ccsegiatn cs stoners ara Per cent.
Acid-insoluble ash.............. Per cent.
ANALYTICAL REPORTS
Form No. II
(No., label, dates, condition of seal and organoleptic tests, as for form I.)
Adjunct tests. Sublimation tests for EISEN ZO1 CHA CHM cise gts a erteraam Nb cede astlpeitetta eats don eta ed
Sali Hi Gravel Gnu sie sasnigs co fe wen ream a Wadiator econ 6 Ney coerce
Boric acid (curcuma thread) Iodine reaction
Microscopical findings. General
Cytometric counts.
DEA east: CCl Sa rercraimsigneawerrae aan aetis oe aan per cc.
Living yeast Celli csi ns once nrg aeons earn amen per cc.
Ba Ctenasincnnc war nea cnwuweres daiagiogn es «aunt per cc.
Mold (hyphal fragments and hyphal clusters). ..per cc.
MOlA SPOKES) ee icos sate manne ERE ROT TA RHEE per cc. Conclusions
19
20 MICRO-ANALYTICAL METHODS
Form No. IIT
Bacteriological Examination
(No., label, dates, condition of seals as for form I.)
1. Direct count. (Thoma-Zeiss hemacytometer with Turck ruling.) 1. Bacilli per ec by COCR PEF Cli nna 54 ens CAR RERSS EENMRE DEHESS SEOERE ES
II. Plate and tube cultures. (Lactose-litmus-agar.)
1. Temperature differential test. a. (20° C.) colonies per cc b. (38° C.) colonies per cc 2. Color differential test. a. Pink or yellow colonies per cc......... b. Not pink or yellow colonies per cc
. Gelatin liquefying colonies per cc
» IAGO TEACTIOR CA eons cssp donk deapie caw deacon oe duane
. Neutral red reduction (+)
, Gas Ciydrogen) formulas... cs neces vane ewan
. Gram stain behavior (+).........0......
. Presumptive colon bacillus test (+).
a. Amounts used
Om An bw
ANALYTICAL REPORTS
We may give an example of a report as follows:
Form No. II Lab. No. 462. Label: Pure currant jelly. Madeby Smith, Jones & Co., Nan- tucket, Wis. Sample received August 5, 1914. Sample examined August 5,
1914.
Condition of seals: Good, unbroken sample. Organoleptic tests: Not conclusive.
Consistency or feel: Poorly jellied.
Color: Normal for eurrant jelly.
Odor: Faint, somewhat disagreeable.
Taste: Not characteristic, bitterish, quite acid.
Adjunct tests. Sublimation tests for Benzoic acid: Negative. Salicylic acid: Very marked. Boric acid (curcuma thread): Negative. Todine reaction: Very marked.
Intracellular: Negative.
Extracellular: Positive, very marked.
Special tests: Salicylic acid color reaction, with ferric chloride, very marked. Microscopical examination.
General. Some apple tissue (window cells and pulp cells) and currant tissue (selerenchyma) present. Added wheat starch about § per cent.
Cytometric counts.
Dead yeast cells, 80,000,000.................... per cc. Living yeast cells; W00C ci .03 ceed Gauiewavhuatias per cc. Bacteria, 660,060,000 ii oe vain da eur eee yee per cc. Mold (hyphal fragments and clusters), 84,000 per cc. Mold Spores, 5,000/000i0.15c0cccaeei naw adeacas per cc. Smut. Spores, Woes vue we a anus axa woe naa k a4 ahs per cc.
Conclusions: Misbranded. Adulterated with apple and with wheat starch and made from fermented and decomposed ma- terial, preserved with salicylic acid. Not fit for human con- sumplion because of the quantity of yeast, mold and bacteria present.
John Doe, Analyst.
21
22 MICRO-ANALYTICAL METHODS
The great advantage of the micro-analytical work as compared with chemical work lies in the fact that small amounts of the substances are used for analysis, the equipment is comparatively inexpensive and the results are quickly attained. From twenty to forty and even sixty samples of simple spices can be examined in one day, from five to twelve samples of powdered vegetable drugs, cocoas, chocolates, flours, meals, etc., and perhaps an equal number of jams, jellies, etc.
Because of the very close relationship between the micro- scopical and bacteriological work, as already explained, certain essentially micro-analytical methods will be given under bac- teriological methods, more especially in Chapter 2 of Part II which deals with the direct bacterial counts, and also under milk analysis, water analysis and meat analysis.
DESCRIPTION OF PLATE I
Fic. 1—Types of Pollen Grains.—1r. Saffron flower. 2. Flax. 3. Pink. 4. Pumpkin and squash. 5. Cloves. Mature pollen grain. 6. Cloves. Immature pollen grain. 7. Onagracee. Circea lutitiana (Enchanter’s Nightshade). 8. Scutellaria. 0. Mallow. Distended by moisture. 1o. Mallow. Normal form. t1. Albuco. 12. Lobelia inflata. 13. Composite, showing one mature and two immature pollen grains. 14. Hibiscus. 15. Pine pollen. 16. Santonica. 17. Mentha species. 18. Hyoscyamus niger.
Fic. 2.-Potato Starch——The granules are large and the markings (hili, lamel- lations) are distinct. The cross bands under the polarizer are very distinct. Potato starch, mounted in water, makes a good test object for judging the resolving power of objectives. Dried and ground potatoes and potato parings are sometimes used for adulterating purposes.
Fic. 3.—Starches.—1. Sago starch from Cycas revoluta (Cycadacee). The commercial article known as sago is usually in the form of small granules (pearl sago). There are many false sagos made from other than Cycad or Palm starch. Much of this false sago is made from corn starch.
2. Canna starch from several species of Canna. The markings are very distinct, the hili being at the larger end asarule. Also called arrowroot (lows le mois arrow- root).
3. Cassava or tapioca starch from the tuberous roots of Manihot utillissima and other species of Manihot. Simple and compound granules; the granules are largely separated in the processing, thus giving the appearance of simple granules. Their compound origin is, however, recognizable by the contact facets.
4. Maranta starch (.Arrowroot starch) from Jfaranta arundinacee (Marantacez). The granules have many of the structural characteristics of potato starch.
5. Yam starch from several species of Dioscorea (Dioscoreacez).
Fic. 4.—Dextrinized Starch.—The process of baking and cooking causes the starch granules to undergo marked structural changes. They become much enlarged, the outline becomes quite indistinct and the hili and lamellations are distorted and correspondingly indistinct. 1. Normal wheat starch granules. 2. Normal rye starch granules. 3. Dextrinized wheat and rye granules. 4. Normal and dextrinized corn starch. 5. Normal and dextrinized bean starch. 6. Normal and dextrinized ginger starch.
PLATE I
DESCRIPTION OF PLATE II
Fic. 5—Types of Crystals of Calcium Occurring in Different Plants.—1. A parenchyma cell containing a bundle of needle shaped (acicular) crystals of calcium oxalate {raphide). 2, 3, 4, Acicular crystals differing in length, as they occur in Scilla and in other representatives of the liliaceous groups of plants. 5. Much elongated prismatic crystals as they occur in Quillaja andin Iris florentina, 6. Prismatic crystals very widely distributed in the plant kingdom. 7. Elongated prismatic crystals. 8. Twin crystals as they occur in Ulmus bark. 9. Very large aggregate crystals as they occur in Rheum and Polygonum species. 10, 11. Smaller aggregate crystals very widely distributed in the vegetable kingdom. 12, 13. Very minute prismatic (pyramidal) crystals as they occur in Belladonna. 14. Prismatic crystals as they occur in Hyoscyamus and in other plant groups.
Calcium oxalate crystals are among the highly diagnostic structural characteris- tics of drug plants and should be studied not only as to form but also as to size. They are not dissolved in the usual mounting media and are not destroyed by heat. They dissolve slowly in the stronger acids (hydrochloric acid).
Fic. 6.—Types of Bast Cells as They Occur in Barks and in Other Plant Parts.—1. Shorter bast cell as they occur in the cinnamon barks. 2. Typical bast cell (showing a portion of a cell only) as they occur in willow bark, in Ulmus, in Mezereon, etc. 3. Branching bast cells as they occur in Quillaja and in Prunus
bark. 4. Greatly thickened sclerenchymatous bast cells as they occur in the Cinchonas.
Fic. 7—Types of Sclerenchyma (Stone) Cells.—1. Typical sclerenchyma cells as they occur in the endocarp of drupaceous fruits and nuts. 2. Elongated bast- like sclerenchyma cells. 3. Thin-walled typical sclerenchyma cell. 4. Scleren- chyma cell with unequally thickened walls as they occur in the cinnamons. 5. Large thin-walled sclerenchyma cells as they occur in the seed coat of Amygdala. 6. Branching sclerenchyma cells as they occur in tea leaves and in peanut exocarp. 7, 8, 9. Forms of sclerenchyma cells.
Fic. 8.—Typical Sclerenchyma Cells (in groups) as they occur in the pulp of the pear.
DESCRIPTION OF PLATE III
Fic. 9.—Buckwheat.—r. Proteid-bearing tissue. 2. Starch-bearing endosperm tissue. Cell walls are very thin and the entire cell lumen is packed with starch granules. 3. Starch granules. The granules resemble those of corn, being some- what smaller. 4. Sclerenchymatous fibers.
Buckwheat is the predominating ingredient of the buckwheat pancake flours and is occasionally used as an adulterant of spices.
Fic. 10.—Tissues of the Pine.—r1. The characteristic tracheids with bordered pits. 2. Bast-like fibers of the bark. 3. Crystal-bearing bark parenchyma cells. 4. Tracheids in radial view. 5. Medullary ray cells in radial view. Pine wood (pulp) is much used in making paper.
Fic. 11.—Sclerenchyma Cells of Olive Pits.—Ground olive pits were, until recently, extensively employed as an adulterant of spices and drugs.
Fic. 12.—Clove Stems.—.\ very common adulterant of cloves and of allspice. 1. Typical sclerenchyma cells. 2. Sclerenchyma cells with unequally thickened walls. 3. Sclerenchymatous bast fibers.
DESCRIPTION OF PLATE IV
Fic. 13.—Cassia Buds and Cassia Stems.—r. Sclerenchymatous fibers of the cassiastems. 2. Bast fibers of cassiastems. Parenchymatous cells of the buds. 4. Trichomes of buds. 5. Thick-walled parenchyma cells. Cassia buds and cassia stems are frequently used in adulterating cloves, allspice and cinnamon.
Fic. 14,.—Coffee Adulterants.—r. Sclerenchyma cells of date pits. 2. Scleren- chyma cells of the walnut shell. 3, 4, 5. Tracheids and inulin-bearing parenchyma cells of chicory. Figs and prunes are also much used as coffee adulterants, also cereals, fleshy roots, acorns, etc.
Fic. 15.—Wheat Tissues.—1. Wheat starch. 2. Trichomes from the bran. 3. Starch-bearing parenchyma. 4. Epicarp cells. 5. Proteid-bearing cells from middlings. Rye histology is similar to that of wheat. Wheat flour is used in macaroni, spaghetti, noodles, etc. Wheat flour, bran and middlings are much used for adulterating purposes. Rye starch differs from that of wheat in the larger size of the granules and the greater prominence of the hili.
Fic. 16.—Rice Tissues.—1. Starch. Single granules and aggregates. These aggregates are characteristic of rice and of oats. 2. Starch-bearing endosperm cells. 3, 4, 5. Epicarp and pericarp cells. In form the starch granules of rice, oat, corn, darnel, millet, fox-tail, buckwheat and chess resemble each other. The size varies very much.
DESCRIPTION OF PLATE V
Fic. 17—Bean Tissues.—1. Epidermal palisade tissue with the crystal-bearing hypoderm. 2. Starch-bearing endosperm tissue. 3. Starch granules with promi- nent fissured hili. 4. Spongy tissue. 5. Epidermal palisade cells-in vertical view. 6. Prismatic crystals of calcium from hypoderm.
Ground beans, peas and lentils are used for adulterating purposes.
Fic. 18.—Histology of Mallow Leaf.—1. Transverse section of leaf showing stellate trichome, epidermal, palisade and spongy tissue cells. Aggregate crystals of calcium oxalate are present. 2. Stellate or aggregate trichomes. 3. Epidermal cells (lower) showing stomata. Mallow leaves are extensively employed for adul- terating leafy spices and drugs.
Fic. 19.—Histology of Corn.—1. Corn starch. 2. Starch-bearing endosperm of corn kernel. 3. Trichomes of the chaff of the corn cob. 4. Sclerenchymatous cells of the corn cob. Ground corn cobs are used for adulterating purposes and also in the manufacture of artificial maple syrup flavor.
Fic. 20.—A Few Types of Trichomes.—1. Branching trichome of mullein. 2. Many-celled simple trichome of henbane showing wart-like marking on outer surface. 3. Simple single-celled trichome as of rye and wheat. 4. Glandular trichome with two secreting cells. 5. Glandular trichome with one secreting cell. 6. Many- celled glandular trichome. 7. Simple, single-celled trichome of Indian hemp. 8. Much elongated and twisted single-celled trichome, as of sage. 9. Sessile glandular trichome (Eriodictyon). 10. Indianhemp. 11. Pyrethrum. 12. Simple trichome.
PLATE V
DESCRIPTION OF PLATE VI
Illustrating the Histology of a Typical Bark Showing all of the Tissues Which May be Found in a Bark.—a, Longitudinal section in the radia) direc- tion but not showing the medullary rays. B, Transverse section. 1. Outer bark. The demarkation between outer and inner bark is not always distinct. 2. Inner bark. 3. Beginning of wood tissue. a, Epidermis. Always wanting in tree trunks and older branches. 6, Cork tissue. c, Bark parenchyma. Cell-walls are usually not suberized and the cells may contain various inclusions such as crystals of calcium oxalate, tannin, starch granules and resin. d, Groups of sclerenchyma cells. These, when present, normally predominate in the outer bark. ¢, Crystal- bearing fibers which usually accompany the bast fibers. f, Bast fibers. These, when present, normally predominate in the inner bark. The fibers may occur singly orin groups. g, Cambium. h, Wood fibers. 7, Ducts. Usually of the typically porous type. k, Medullary rays.
An excellent typical bark having all of the histological elements indicated in Plate VI is Rhamnus purshiana. The demarkation between outer bark and inner bark is well defined in Ulmus and Quillaja.
Pirate VI
Fic. 21.
II
BACTERIOLOGICAL METHODS IN FOOD AND DRUGS LABORATORIES
1. Introduction
The study of the significance of bacteria in foods of all kinds is one of the most important and interesting of scientific subjects and one which has received much attention ever since the science of bacteriology has become more highly developed as the result of the perfection of the compound microscope. For a long period of time the popular notion has prevailed that bacteria were essentially harmful and to designate any substance as bacterially contaminated was to pronounce it dangerous and to condemn it without trial. We now know that many, in fact most bacteria, are beneficent rather than harmful, and that many different species of bacteria are concerned in the preparation of food substances. It cannot be denied, however, that many kinds of bacteria as well as other organisms are concerned in the pro- duction of changes in food substances which we know to be highly detrimental to the well being of the human race. It is the duty of modern sanitary science to guard against disease and the contamination of food substances through the invasion of patho- genic and otherwise objectionable organisms. It is the work of the food bacteriologist to detect objectionable contaminations in foods and to aid in developing those processes and methods of food preparation and manufacture which will prevent the re- currence of such contamination. The food bacteriologist will center his attention on the following:
23
24 BACTERIOLOGICAL METHODS
1. Chemical (decomposition) changes in foods and drugs induced by the various organic infecting agents, as bacteria and other living organisms, which render such substances unfit for human use or which render them dangerous for human use.
2. Foods and drugs as actual or possible carriers of infecting agents which are or may be dangerous to life or which may or might be injurious to the physical
well-being of the human species.
It goes without saying that the food bacteriologist must pro- ceed carefully in order that there may be no hasty decisions re- sulting in the condemnation of food products which are not in- jurious. There is, however, little excuse for hasty or unjustifi- able passing of judgments as regards the quality of food. Bac- teriological and toxicological methods have been sufficiently perfected so that the careful analyst need not make unfair or unwarranted decisions. The men entrusted with the critical examination of foods and drugs as to their fitness for human use should be investigators of authority and should have had wide range of practical as well as laboratory experience, and they should furthermore be possessed of good judgment. While the condemnation of food materials should not be hasty it should on the other hand not be too tardy or conservative. The prime object of the work by the food bacteriologist is to protect the consumer, not the dealer or manufacturer. This very important point is most unfortunately not properly heeded with the result that some of the work done in the administrative laboratories is, or appears to be, in the interests of the dealer or manufacturer.
A goodly number of infections enter the human system by way of the mouth with the ingested foods and drinks. Food substances form excellent pabula for the bacteria and other parasitic agents which enter the digestive tract or which may already have entered. Foods and drinks are exposed to infection in a great variety of ways. For purposes of illustration we may cite bread, the so- called “‘staff of life,” as one of the foods which is liable to infec- tion. It may be assumed that the loaf of bread, when it is taken from the oven, is entirely sterile and free from living organisms of all kinds. Just as soon as the loaf is cool enough to permit it,
INTRODUCTION 25
the promiscuous manipulation begins and is continued until the bread enters the digestive tract of the consumers. The loaves are handled by the dirty, sweaty and oftentimes diseased hands of the baker or his helper. Basketfuls of uncovered bread are dragged over the dirty floors, over sidewalks, and through the filthy alleys. The uncovered loaves are repeatedly handled by the bakery drivers whose hands and clothing are generally very filthy. The uncovered loaves are left on doorsteps and other exposed places on the premises of the consumer. This much- handled bread is finally eaten, crust and all, without any attempt at sterilization. Such bread may be contaminated with a great variety of disease germs. Infections from hands, disease-bearing dust from the streets and alleys, excreta from disease-carrying flies, excreta from the intestinal tract of man and of animals are among the deposits which have been found on the exterior of bread. Miss Katherine Howell has traced an epidemic of typhoid fever to the consumption of contaminated bread and she has demonstrated the presence of typhoid fever germs and of in- testinal bacteria on numerous loaves of bread. Edward Bartow, director of the Illinois State Water Survey, has also demonstrated a bread-borne typhoid epidemic in Rockford, Illinois. Colon bacilli are usually found in considerable numbers on every loaf of unwrapped bread. Every loaf of bread from the public bakeries should be wrapped in sterilized paper bags just as soon as it leaves the oven and it should remain in these bags until ready to be placed before the consumer.
Polluted water may carry the germs of dysentery, of cholera, of typhoid fever and the larve of intestinal and other parasites. Clams and oysters have caused typhoid epidemics. Fruits and vegetables are frequently polluted with fertilizer, especially where human fertilizer is used, as is the custom with the Chinese truck gardeners not only in China but also in other lands where the Chinese are found. Using human excrement as a fertilizer of soil should be prohibited by law. American army surgeons at
26 BACTERIOLOGICAL METHODS
the time of the American occupation of Cuba made the filthy farming customs of the Chinese the object of a special report but apparently nothing ever came of the recommendations made. The Chinese also import dried human feces and dried human urine for medicinal purposes and a recommendation was made to Washington to prohibit such importations but apparently nothing has ever been done about it.
Pollution of fruits and berries of all kinds may come from the hands of pickers. Gathering of fruit is usually done by the very ignorant, those who have no proper conception of personal cleanliness and of sanitation. Entire families, men, women, and children, migrate to the fields and work during the hottest part of the season. ‘They live in the open or in tents or perhaps in covered wagons. The environment of these temporary abiding places is anything but sanitary. Sickness often prevails in these camps, such as typhoid fever, scarlet fever, measles and dysentery, to say nothing of the more common body and intestinal parasites which infest many of the laborers. These multitudinous infections are brought in contact with the fruit, berries, peas, beans, lettuce, cabbage, cucumbers, etc., etc. The products of the field are then carried to the consumer by a driver who disseminates the contami- nation by mixing and frequent handling. And in spite of all this there are those who insist on eating berries unwashed because they might lose some of the natural flavor.
Next to bread, milk is the most popular food substance. Most unfortunately milk is also one of the best food substances for all manner of germs, harmful and harmless. Sickness in those employed about the dairying establishment has time and again caused epidemics, such as diphtheria, typhoid fever, scarlet fever, tuberculosis, dysentery, and streptococcic tonsillitis. Diseased animals transmit infection to humans, as will be more fully ex- plained in the chapters following.
It is generally believed that the usual processes of baking and cooking as practised in the household are a sure guarantee that
INTRODUCTION 27
the foods so prepared are entirely free from living bacteria! infection of all kinds. This is true of some foods but not by any means of all of them. Dr. W. A. Sawyer, Director of the California State Hygienic Laboratory, in reporting upon an epidemic of 93 cases of typhoid fever (at Hanford, California) due to a single carrier, traced the source of the infection to cooked Spanish spaghetti, prepared by the typhoid carrier. The following tests were made in the California State Hygienic Laboratory to ascertain the effects of baking on the presence of typhoid fever germs in the interior of a mass of spaghetti.
‘““A large hot-air sterilizer was heated and kept between 160° and 170° C. (320° and 338° F.). The pan of spaghetti was in- troduced and subjected to this heat for 30 min. When the dish was removed the surface was of a golden brown color. The ap- pearance and aroma suggested that the spaghetti was thoroughly cooked and very hot. The temperature near the top was 54° C. (129.2° F.) and at the middle, 23° C. (73.4° F.). Ten minutes later the temperature at the middle was 24° C. (75.2° F.) and the dish was then returned to the oven. Cultures taken at various levels showed that the typhoid bacilli were alive even close to the surface.
“In the next baking the oven was kept at temperatures ranging between 207° and 214° C. (405° to 417° F.). After half an hour the pan was removed. The surface was dark brown and the points sticking up from it were charred. The liquid around the margin was boiling vigorously and the whole dish was sizzling. The temperature just under the surface was 83° C. (181.4° F.). At the middle it was 28° C. (82.4° F.) and near the bottom it was 48° C. (118.4° F.). An hour later the temperatures had be- come nearly equalized and were 46° C. (114.8° F.) near the top, 42.5° C. (108.5° F.) at the middle, and 43° C. (109.4° F.) near the bottom. This showed that the interior of the dish did not reach even a pasteurizing temperature.
“Cultures taken at the surface soon after the pan had been
28 BACTERIOLOGICAL METHODS
removed from the oven showed no typhoid colonies and very few of other kinds. Cultures taken at a distance of half an inch from the surface showed a few colonies of the typhoid bacillus, most of the organisms having been killed. Cultures from a depth of 216 in. showed abundant colonies of typhoid bacilli. In these cultures the typhoid colonies were identified by their appearance on Endo medium and Russell medium and also by agglutination by anti-typhoid serum.”
Dr. Sawyer sums up the experimental evidence as follows:
“The laboratory experiments completed the explanation of the Hanford outbreak by showing that the sauce used in making the Spanish spaghetti was a good culture medium and that the dish had not been sterilized after leaving the house of the typhoid carrier.
“Moreover, it was demonstrated that cooked dishes must be considered as possible conveyors of infection unless it can be shown that the method of cooking would produce complete sterilization. The slowness with which heat penetrates dishes like the Spanish spaghetti shows that very prolonged heating would be necessary for sterilization of large dishes of such food. Ordinary baking merely incubates the interior of these masses of food.”
This report by Dr. Sawyer is of special significance to the food bacteriologist as it illustrates two very important factors concerned in the study of food sanitation: First, the possible contamination of food materials through carriers of disease, and secondly, the necessity of studying more carefully our pres- ent methods of sterilization (of food materials) through the agency of heat. As will be more fully set forth in subsequent chapters, the examination of canned foodstuffs shows that sterili- zation is far from complete in the great majority of cases.
In addition to the more or less acute infections traceable to the consumption of contaminated food products, there are the multitudinous infections which are of slow development or
INTRODUCTION 209
chronic in character. In many of these cases it is not possible to acertain definitely how the infection entered the system. There are numerous so-called autointoxications which are said to be due to autolytic changes in the ingested food substances resulting in the formation of toxins which often give rise to very serious and even fatal poisoning. As is generally known, certain toxin-forming bacteria after once gaining access to the intestinal tract may remain there for years feeding upon the contents of the intestines and producing enough of the toxin to give rise to symptoms of poisoning of a more or less chronic character. In some instances the toxin-forming bacteria are not present in suf- ficient numbers or do not multiply in sufficient numbers to give rise to any marked symptoms, and in still other cases the originally pathogenic or toxin-forming bacteria lose their virulency after having lived in the intestinal tract for some time. As is known, there is constant warfare in the intestinal tract between the harm- ful and the really beneficent bacteria, and it is this discovery which has led Metschnikoff and other bacteriologists to find germs which upon being introduced into the intestinal tract would overcome or crowd out the objectionable toxin formers.
Food poisoning has received considerable attention in re- cent years. Vaughan and Novy have suggested a nomenclature applicable to certain recognizable forms of poisonings traceable to foods, as:
Bromatotoxismus or food poisoning. Galactotoxismus or milk poisoning. Tyrotoxismus or cheese poisoning. Kreatoxismus or meat poisoning. Ichthyotoxismus or fish poisoning. Mytilotoxismus or mussel poisoning. Sitotoxismus or cereal poisoning.
The poisonings mentioned are generally due to toxins or related products elaborated by bacteria, but in some instances the exact species responsible for such toxin formation have not yet been determined. The identification of the species of bacteria
30 BACTERIOLOGICAL METHODS
responsible for the poisoning of foods and drinks is of minor im- portance. What is of prime importance to the food bacteriologist is to find the poison and if possible to ascertain the manner in which the poison gained access to the food substance, in order that methods may be devised to guard against the recurrence of such contamination. It may also be stated that in the great majority of cases of food poisoning the nature of the poison and its source have already been determined and means are available to pro- tect the consumer. If the manufacturers of foods and of food products would give proper attention to the modern methods of manufacture, then poisonings due to the eating of such prod- ucts will be a rare occurrence indeed. It is regrettable that so many of the smaller establishments engaged in the manufacture of food products are not better informed regarding the available modern methods of preparing and storing food substances in such a manner as to guard against infection and contamination. It is also regrettable that the various pure food and drugs laws and regulations intended to protect the consumer are not more eff- ciently and more strictly enforced.
We have already suggested a more efficient coordination of the chemical, microscopical and bacteriological methods of analy- sis in our food and drugs laboratories—federal, state, munici- pal and private. The following are the bacteriological methods applicable in the examination of foods and drugs as to quality and purity. It is hoped that the suggestions offered may serve as a basis for establishing more complete practical working methods and at the same time indicate lines for further research.
Just what bacteriological analyses and tests should be made in pure food and drugs laboratories has as yet not been decided upon. However, based upon the present purpose and scope of such laboratories, we submit the following outline as covering the field fairly well and which outline will be followed quite closely in the text, however not necessarily adhering to the same sequence of the subject-matter.
INTRODUCTION 31
QUANTITATIVE AND QUALITATIVE DETERMINATIONS OF ORGANISMS In Foops anp Drucs
Substances to be analyzed.
Liquids of all kinds.
Semiliquids and semisolids miscible with water.
Solids of all kinds.
Numerical and quantitative limits of contamination in different substances.
For molds—quantity of spores and hyphe.
For yeasts—number and kind.
For bacteria—number and kind.
For pus, dirt, sand, etc.
Methods.
Making concentrations.
Making dilutions.
Making the counts and estimates. Bacteria.
Yeasts.
Mold spores and mold hyphe. Alge, in drinking waters, etc. Protozoa.
Pus cells, in milk, etc.
Dirt, sand, etc.
Plate counts—Petri dish cultures. Culture media used. Optimum temperature.
Time of incubation. Qualitative determinations.
Apparatus.
Culture media.
Stains.
Special methods.
Colon group of bacilli. Presumptive colon bacillus test. Sewage streptococci. Dysentery bacilli and amebe. Bacillus typhosus. Paratyphoid group.
Cholera vibrio.
Yeasts.
Molds.
Animal parasites.
Larve, ove, etc.
32 BACTERIOLOGICAL METHODS
Biological water analysis. Bacteria, number and kind. Diatoms. Desmids. Nostoc. Other alge. Molds; significance of. Bacteriological milk analysis. Quantitative. Standards for different geographic areas. Summer and winter standards—temperature standards. Qualitative. Pus and blood corpuscles; significance of. Milk diseases. Blue milk. Ropy milk. Bad odors, bad taste, etc. Sour milk. “Buttermilk”’ tablets. Kefir, koumys, etc. Bacteriological examination of shellfish. The bacteriological and toxicological examination of meat and meat products. The bacteriological examination of eggs and of egg products. Bacteriological examination of mineral waters. Bacteriological examination of pharmaceuticals. Bacteriological examination of sera, vaccines, bacterins, etc. The microscopical and bacteriological examination of syrups. Standardization of disinfectants. Phenol coefficient. Albumen coagulation coefficient. Toxic coefficient. The efficiency value of disinfectants. Biological toxicity tests.
Upon first consideration it would appear that the bacteriolog- ical methods in food and drugs laboratories might be closely simi- lar to those in hygienic laboratories. Such is the case in a gen- eral way, however, with certain well-defined differences. Whereas the bacteriological work in hygienic laboratories pertains to the prevention of disease and finding the primary causes of disease, the work in the food and drugs laboratories has to do with the
INTRODUCTION 33
investigation of the biological factors influencing the quality of food and drugs and the significance of pure food and drugs in the maintenance of the public health and the physical well-being of the human race, as against the pernicious effects of contami- nated foods and drugs.
The question for first consideration is what bacteriological methods are necessary and practicably applicable in testing foods and drugs? This phase of the subject is comparatively new and accordingly there are but few food and drugs bacteri- ologists who have had any considerable general range of ex- perience, and as a consequence there are comparatively few methods fully worked out. Most of the bacteriological in- vestigations and researches pertaining to foods have been along special lines, and indeed much valuable information and useful data have been brought together. Within the last 10 years the work on the sanitary examination of milk and of water supplies has become monumental in volume as well as in importance. Numerous methods have been tried, some to be entirely abandoned after being for a time heralded as the final word in determining the potability of water supplies. The same may be said of the development of the bacteriological examinations of milk supplies.
Quite recently bacteriologists have given considerable atten- tion to the sanitary examination of shellfish, more especially with reference to sewage contamination. In this investigation American bacteriologists have taken the lead. European bac- teriologists have done an enormous amount of work in the ex- amination of sewage and of sewage disposal, to say nothing of the classical researches on yeasts and on fermentation in general. However, the general bacteriology of foods and of drugs is as yet an unexploited field. It is true, the Bureau of Chemistry of the Department of Agriculture has, within recent years (since 1906), done considerable work on the sewage contamination of oysters and other shellfish (Bulletin No. 136, Bureau of Chemistry. U. S. Department of Agriculture, by George W. Stiles) and in
34 BACTERIOLOGICAL METHODS
the quantitative estimation of the microbic contamination of certain food supplies, and still more recently the laboratory division of the U. S. Public Health Service has done much efficient © work on the standardization of disinfectants. We must also mention the work on milk, meat inspection, etc., by the Bureau of Animal Industry and the work on sanitation and related sub- jects by the U. S. Public Health Service, not forgetting to men- tion the vast amount of routine analyses in state and municipal health laboratories and the sporadic research work in the bio- logical and bacteriological laboratories of our colleges and uni- versities and the individual investigations of food and drug contamination on the part of afew of the more enterprising state and municipal health officers. Very recently the sanitary study of mineral waters has received a great deal of attention on the part of individual workers. The Committee of the Laboratory Section of the American Public Health Association has prepared a report covering the general conclusions regarding some of the methods of analysis.
The purely microscopical examination of food substances and of drugs, with reference to contamination by mold, yeast and bacteria, should be a part of the work of the bacteriologist rather than that of the chemist. Therefore, for the sake of completeness, this phase of the subject is included in the present report. We shall now proceed with the discussion of the bacteriological method applicable in food and drugs laboratories, giving only the essential details, however adding certain suggestions intended as a guide for further investigation with a view to the improvement of the present working methods. Detailed description of apparatus and of technique will be given only when thought necessary.
For all practical purposes, the examination of foods and drugs for the presence of biologic contamination (inclusive of bacteria, yeasts, molds, protozoa, ova and larve of higher animal parasites, etc., etc.) is either made directly or indirectly. That is, the sub- stance is either placed on a slide or counting apparatus and the
INTRODUCTION 35
quantitative or qualitative determinations made directly under the suitable power of the compound microscope; or, certain quantities of the substances are placed in or upon certain culture media (Petri dish cultures, tube cultures, etc.) in order to bring out the biological and biochemical characteristics of the contaminating ‘organisms, whereupon the cultural products are examined micro- scopically. In this latter instance the microscopical examination may even be entirely omitted.
The direct microscopical method has some very marked ad- vantages and should be carried out whenever feasible, particularly whert purely quantitative results or estimates is the main object sought after. In other instances the direct method must be combined with cultural tests and the two are often checks upon each other.
2. Direct Bacteriological Examinations—Quantitative Tests
Substances to be examined include waters and liquids of all kinds; sewage; milk! and cream; ice cream, liquid pharmaceuti- cals and medicamenta, oils, catsups, beverages of all kinds, all semisolids such as pastes, jams, jellies, etc., other semiliquids and semisolids which may be readily diluted with water if necessary; solids as powders, pills, tablets, soils, clays, meats, starches, dextrins, flours, meals, dried fruits, dried eggs, dried albumen, sugar, etc. In fact all substances which are in any way liable to contamination by micro-organisms.
The following is an outline of the methods of making determina- tions of the number of organisms in food and drugs.
a. Substances Requiring Concentration——Certain substances which contain comparatively few micro-organisms, as drinking waters, mineral waters, beverages generally, tinctures, fluid ex-
1Tn the case of milk, the centrifuge is first used to separate out the fat as much as may be necessary to make the ready counting of the organisms possible. (See also Chapter on Milk Analysis.)
4
36 BACTERIOLOGICAL METHODS
tracts, aque, etc., must be subjected to processes which will con- centrate the organisms, as by passing the liquid through a filter in which the pores are sufficiently small to leave the organisms behind, as for example a Berkefeld or Chamberland clay tube. In addition to the filter, the centrifuge will be found useful as will be explained later.
Any liquid containing not more than from 100 to 1,000,000 organisms per cc. does not lend itself to direct examination quanti- tatively without concentration. The amount or volume of sub- stance (liquid) to be passed through the filter will depend upon the degree of concentration required. Since the Thoma-Zeiss hemacytometer (with Turck ruling) is to be used in making the counts, the organisms should average at least 4 to 5 in the lgs9 c.mm. areas of the counting apparatus, or 1,000,000 to 1,250,000 organisms per cc. Let us suppose that a direct count is to be made of a drinking water which is very pure, having not more than from 50 to 500 bacteria per cc. In order to make direct counting with the hemacytometer possible, it would be necessary to pass from 20 to 30 liters of the water through the clay filter and thoroughly mix the organisms left in the tube with ro or even 1 cc. of filtered sterile water. To filter that amount of water requires too much time unless a large specially constructed apparatus is installed. For practical purposes, 1 liter is the largest amount of liquid that it will be necessary to filter and reduce to 1 cc., making a concentration of 1ooo. For special purposes the 1 cc. may be further concentrated in the centrifugal tube described in Fig. 3. Weaker concentrates may answer the purpose in some cases, as ten or one hundred, as in sewage, badly contaminated milk and in other liquids in which the number of organisms present may range from 100,000 to 1,000,000 per cc.
The special centrifugal tube described in Fig. 3 is used as follows: After passing a liter of the liquid to be examined through the clay filter tube and thoroughly washing out the organisms and other particles left in the tube, pour the contents into the special
DIRECT EXAMINATION 37
9) A
——— Occ:
-=-- fee,
Mi
ei
SH
Fic. 2. Fic 3.
Fic. 2.—Kitasato filtering outfit ready to be attached to the exhaust pump. A two-opening flask or bottle is interpolated to receive the backflow water, should there be any. Various types of clay bougies may be used with this filter. The rubber tubing for the connection must be heavy so as to prevent collapse by the ex- haust pressure.—(Pitfield.)
Fic. 3.—Special centrifugal tube (A) for concentrating bacteria and other micro- organisms in liquids and also used in isolating or separating motile bacteria from those which are not motile, as is explained under water analysis. The tube has a capacity of 15 cc. with 1 cc. and rocc. marks. The tube isin two parts. The lower narrowed end, having a capacity of 1 cc., is attached to the larger part by means of a rubber-band ring. Centrifugalization is done at high speed. After centrifugali- zation, the 1 cc. tube is removed and the contents thoroughly mixed by means of a platinum wire loop. To avoid loss of the contents of the tube during the mixing, attach the rubber ring. After the mixing the material is ready for the microscopical counting and other examination.
A suitable stopper attached to a brass or other metal rod (B) may be inserted into the narrowed portion of the upper part of the tube in order to prevent mixing of contents when removing the 1 cc. tube. These tubes will also be found useful in measuring the amount of sediment in milk, water and other liquids. For this purpose the 1 cc. portion should be graduated into tenths and hundredths.
3 8 BACTERIOLOGICAL METHODS
centrifugal tube. For washing use about 10 cc. of filtered sterile water, adding up to the ro cc. mark if necessary. Centrifugalize at high speed for 30 min., which will throw the bacteria and other solids down into the narrow 1 cc. end of the tube.
The following is a brief outline of the method of procedure: Use a Kitasato filter with the usual hydrant suction pump attach- ment. Pass a liter of the liquid to be examined through the filter, continuing suction until nearly all of the liquid has passed through. Remove the clay bougie and wash down the organ- isms clinging to the sides of the tube with not more than ro cc. of distilled water which has been filtered and boiled. Place thumb over the opening of the tube and mix the contents thoroughly by shaking for 20 sec., then pour the thoroughly mixed contents into a sterile cylindrical graduate and add sterile distilled and filtered water up to the 10 cc. mark, shake thoroughly and make the counts at once by means of the hemacytometer. This pro- cedure gives a concentration of roo. By means of the special centrifugal tube the concentration may be increased to 1000, as already explained. The method gives approximate results only, the counts as a rule being less than the actual number of organisms present in the liquid, a difference due to three chief sources of error: First, a small number of bacteria (especially the smaller motile forms) will pass through the clay filtering tube; second, some of the smaller bacteria are caught and held in the pores of the clay tube; and third, some organisms will remain clinging to the inner surface of the tube after the mixed contents are poured out for counting purposes. These sources of error are, however, not great, perhaps not exceeding 8 to ro per cent., and are on the side of conservative estimates. The clay bougies used should be of the finest quality and should be of uniform and standard thickness. The sources of error by the direct method are perhaps not as great, certainly not greater, than by the usual plating methods and offer some very decided advantages. The concentrates show, in addition to the bacteria, other organisms
DIRECT EXAMINATION 39
Fic. 4.—Apparatus for fractional filtration, designed for use with Pasteur- Chamberland or Berkefeld filters. a, Glass mantle surrounding filter; 6, Chamber- land filter; c, paraffin joint; d and e, rubber stoppers; f, double side-arm suction flask; g, pinchcock controlling outlet from suction flask; 4, outlet tube surrounded by glass shield and attached to lower end of suction flask by means of short rubber tubing; i, glass shield fused to and surrounding outlet tube as a protection against contamination when the filtrates are drawn off; /, glass inlet tube plugged with cotton, for admitting air into suction flask; k, pinchcock governing the admission of air into flask; 7, vacuum gauge; m, stopcock connected with vacuum pump.—(U. S$ Dept. of Agriculture, Bureau of Animal Industry, Bull. 113.)
40 BACTERIOLOGICAL METHODS
as mold hyphe, mold spores, protozoa, diatoms, etc., besides dirt particles, sand particles, starch granules, body cells, pus cells, etc., etc., which would be lost or rather which would not appear in the plating method. Furthermore, the counts can be made with a great saving of time—in a few hours as against 24 to 48 hr., and longer, by the plate cultural method. It is true that in many instances the direct method must be supplemented by the cultural methods when, in the judgment of the analyst, this becomes desirable or ng@ggsary.
Concentrates may alsojf de by evaporation under reduced pressure. With a little i y a suitable equipment may be constructed in the labora any The container of suitable ca- pacity (z liter and more) igittgenected with an exhaust pump which lowers the pressure sufficiently to cause boiling at a tem- perature not to exceed 37° C.; 24 hr. is usually sufficient time to evaporate the liquid to nearly dryness. After the evaporat- ing process has continued for several hours, various enrichment media may be added to the liquid to be evaporated, which will of course aid the intended isolation and development of the de- sired bacteria. If the enrichment medium is added from the first, annoying bubbling and frothing may take place. This method is especially useful in isolating the typhoid bacillus, the paratyphoid group and the intestinal bacteria in general.
b. Substances Which do not Require Concentration—Badly contaminated substances as sewage, milk from badly managed dairying establishments, badly contaminated liquids of all kinds, soups, broths, beer, wines and such products as tomato catsups, jams, jellies, canned oysters, etc., may be examined directly without the necessity of making concentrations, or of centrifugali- zation, in order to make quantitative and certain qualitative estimates. The substances of this class may be divided as follows, based upon the approximate number of organisms per cc. as determined by means of the spore and yeast counter described under Fig. 5, and the Thoma-Zeiss hemacytometer.
DIRECT EXAMINATION 4I
COUNTING APPARATUS ioe Sor
MOLDS end SPORES
A|B /23456|//23456
m& AL& & S
c;D
Fic. s.—A, Counting apparatus for molds (hyphz and spores) and yeasts. The rulings are 75 sq. mm., 25 sq. mm., I sq. mm. and }g5sq.mm. On either side of the ruled area are glass slips 0.2 mm. thick, so that the entire capacity of the space within the ruled area is 15 c.mm., subdivided into 5 cmm., 4% c.mm. and Mos c.mm. areas.
42 BACTERIOLOGICAL METHODS
1. Substances in which the organisms are not too numerous to permit the use of the 145 sq. mm. areas without making dilutions. That is, substances in which the number of organisms does not exceed 10,000,000 per cc., hence the number of yeast cells, spores, bacteria, etc., may not exceed forty in one of the 4459 ¢c.mm. areas of the hemacytometer. The limit for the spore and yeast counter would be 5,000,000, before making the dilution is necessary.
2. Substances in which the number of organisms and spores are too numerous to permit the use of the 145 sq. mm. areas of the hemacytometer, but permitting the use of the 1499 sq. mm. areas without making dilutions. The total number of spores, bacteria and other organisms may range from 10,000,000 to 100,000,000 per cc., numbers derived from finding on an average from 2.5 to 25 organisms in one of the 14999 c.mm. areas of the hemacytometer.
The counter is used as follows: A bit of the thoroughly mixed substance, as jam, jelly, tomato paste, catsup, etc., is placed on the slide in the ruled areas and covered with a rectangular cover glass (No. 2). Slight pressure may be necessary to make the cover glass rest evenly on the two slips. The counting is done in areas entirely filled (from slide to cover glass) by the substance mounted. The larger areas may prove useful in estimating the amount of sand particles, dirt, etc., present. The 25 c.mm. areas will be used in counting spores, yeast cells and mold hyphe and similar contaminations. It is possible to make counts without dilutions as long as the number of organisms in the areas does not exceed forty. If more organisms are present in one area dilution becomes necessary, as already ex- plained. Making dilutions of 1-10, 1-100 and 1-1000 makes the counting limits 50,000,000, 50,000,000 and §5,000,000,000 per cc. The 1495 c.mm. areas are also used in estimating the quantity of mold hyphe present. Finding clusters of mold hyphe in 25 per cent. of these smallest areas is presumptive proof that the substance is unfit for human consumption. Naturally the more finely divided the substance is the more numerous are the mold fragments. or making mold counts the material to be examined should be reduced to uniform fineness. This could be accomplished by rubbing a thoroughly mixed sample through a sieve of standard mesh, say 14 mm.
B, a simplified modification of the counting apparatus just described, is made as follows: The two slips 0.2 mm. thick are placed in position, but the ruling is omitted and in place thereof an eye-piece scale C is used, the measuring value of which has been carefully determined by means of the stage micrometer. The rulings on the eye-piece must be delicate and the analyst must be careful not to move the eye or change the direction of his vision while making counts.
The ruled slide (D) will be found useful for making quantitative estimates of seeds, sand particles, dirt, larger parasites such as vinegar eels, ova of intestinal parasites, etc., in catsups, crushed berries (strawberries, raspberries, loganberries, etc.), jams and in other vegetable substances. Definite quantities of the substance to be examined are placed upon the ruled area of the slide by means of a small measuring spoon (0.25 gram, 0.5 gram, 1 gram), spread and covered with a suitable cover glass and the counts made in the entire amount placed on the slide, using the low power (80 diam.) of the compound microscope.
DIRECT EXAMINATION 43
3. Substances in which the organisms are too numerous to permit ready counting by means of the 0.004 c.mm. areas of the Thoma-Zeiss hemacytometer. It now becomes necessary to use dilutions, which are made as follows.
Making the Dilutions.—The dilutions generally used are 1-10. Rarely will it be found necessary to use higher dilutions. Should this, however, become desirable, a dilution of 1-100 is to be made. The highest counts so far recorded were in the case of two tomato pastes which showed 2,400,000,000 and 4,000,000,000 bacilli per cc. In these instances dilutions of 1-10 were used and proved quite satisfactory, though it was evident that a greater number of
Fic. 6.—Thoma-Zeiss hemacytometer. Complete equipment for blood count- ing. This is very convenient for making bacterial counts in catsups, jams, jellies and other vegetable foods and also in animal food substances.
bacilli per cc. would have necessitated the use of a dilution of 1-100. However, a dilution of 1-10 is all that is required for practical purposes, as a bacterial count of 4,000,000,000 and more per cc. would indicate the decomposed condition of the food substance and its unfitness for human consumption.
In case of liquids and near liquids, 9 cc. of distilled water is added to 1 cc. of the substance, and in the case of pastes and similar products, g cc. of distilled water is added to 1 gram (or 1 cc. semiliquid) of the substance. The dilutions are made in 25 cc. graduated cylinders, which answer the purpose very well. Or roo cc. graduates may be used for making the dilutions, adding
44 BACTERIOLOGICAL METHODS
90 cc. of distilled water to 10 grams (or 10 cc.) of the substance. Place the thumb firmly over the opening of the graduate; the con- tents are thoroughly mixed by shaking vigorously for about 20 sec. By means of a slender glass rod slipped well into the mixture, take up a droplet of the mixed material and touch the end of the rod lightly and quickly upon the middle of the ruled area of the hem- acytometer. All this must be done rapidly, before the organisms
C.ZEISS, TENA ©.ZEISS,JENA
Fic. 7. Fic. 8.
Fic. 7.—Zappert ruling of the Thoma-Zeiss hemacytometer. This form of ruling is especially convenient for making bacterial counts and counts of fat globules in milk.—(Carl Zeiss.)
Fic. 8.—Turck ruling of the Thoma-Zeiss hemacytometer. This is especially useful if it is desired to combine the bacterial count with the spore and yeast count. The smaller areas (1-400 sq. mm.) may be used for making the bacterial counts, while the larger areas (1-25 sq. mm.) may be used for making the spore and yeast counts.— (Carl Zeiss.)
have had time to settle to the bottom of the graduate, and before they have had time to accumulate at the end of the glass rod. Making the Count.—After having cleaned the hemacytometer (do not use alcohol), it is sometimes desirable to rub a very soft, grit-free graphite pencil over the ruled area so as to render the lines more readily visible. Usually, however, this is not necessary. After placing the droplet of material as above described, cover with a No. 2 cover glass and orientate the ruled area by
DIRECT EXAMINATION 45
means of the low power and make counts under the suitable high powers. From ten to twenty of the ruled areas should be counted and from these countings figure the average. It is desirable to make two or three mounts of each sample, thus giving the average of from twenty to thirty areas counted. The countings are to be made in areas free from pulp fragments and including all organ- isms lying within the ruled bound- ing lines and inclusive of half averages of those organisms which lie across the rulings. All count- ings which present characters of doubt are omitted from the final estimates.
Those organisms which occur within the cell-lumen of the vege- [Raia table tissues are not to be counted. .2EISS,3ENA To do so is practicably impossible 5, or ititeer ruling, ueehel de and such countings, even if pos- making counts of milk fat globules, sible, would add nothing to the SPC tulyet cls. The amaze value of the findings. In case the cells contain numerous bacteria this should be noted in the report, as it certainly indicates decomposition of the material. The prin- cipal decomposition changes due to the invasion of bacteria and other organisms are, however, largely limited to the exterior of cells, especially by those organisms which develop during or after the factory processing. ‘The numerical determinations are there- fore limited to organisms which occur in the matrix and those which have been washed from the exterior of cells by the thorough mixing. The thorough mixing of the samples is a very important part of the procedure. In the case of liquids and semiliquids, mixing is done by thorough shaking, and in the case of pastes and similar materials, by means of a spatula or a small spoon.
In making counts of very small or comparatively short bacilli,
46 BACTERIOLOGICAL METHODS
some difficulty is caused by those organisms which happen to be vertically suspended in the counting chamber, thus presenting an end view which gives the appearance of small granules or spherical particles which the comparatively inexperienced observer may not recognize, or which may be mistaken for inorganic particles or organic particles other than microbic. In case of doubt, allow the
Fic. 10.—Tomato pulp cells in normal catsup. The cells are large, thin-walled, containing granular particles. The coloring matter of the tomato frequently ap- pears as deep scarlet-red crystalline particles usually arranged in groups within the cell.—(Howard, Yearbook U.S. Dept. of Agriculture, 1911.)
mount to remain at rest for to or 15 min., thus allowing the bacilli to settle to the bottom of the cell where they will assume the horizontal position, thus presenting the long axis to view and making counting easier.
In order that all of the cells (individuals) of the bacilli may be counted, it is necessary to use a high power (480 to 500 diam.). Lower powers are not satisfactory for counting bacteria. For
DIRECT EXAMINATION 47
counting spores and yeast cells a magnification of 180 diam. would prove very satisfactory, especially with a well-corrected wide aperture objective. The counting of cocci is more confusing than the counting of bacilli, but fortunately the microbic contami-
Fic. 11.—Cluster of mold hyphz in granular (decomposed) tomato pulp. This type of mold is traceable to field-rotted tomatoes. The finding of hyphe of this type in tomato catsup indicates the use of rotted tomatoes, therefore, indicates inadequate culling at the factory—(Howard, Yearbook U.S. Dept. of Agriculture,
Ig1t.)
nations of most vegetable substances are bacillar, though there are some notable exceptions.
Mold Counting.—Thus far no satisfactory method for making estimates of the amount of mold hyphe present in fruit and in animal products has come into use. The method recommended by B. J. Howard, Chief of the Micro-chemical Laboratory of the U.S. Bureau of Chemistry, namely, determining the degree of
48 BACTERIOLOGICAL METHODS
mold contamination from the number of microscopic fields of the compound microscope which show the presence of hyphal clusters, is far from satisfactory. It indicates the amount of contamination in a general way only. More reliable and more accurate estimates could be obtained through the use of a counting apparatus in which the number of hyphal clusters could be ascertained in definite quantities of the material under examination. The hemacy- tometer already mentioned does not serve the purpose because of the smallness of the counting areas. The special counter described in Fig. 5 would serve the purpose very well. It is furthermore necessary to reduce the material to a uniform and standard fineness by rubbing it through a sieve. A very small standard mesh sieve would answer the purpose. Take1 gram of the thoroughly mixed material and by means of a small spatula rub all of it through the sieve and make the estimations from the pulp which has been passed through the meshes of the sieve.
Precautions.—The following are some of the factors which necessitate caution in making counts of microbes, yeast cells, spores and mold fragments.
a. Badly decomposed factory pulp which compels prolonged heating in order to render it suitable for canning, often presents such a granular appearance as to make accurate counting of the microbes rather difficult. In such materials many of the more or less disintegrated pulp cells are filled with bacteria and these cannot be included in the count. Commonly in such substances many of the mold fragments are also very much disintegrated through decomposition changes, probably initiated by enzymes formed by the bacteria and other organisms.
b. While it is quite easy to distinguish between living yeast cells, dead yeast cells and spores, it is not thought advisable to attempt such differentiation in routine laboratory practice, ex- cepting in cases where identification is simple and where there is very little room for doubt. One of the first important problems for the food and drugs bacteriologist to solve is the identification
DIRECT EXAMINATION 49
of those micro-organisms which commonly attack foods and drugs, more especially the molds and yeasts.
c. It is neither practicable nor necessary to differentiate between the different kinds of spores which may be present in a product, excepting as suggested under (0).
d. In many instances it would be desirable to resort to plat- ing methods in order to determine the number of viable organ- isms present. This would be simple for bacteria and mold spores, but more difficult for yeasts.
Differentiating between Living and Dead Bacteria and other Low Forms of Organisms.—It would be most desirable to deter- mine some practical working method for distinguishing between living and dead bacteria in foods and drugs. Often the question arises as to the time and place source of the contamination. Did the organisms present develop in the fruit, in the pulped material during the processing or in the containers after manufacture? Again, are the organisms estimated by the direct count dead or alive?
Several investigators have stated that dead and living bacteria react differently with certain stains. For example G. Broca, an Italian bacteriologist, declares that the use of the following mixed stain will serve this purpose. To 8 cc. of concentrated carbol- fuchsin add 100 cc. of Loeffler’s methylene blue. Let the mixture. stand for 24 hr. before using. Exposed to this stain, dead bacteria (killed by heat or by disinfectants) are colored red while living bacteria are colored blue. It is declared that other stains, as Giemsa’s, will react in a similar manner.
More recent experiments would indicate that selenium and tellurium compounds will serve to differentiate living bacterial contaminations. It would appear that these substances are decomposed into metallic tellurium and selenium when brought in contact with living organisms. Much experimental work along this line has been done by Hansen, Gmelin, Gosio and others, and still more recently (1913) by King and Davis of the
50 BACTERIOLOGICAL METHODS
Research Laboratory of Parke, Davis and Company. Potassium tellurite is said to be the most satisfactory reagent. In dilutions of 1:50,000 this substance forms characteristic black compounds with all of the more common micro-organisms when in the living state. The reaction does not take place in the presence of dead micro-organisms and the different organisms do not all react in the same degree or manner, Some are much more susceptible than others. The Bacillus coli appears to be the most sensitive to the reagent. With most species of bacteria the time re- quired to produce the characteristic color and precipitation reac- tion ranges from 12 to 96 hr. at a temperature of 37° C., but with the colon bacillus a distinct coloration or color ring becomes visible several minutes after the reagent is added. King and Davis summarize the experimental results as follows:
1. Nearly all of the more common micro-organisms (bacteria and yeasts) react with potassium tellurite, forming characteristic, black compounds.
' 2, This capacity depends on an active stage of metabolism of the reacting organism, and the action is, in all probability, a reduction of the tellurite.
3. The “‘tellurite reaction”? can be used as an indicator of microbial life, and is especially suitable for revealing microbic contamination.
4. A dilution of 1 : 50,000 of the salt seems to be most suitable for its action as a general microbic indicator. In this concentration, it produces no irritative action when introduced into test animals.
5. The bacteria of the ‘“colon-typhoid group” show differences in resistance to the antiseptic action of potassium tellurite and in the appearance of their reaction with this salt. These variations are sufficient to suggest the experimental use of potassium tellurite for differential diagnosis in the group.
6. The intensity of bacterial action on potassium tellurite depends upon the individual resistance of the bacterium and the concentration of the salt present. The velocity of reduction of the tellurite is apparently a specific function of an organ- ism, apart from its resistance to antiseptic action. With the colon bacillus, the “‘tellurite reaction” is almost instantaneous.
7. Treatment with potassium tellurite has practically no influence on the bio- logical characteristics of an organism.
3. Numerical Limits of Micro-organisms in Foods and Drugs
What should be the maximum iimit of the number of bacteria and other micro-organisms in food and drugs within the intent
DIRECT. EXAMINATION 51
of the U. S. Pure Food and Drugs Act? This is as yet an un- settled question and one that requires further careful considera- tion, even calling for some extensive investigation in order that certain disputed points may be finally settled. There are, however, certain results based upon extensive observation which
Fic. 12.—Type of mold development in the tomato pulp during and after the processing. According to tests made by B. J. Howard of the Bureau of Chemistry, mold will develop in tomato catsup containing 0.1 per cent. sodium benzoate. Com- pare the hyph with those shown in Fig. 11. They are much larger in transverse diameter and the walls of the cells are much thinner.—(Bitting, Bull. 119, Bureau of Chemistry, U. S. Dept. of Agriculture.)
may be set down as conclusive. The organisms of all kinds which
may occur in and upon clean and uncontaminated ripe fruit,
for example, are negligible quantitatively as well as qualitatively.
Such organisms as do occur are limited to the exterior. Only
under abnormal conditions do micro-organisms find their way 5
52 BACTERIOLOGICAL METHODS
into the tissues beneath the epidermis and into the parenchyma- tous cells of whole fruits. It would be interesting to determine the average number of bacteria on the exterior of such fruits as the apple, the peach, the pear, the apricot, the tomato, the cucumber, etc., and from these figures to estimate the number of organisms per cc. of the fruit substance. The practical value of such information would, however, not be great, as may be understood from the statements already made. It must be admitted without question or doubt that fruit products of any kind, which contain only such organisms as normally occur on clean uncontaminated ripe fruit, will never come under the ban of the pure food and drugs act. This also applies to foods and drugs in general. The organisms which concern the analyst are those which occur in and upon contaminated and diseased fruits and those which are in- troduced or added or allowed to develop and multiply during the processing, and afterward. We may therefore make the following postulate: All fruit products from clean uncontami- nated fruit (ripe or green), prepared under modern sanitary con- ditions, contain micro-organisms in negligible quantities only. It is true that the ideal conditions implied in this postulate may not always be attained in practice, yet we are warranted in making a second postulate, namely: that the number of organ- isms present in fruit products, over and above the negligible quantities mentioned, are in direct proportion to the careless- ness in the various steps of the processing. Stating it conversely, as the manufacturers of food products attain the practically ideal conditions, the number of organisms in their products will become gradually negligible. That such conditions are attainable is clearly shown by the canned products of the careful housewife and of the careful manufacturer. What may be done by the careful housewife may be done even better by the careful manufacturer, because the latter can employ the most approved modern methods, aided by special machinery, which are not at the disposal of the housewife or even of the small manufacturer.
DIRECT EXAMINATION 53
In a general way, the number of micro-organisms in food prod- ucts and in liquids intended for internal use, not including the fer- mented products, is negligible when they do not exceed 250,000 per cc. (ranging from 5000 per cc. to the maximum). In fer-
ad
Fic. 13.—Various stages in the germination of spores in catsups. Note trans- verse septation and branching of the hyphe. Germinating spores may be traceable to the tomato from the field or they may be from spoiling factory pulp.—(Bitting, Bull. t19, Bureau of Chemistry, U. S. Dept. ot Agriculture.)
mented products, as cider, vinegar, wines, beer, etc., the number of organisms present may be much greater, but even here the quantitative estimates generally become negligible if the modern methods of purifying or clarifying (through sedimentation, the use of albumen, gelatin, casein, etc.), filtration, centrifugalization, and sterilization are carried out. Of course, in such products as
54 BACTERIOLOGICAL METHODS
sour milk, ripened cream, ripened cheese, sauerkraut, pickles, etc., the processes of clarification are not applicable, and hence we always find a large number of certain predominating types or species of organisms present.
The microscopical examination of products which have under- gone normal fermentation shows that the number of organisms present is quite variable, depending upon a variety of causes and conditions. This can readily be ascertained from the examination
Fic. 14.—Substances frequently found in tomato catsup. a, Heat dextrinized corn starch. Starch is frequently used as a filler or stiffening agent. 6, bacteria which frequently appear in great numbers. c, Vinegar eels derived from cider or wine vinegar. Soil nematodes may also be found, indicating gross soil contamina- tion and inadequate washing at the cannery. d, Nematode larvae derived from the soil. e, f, g, k, Spore types frequently met with in catsups. 7, Yeast cells.
of such common household products as vinegar, sour cream, cider, apple butter, sour milk, etc. It would be most desirable to de- termine the exact identity of the organisms which produce the most favorable fermentation changes in fermented food products. This has been done in some cases and pure cultures of the specific organisms are used for manufacturing purposes, resulting in the production of superior food articles. When the fermentation
DIRECT EXAMINATION 55
processes are left to nature the result is not by any means uniform and we have products which are often so vitiated by the develop- ment of undesirable associated organisms as to make the food unfit for use. There is a definite biological relationship between those organisms which initiate desirable fermentations and those which are objectionable; both kinds are generally present, but fortunately
Fic. 15.—Vinegar eels from decomposed blackberry pulp. The small particles scattered through the field are yeast cells. Bacteria were also present but they do not show in the illustration —(Howard, Yearbook U. S. Dept. of Agriculture, 1911.)
the desirable or beneficent forms overgrow the objectionable forms very rapidly, but not always. It should be one of the prin- cipal efforts of the food and drugs bacteriologist to isolate and identify the organisms which are desirable in the production of fermented food products and those which are unquestionably undesirable and objectionable, for in these products it is not a question so much of quantity as of quality of the organisms present.
56 BACTERIOLOGICAL METHODS
This is by no means a simple problem. Much of this field of work is as yet untouched, and it is not likely that definite conclusions will be reached in the very near future. It means an investigation of those conditions which are recognized as diseases in industrial or manufactured products, characterized by unaccountable de-
Fic. 16.—Mold from decomposing plum.—(Howard, Yearbook U.S. Dept. of Agriculture, 1911.)
teriorations in flavor, in taste, in color, in nutritive value, etc. It means a very careful study of organisms which are similar in morphology and yet quite different in specific functional activities, giving rise to objectionable fermentation products.
The following tables will give some idea of the number of organ- isms which occur in certain canned food products. Animal food
DIRECT EXAMINATION 57
products are not included in Tables II and III because there are not sufficient data available on which to base suggestions. There appears to be no plausible reason why canned animal products should not be subjected to the same method of examination as vegetable substances, particularly sausage meats, canned meats, canned oysters and shellfish generally, canned eggs and canned soup stocks. Pickled herring which shows 8,000,000,000 bacteria per cc. in the liquor is certainly a questionable food article. In
Fic. 17.—Spores and hyphal fragments from decaying sweet pepper. ‘Dry rot”’ fungus.—(Howard, Yearbook U.S. Dept. of Agriculture, 1911.)
this particular instance there was no objectionable odor noticeable, but the meat of the herring was somewhat soft. Smoked meats and fish should be examined for mold in addition to bacteria. This subject should receive immediate careful consideration on the part of food bacteriologists.
Table I shows the number of organisms which may occur in some of the more common household food substances, fermented and unfermented. The figures are based upon direct counts. Table II is based upon the examination of factory products ob- tained in the open market. The numerical extremes in the micro-organisms given in Table II, are in direct ratio to the relative
58
BACTERIOLOGICAL METHODS
Taste I,
Name of Substance
| |
Blackberry jam. | Blackberry jelly. ., Cheese, California
Cider vinegar... . Currant jelly..... Fruits, canned... Herring, pickled..
Meat, sausage.... Milk, ordinary... Milk, certified.... Milk, sour....... Plum preserve.... Plum relish......
Water, drinking (San F.).
Number of Organisms per Cc.
Bacteria
80,000,000
50,000 to 500,000 1,000,000
1,000,000 to 150,000,000 25,000 to 2,000,000 1,000 to 15,000 2,000,000,000 to 7,000,000,000 100,000,000 800 to 32,000,000
z, & — = Hyphe Yeasts Spores
ed ge |
ican tosegerio wo eve axel ack dasa Sin Few
sn ye Wd Re Wada ea wean swe Few.
RW vnarscoeuee| owe wateequncher Entirely _ per-
meated. §0:066-16 | acc cs sn aWm yy akoere sew eae 30,000,000
CWS rs icesnctepscata ele weaesitae techn siete nl ard naeca alone: a ttauianecly
POWs exwiuwionen site ean y pads whe canna 9 ceca d
PE Wie. s sseicersue e's BOWs 2c ¥Rae Few
TRE Wie scaucrnconcoas a Sl waders ecko ib he Bata Rca sce eas ORT
PeWsceseegeege E@Wijsgogge ae Few.
F@Way ae y sean 2,000,000 | Some.
unsanitary conditions in the factories. the products of the manufacturers who employ modern methods are fully up to the quality of those prepared by the careful housewife.
It is quite evident that
‘The counts recorded in Tables I, II, and III were made by the direct method
using the hemacytometer.
In the case of the sausage meat some of the counts were
checked by the plating method and it was found that the count by the plating method was invariably higher than by the direct method. Other investigators have
noted similar discrepancies.
The direct examination of meats for bacteria is occasion-
ally unsatisfactory because of the confusion due to granular fragments traceable to broken up blood corpuscles, fragments of coagulated albumen, etc.
Name of Substance ,
Apricot jam...... Blackberry with apple.
Cherry jam with apple. Currant jam.... Currant jam.... Loganberry jam... Loganberry jam...) Orange marma- lade Plum jam....... Peach jam....... Strawberry jam.. Strawberry jam.. Tomatoes........ Tomatoes........ Tomato paste... .| Tomato paste... .: Tomato paste.... Tomato paste.... Tomato paste.... Tomato paste... .'
DIRECT EXAMINATION 59 Taste II. Number of Organisms per Cc. Hyphe
Bacteria Yeasts Spores Se ee ree 3,750,000° | Few........| Some.
500,000 AOASOOO. | ost canaear ands Reeea aged eae 400,000 9,250,000 Some....... Some.
BS COG haan gen eis Goe4 None....... None.
40,000 1,728,000 Numerous. .| Very abundant.
GiOCOf00O sexs vain ee a Mes seared eeeales Abundant.
44O;000;000 | gai dad resamews 5,000,000 | Very abundant. SOO;06GjO60. icc ccce sa caaiae's 27,500,000 | Very abundant. SOG;O00,006" || fn tswivnacwaaaw's 20,000,000 | Entirely _ per- meated. 200,000,000 12,000,000 1,500,000 | Very abundant. §;000;000: — hess se een sea as Fe wes cciners Trace. 89;000,000 asauctwsscewins 5,000,000 | Very abundant. 4O0j000,000 fae eck cere es 7,500,000 | Very abundant.
5,000,000 1,000,000 200,000 | Very abundant. nee iuk, bseeLre bak AOi0O0j;006; nvidia egdeuieand| ee hamaodeud Lae eee ree tads erie 45,000 iG Rombaalieal| wad Wace cua
1,000,000 1,250,000 hin can aoas <|deiaewennta meas FeWseseseecees GyZ5000O | wricensa eatesrns Some.
2,500,000 8,750,000 |nvaceeneustegs Quite abundant.
500,000 500,000 10,000 | Some.
1,250,000 TEOOOO™ | ‘Iiniadcdesn tne 4 |etuadneal mands Se ee 4,500,000 Few........| Some. vegiemelsss ag cis 500,000 750,000 | Abundant.
¥2;000j000 | cisinve vy ceaning 1,400,000 | Very abundant. 2;000;000j000 | aa cveaaveds seem 4,000,000 | Very abundant. 2,000,000,000 |v. scciurs ane aaus 1,000,000 | Very abundant. BiGO0;600,000) |e ecicicns dn ees cia 1,200,000 | Very abundant. 1,400,000,600: | oc casawsacasane 5,000,000 | Very abundant. 4;000,000,000: | js acey sagas sus 6,000,000 | Very abundant. TjOOOjOCO}COO” | weds ev ae gn on I,000,000 | Quite abundant. 2,000,000,000 80,000,000 +=|100,000,000 | Entirely _ per-
meated.
60 BACTERIOLOGICAL METHODS
TABLE II.—(Continued)
Number of Organisms per Cc.
Name of Substance aed = Hyphe Bacteria | Yeasts Spores Tomato pulp!....| Less than as Less than | Practically none GiOOOOOO! | nacemie ax aetna va a 500,000 (1-3 per cent. | of fields). Tomato pulp!....|1,900,000,000 |..........2.00- | 37,000,000 | Entirely __ per- | meated (100 per cent.), Imitation jam.... Few........... | 30,000,000 FEW scone d os | Few. | ‘ TaBie III Maximum No. of Organisms per Cc. | Name of Substance —_ eee — Hyphe? Bacteria Yeasts | Spores | 3 i j 2 eee — Apple butter..... 5,000 to | TYOOGOOO LOU yee 9 4 gant gaa gitar vcerrmersincinecern soa 1,000,000 10,000,000 Berries..........| Few...........| 500,000 $00,000 — 15 per cent. Catsup......... = 10,000,000 to | Few........... 500,000 | 18 per cent. 50,000,000 | : CHO, cco esis ws 500,000 to | ES OCOOO LOM: cane aarsmrmacts aan Hm acmMna cates 2,000,000 5,000,000 FEULES 4 vswncenn ys REWeagsnemnesicel 50,000 to ~—- 500,000 to’: 10 to 12 percent. \ 500,000 ' 1,000,000 JOM Sixes eterna a a : 1,000,000 1,000,000 to 500,000 Io per cent. 10,000,000! | HOMES) sisi actos: all: BOW ca snmnewese es 1,000,000 PO Wiicccsdree | 1 to 5 per cent. Marmalade’..... | Po Renin aratvaniaral ora haotagee qa mug gllusatipaiatanenthe oad N elnsee, Seti biStedoith Sere Tomato pastes. . 4 500,000,000 Few 2,000,000 | 20 to 25 per | cent. Vinegars (fruit). . 5,000,000 AW Gala tecle lcsslg ache eg cles Gah Ros Aces | Wash’ Beco ineagraah a |
1 Both samples were from large factories and represent the extremes in the factory
conditions. should be, the second from a factory where the conditions are just
The first sample is from a factory where the conditions are what they
the reverse.
* Percentages given this column refer to the number of the 1/125 c.mm. areas of
the mold counter described in Fig.*s which‘contain’hyphal'clusters.
Asarule abun-
dant spores indicate the presence of abundant hyphal tissue, and vice versa. * The organism in orange marmalade, under ordinary conditions of manufacture,
are necliathle in amannt
DIRECT EXAMINATION 61
Other manufacturers, either through greed, ignorance or careless- ness, or through all three causes combined, refuse to employ modern methods and as a result their products are very often in an undescribably filthy condition, wholly unfit for consumption. In addition to the bacteria, yeast cells, mold, sand and dirt particles present in the inferior grades of catsup, jams, jellies, etc., there are found insect remnants (flies, aphides, beetles), vinegar eels, larve of various nematodes (from soil), etc. The presence of numerous fly remnants is certainly an indication of highly unsanitary factory conditions. The presence of vinegar eels indicates the use of bad vinegar and the presence of soil nematodes and of sand and dirt particles indicates insufficient or no washing. Laboratory experience has demonstrated that there is a definite relationship between the number of bacteria and other organisms and the amount of dirt and other impurities present in factory products. Unsanitary factory conditions en- courage a certain recklessness in such factories, inducing the laborers about the place to even go out of the way to add more filth. Thus shovelfuls of refuse are taken up from the filth-coated floors and thrown into the mixing vats, the idea evidently being that it will add to the bulk and that no one will know the difference. Vats are often not cleaned until the conditions are almost unde- scribable. Refuse is added, often of such a character as to be un- fit as food even for animals. This criminal negligence, care- lessness and indifference is too frequently engendered by ignor- ance which, gives heed to nothing else than a strict enforcement of the law.
The filthy condition of some of these products is very generally not apparent to the layman because of certain methods employed primarily intended to hide or mask such defects. The odors of decomposition are quite effectually dissipated by the steaming and cooking process. The vitiated taste is quite effectually masked by the heavy spicing. Any appreciable change in color is
62 BACTERIOLOGICAL METHODS
restored by means of added coloring substances. Any change in consistency is corrected by adding fillers, such as starch, gelatin and agar. The unscrupulous manufacturer will work up a supply of spoilt canning tomatoes, including rejected “swells” and “leaks,” making them into catsup or paste. Overripe and par- tially decomposed fruits (culls and rejects) are worked up into jams preserves and into combinations in which the objectionable character and appearance are hidden or lost sight of.
We are justified in the conclusion that the number of micro- organisms in food products is a reliable guide to the wholesomeness and sanitary quality of such products and the very natural ques- tion arises, what are the maximum numbers of bacteria, yeast cells and mold spores (including mold hyphz) permissible under reasonable and practicable sanitary conditions. While ideal factory conditions may not always be practicably attainable, yet it is wholly reasonable to expect the operation or methods which will bring the maximum quantitative counts per cc. within the numerical limits given in Table III. These proposed maximum numerical limits are tentative only. As the sanitary conditions in the canneries are improved, as they undoubtedly will be, the limits can be correspondingly decreased, finally reaching the negligible quantities as already explained. Where numbers are omitted in the tables it indicates that the quantity of organisms is negligible. “Few,” indicates that the number of organisms is somewhat more than in negligible amounts, yet not sufficient to make counting necessary or to question the suitableness of the article for food purposes.
It is quite evident that different numerical limits must be adopted for different classes or kinds of food products. This can be seen from a study of the tables. Some fruits and fruit products are more susceptible to the attacks by bacteria, yeasts and molds, than others. Acid fruits, as the cherry, the plum, tomatoes, loganberries, blackberries, etc., are much more likely to be attacked
DIRECT EXAMINATION 63
by molds than are apples, peaches, pears and apricots. Yeasts very rarely appear in the whole fruit, but they develop very rapidly in fruit pulps which contain sugar (natural or added). Yeasts require in addition to sugar, a high percentage of moisture for their active growth, including an ample supply of oxygen (air). The presence in canned fruit products of numerous yeast cells indicates fermentation during the processing. The presence of numerous bacteria in fruit products indicates the use of rotted (bacterially) fruit or bacterial contamination and development during the processing, or both.
It would appear that most of the bacteria which develop in fruit pulps, especially those from fruits which are quite acid, as for ex- ample tomato pulps, belong to the lactic acid group. Numerous tests in the laboratories of the Bureau of Chemistry show a paral- lelism between the number of bacteria and the amount or per- centage of lactic acid present in tomato catsups. The usual rotting bacteria require more air (oxygen) then is present in the pulp mass and as a result these are soon overgrown by the lactic acid bacilli, if the pulp is allowed to stand fora time without steril- ization. It is, however, very evident that the contamination of such products as catsups, tomato pastes and tomato purees is never wholly limited to lactic acid bacilli. The inclusion of field rotted tomatoes and the rotted pulp material from filthy mixing vats and other parts of the machinery of the unsanitary factories, adds a sufficient number of rotting bacteria to render the article dangerous to health, if consumed. Ravenel and other investiga- tors have shown that when certain food products, as cream and milk, are kept in cold storage, particularly after pasteurization or incomplete sterilization, the development of lactic acid bacilli is checked and the growth of toxin forming bacteria is encouraged, resulting in occasional poisoning to the consumer. It is very likely that similar conditions may exist in some of the incompletely sterilized canned food products (vegetable as well as animal) which have been stored for some time at a comparatively low temperature.
64 BACTERIOLOGICAL METHODS
The question is frequently asked, what percentage of rotten or moldy fruit must be present to render the product unfit for human consumption? This question cannot be answered definitely. In a general way, it may be stated that where there is not over 5 per cent. of rotted or moldy fruit used, the number of organisms in the finished products will not reach the maximum limits given in Table
Fic. 18.—A type of mold, Spicaria sp., very frequently found on decaying tomatoes. Some of the filaments and numbers of spores are shown.—(Howard, Yearbook U.S. Dept. of Agriculture, 1911.)
ILI, in fact the counts will in all probability be considerably less. A careful culling of spoilt fruit in the field and at the factory, coupled with reasonably sanitary factory methods and modern methods of sterilization, will furnish products which will meet all of the requirements of any pure food law.
The statement is frequently made by manufacturers that even
DIRECT EXAMINATION 65
though bacteria, yeasts and mold are present in considerable numbers, they are harmless and do not produce toxic effects when introduced into the digestive tract. This statement is wholly without foundation in fact. On the contrary it is known that certain bacteria, yeasts and molds do cause disease and more or less severe intoxications and intestinal disturbances. The objectionable character of mold is universally recognized
Fic. 19.—Mold colonies in gelatin seen under the low power of the microscope (X 80). This mold developed in the gelatin after it was spread on the screen to dry. This gelatin also contained numerous bacteria. Gelatin thus infected is not suitable for bacteriological purposes neither is it suitable for use as food.
and nearly all animals refuse to eat moldy and mold contaminated food materials. Various ulcerative diseases of the skin and of the digestive tract are caused by mold organisms. While many of the yeasts are entirely harmless and cause very important fermentative changes, some of them are pathogenic to man while others initiate objectionable fermentation changes in the food substances.
66 BACTERIOLOGICAL METHODS As already indicated the number of organisms in food sub- stances is in direct ratio to the following conditions:
1. Insufficient culling of partially and wholly decomposed fruits. 2. Unsanitary factory conditions and unsuitable methods.
. . . : : Ova of the Parasitic Worms or Man
TREMATODA DRAW N TO SCALE x 1600
Heterophyes ZS =e heterophyes fe “© a Fie
0 ‘ is
5 Opistho
e FELMEUS (e11erL0051908)
po 4 a :
z [a es ; : 6 each Clonorchis Clonorchis lanceatum, | __ Sinensis, endemicus
oe Fasciola, - Fasciolopsis
POLCO —— HUSK (nertoosaiya)
e (after Loons, 1905)
4 - 2 if Schistosoma pgragonimus Fascioletta iH _ Som Re ite westermani ongiey Seen : casi tiivitetpexiil «AY te ore g ORI, ade US Naval Medical School.
Fic. 20.—Intestinal ova. Trematodes. Ova of intestinal parasites may possibly occur in foods of vegetable origin contaminated by soil, sewage and fecal matter. Note comparative size and the actual measurements according to the scale. It may be mentioned that the extremely small seeds of Vanilla planifolia have been mistaken for ova of intestinal parasites.—(Stitt.)
We are warranted in establishing a maximum limit as to the number of organisms permissible in food substances. The method of estimating the quality of foods based upon the number of micro-organisms present has been tested out in different coun- tries and has proven very reliable and satisfactory; and those who
DIRECT EXAMINATION 67
are entrusted with the enforcement of the laws governing the physical well-being of the people are most emphatically in the right when they insist that the sanitation in and about our factories should be of a high order.
In addition to the purely quantitative estimates of micro- organisms based upon direct examination, the analyst is enabled
Ova or the Parasitic Worms or Man CE ST ODA
DRAWN BCALE 1600
100 MICRONS
Toe Diniot ds th solium, Diplo ibothrio (efter Looss. 1405) Gonoporus cephalus ” Homencienis nana
9rd dis ] US {acer Looss.1 ioe) Gree Seema sae @ jan ‘ter Looss.1905)
go=~ Hymenolepis
m0) eas “ Teenia Wy (ee 9) ff i DD \, \ Gu +1008) saginata 9.1006) i. a ¥ .
Dipylidium
CONINUM Gores sines 00) ta Cestode segments
DRAWN TO SCALE
Re Te) 10 MILLIMETERS avaineo : =
meee ers ‘ariensis Hymeno-
lépis
Giminyte,, ‘Teenin Di lidium ¢ Teenia solium pylon Dibothriocephatus latus H.nana § saginata.
rit iar OS Naval Medical Schioal,
Fic. 21.—Intestinal ova. Cestodes.—(Stilt.)
to form certain opinions and conclusions regarding the source of the contamination. For example, the hyphal development in mold infested fruit.is in marked contrast to the hyphal develop- ment in the fruit pulp, due to unsanitary factory conditions. This difference in hyphal structure is due to a difference in the
amount of oxygen (air) supply, of moisture, of light and the 6
68 BACTERIOLOGICAL METHODS
added ingredients (spices, sugar, vinegar, etc.), of the canning product. The analyst can thus determine approximately how much of the hyphal tissue present is derived from the use of moldy fruit and how much is traceable to unsanitary factory conditions. Again, the presence of one or more ova or larve of intestinal parasites, as the tape worm, would indicate sewage contamination or contamination with fecal matter. Sand and dirt particles indicate insufficient washing, etc. It is self evident that the value of the report by the analyst depends upon his knowledge of the subject and the range of his experience. Until the work is well under way and the methods are perfected, there is no place for inexperienced analysts in our food and drugs laboratories.
4. Quantitative Estimations by the Cultural Methods
Estimating the number of bacteria per cc. in foods and drugs, etc., by planting or plating definite amounts of the substances into plate (Petri dish) culture media, is a well-known and standard procedure. The general and special technique of the plating method is described in the various text-books and manuals on bacteriology. Some of the details of the method are standard, in so far as they are generally adopted by investigators, such as the preparation of certain culture media, making the dilutions, counting, etc.; in other regards there is anything but uniformity. It is generally admitted that the results of different investigators differ widely but there appears little unanimity of opinion as to the factors which are responsible for these variations in quanti- tative results.
Micro-organisms are sensitive to a degree and they respond readily to the slightest variations in moisture, temperature and food’ supply. A failure to recognize this fully in laboratory practice leads to confusion and erroneous results. The following are some of the more important factors which are responsible for errors and variations in results.
CULTURE MEDIA 69
1. Culture Media.—Differences in the quality of the meat used in making the meat infusions has given some marked varia- tions in the quantitative results. Meat extracts from younger animals give higher counts than do extracts from the meat of older animals. Again, the prepared extracts of the different packing houses give different results and the results obtained
] Ova of the Parasitic Worms or Man “4 NE M ATODA = ORAWN SCALE x 1600 Agog EN metrically fee convex (after Looss,: w/ Oxyu Knerurat focus B-Surface focus Vermicularis A. B.
Modined f1 (Mosthes from Stllessans) (after Stiles. 1902)
sie: lus D-Atypicai. unfertilized SU bt ilis fer; — Aer egg (att cr Looss,190) (after Looss, 190) ‘ty Oes. ; Jot 4 \
C-witnout outer aed Necator \ od
ghvelope (Modified americanus en Looss,1908) \ = 7 Pon cenit les, uz da an mb?
tot iter iatoas Ascaris - Apchvlostoma Agehylostome lumbricoides “ease” ,AUOdenaE enwryol | duodenale AB Go tee erie: Strongyloides _ . iOriginal) . SLETCOTLIS LMVEL iy OLAava/ Medical School.
Fic. 22.—Intestinal ova. Nematodes.—(Stitt.)
from the use of media made with manufactured meat extracts differ from those obtained from the use of the laboratory made meat infusions. In fact most workers are opposed to the use of the manufactured meat extracts because of the fact that they are mixed products and also because of the uncertainty of the amount and number of the added ingredients any or all of which may
7° BACTERIOLOGICAL METHODS
interfere with the growth and development of certain bacteria. Investigators have also noted great variations in results with different brands or makes of peptones used, the quantitative differences amounting to 50 per cent. in some cases. Equally remarkable are the differences due to the kinds of water used in the preparation of the culture media. For instance it is known that agar made up with sewage encourages the development of sewage organisms while the same medium made up with tap water encourages the growth of bacteria predominating in such tap water.
The gelatine used is yet another important factor in cultura] results, depending upon the age of the gelatine, its purity, the de- gree of heating to which it has been exposed, its origin, possible contamination with arsenic, with bacteria and mold. Other ingredients used in the preparations of culture media cause more or less marked variation in comparable results. The above statements make it evident that it is absolutely necessary to adopt and to adhere to uniform methods in order that the com- parable results may be approximately uniform.
2. Glassware.—Different investigators have found that the number of bacteria in and upon culture media varied with the composition of the glass containers used. The comparatively soluble glass, for example, yielded enough free alkali to inhibit the development of the more sensitive bacteria. The size of the containers and the thickness of the glass yielded differences in the results. It is therefore very desirable to adopt Petri dishes and test-tubes of standard form and thickness of standard cubic contents. :
3. Other Factors.—The form and size of the incubating chamber, the degree of ventilation, degree of darkness, amount of oxygen present, etc., cause variations in the results.
Of even greater importance than any of the factors so far mentioned, is the personal equation in the laboratory technique. No two workers follow out the same details in the different steps
CULTURE MEDIA 71
of the laboratory procedure and very frequently proper judgment is lacking in the application of certain details of the methods. For example, there is lack of uniformity in the degree of heat to be used in melting gelatin media preparatory to planting, in the amount of material to be planted in each Petri dish, manner of planting, time of incubating, etc.
We hereby submit the following technique in the preparation of culture media and in the methods of making cultures in plates as well as in test-tubes, following very generally the suggestions as given in the report of the Committee of the American Health Association.
5. Preparation of Standard Cultural Media, General Suggestions
1. Ingredients.— Distilled water is to be used in the prepara- tion of all of the standard media. The distilled water must be comparatively free from bacteria and must be kept in clean sterilized containers and as free as possible from mineral and organic impurities. If other than distilled water is used, this is to be stated and the special reasons for using it indicated.
For making meat infusions, fresh lean meat is to be used, from comparatively young animals, free from disease. Meat extracts may be used in place of the meat infusion.
Unless otherwise specified, the peptone used should be made from fresh beef by pancreatic digestion. It should be dry and recently made. Workers should be sure to specify the kind of peptone desired. Egg albumen or fibrin peptone is not to be used in any of the standard media. The article should be secured from some reliable house.
The gelatin to be used in the preparation of the standard media should be of the best obtainable, the so-called French brand be- ing, as a rule preferred. A io per cent. solution should not soften when kept at a temperature of 25° C. It should be en- tirely free from arsenic and as free as possible from acids, micro- organisms, molds, and other impurities. A good grade of gelatin
72 BACTERIOLOGICAL METHODS
should respond to the following test: Place 0.30 gram of the gelatin in a medium sized test-tube and add 15 cc. of distilled water, let stand for half an hour, warm gently until all of the gelatin has dissolved, then place the tube in water at a tempera- ture of 15.5° C. and leave undisturbed for half an hour. The solution should remain in place when the tube is inverted.
The commercial gelatin is a variable product, being made from varying proportions of animal tissues as hides, ligaments, bone and bone cartilage. The purest and best gelatin is made from liga- ments and this kind would no doubt give the most uniform re- sults in bacteriological work, but it is apparently not possible to obtain such gelatin in the market. The next best grade (prac- tically obtainable) would be that made from hides of compara- tively young domestic cows free from all foreign additions as salt, arsenic and other hide preservatives.
Each lot of gelatin should be examined microscopically before making it into culture media. Old yellowed and brittle material should not be used. Examine from five to six sheets from each pound package, using the low power of the compound microscope. The examinations are made directly without mounting. If numer- ous mold colonies are found as shown in Fig. 19, or numerous mold filaments more or less scattered through the mass, it is unfit for use as a culture medium. Numerous formed mold colonies in the matrix indicate growth during the drying process after the gelatin was spread on the drying screens. More or less torn and disintegrated hyphal fragments unequally distributed through the mass indicate infection and growth before the gelatin was spread for drying. To examine for bacteria, mount small bits of the sheet on a slide in water covered with cover glass. If bacteria are numerous, approximating 10,000,000 per cc. and more, it should not be used. In order to make more accurate counts, take 1 gram of the gelatin and rub up in g cc. of boiled distilled water and make the counts of the thoroughly mixed sample by means of the hemacytometer. As a rule it is
STERILIZATION 73
not necessary to make plate or tube cultures to determine the fitness of the gelatin for bacteriological work. Incidentally it may be remarked that a gelatin which is unsuitable for bacterio- logical work is also unfit for use as human food.
The agar should be the highest grade obtainable, and if the shredded form is used it should always be washed in sterilized distilled water before making into culture media.
With regard to the other ingredients required in making cul- ture media, such as dextrose, lactose, maltose, saccharose, glycerin, salt litmus, etc., etc., special efforts should be made to get these as pure as possible. The degree of purity should be determined by actual tests.
2. Sterilization.— Thorough sterilization of all culture media is absolutely necessary. It is, however, known that heating pro- duces some marked changes in the molecular composition of the media, even inducing actual chemical decomposition. It is therefore desirable to make the time of heat exposure as brief as possible. Ordinarily it is therefore preferable to use the auto- clave, bringing the temperature up to 120° C. (15 lb. pressure) for a period of 15 min. This temperature will sterilize all media. A shorter period does not insure complete sterilization and a longer exposure is apt to produce inversion of the sugars used and also permanently lower the melting point of the gelatin. Solid media as gelatin and agar should be liquefied before placing in the autoclave.
The following rules should be strictly observed in using the autoclave:
a. The sterilizer should be hot when the media are introduced. About 100° C. Let all air escape from chamber.
b. At the end of the period of sterilization (15 min.), remove the media and cool them as rapidly as possible.
Compliance with these rules will reduce to a minimum the tendency toward liquefaction of the gelatin and a tendency to decompose the various chemicals used, due to prolonged heating.
74 BACTERIOLOGICAL METHODS
If streaming or live steam is to be used in place of the steam under pressure in the autoclave, intermittent sterilization is to be practised. Place the media in the steam sterilizer for 30 min. on each of 3 successive days. Wait until the tem- perature in the sterilizer has risen to approximately 100° C. before placing the media therein. Agar media should first be liquefied. At the end of each period, remove the media and cool as rapidly as possible for reasons already given.
When media are prepared under the proper laboratory condi- tions and sterilized as above suggested, they are as a rule free from all living germs. However, if practicable, the media should be watched for a period of 2 days, stored in a room at ordinary temperature, in order to note possible bacterial developments.
3. Adjustment of Reaction of the Media.—<As a rule bacteria develop most actively in media which are slightly alkaline to litmus and since certain media are quite acid in reaction (gelatin in particular) it becomes necessary to reduce them to a standard reaction. The standard indicator to be used is phenolphthalein. When phenolphthalein is not obtainable, litmus paper (or a 1 per cent. aqueous solution of Kahlbaum’s azolitmin) may be used. The reaction adjustments are to be made as follows:
Place 5 cc. of the medium to be tested in 45 cc. of distilled water (making a dilution of 1-10). Boil briskly for 1 min., with stirring or rotary shaking. Add tr cc. of the phenolphthalein solution (made by dissolving 5 grams of the salt in 1 liter of 50 per cent. alcohol). Titrate while hot with N/2o caustic soda solution (in distilled water). A distinct pink coloration marks the proper reaction. To be more precise, the pink should correspond to a mixture or combination of 25 per cent. red and 75 per cent. white of the color top recommended by the Com- mittee on Standard Methods of the American Health Associa- tion. The reactions of the media are stated in terms of the percentages of normal acid or alkaline solutions required to neutralize them. Alkalinity is indicated by the minus (— )
STANDARD MEDIA 75
sign and acidity by the plus (+) sign. Thus, if the reaction of a medium is given as + 1.00 it indicates that it would be necessary to add 1 per cent. of normal sodic hydrate solution to the medium in order to bring it to the neutral point (to phenol- phthalein). It will be observed that while the titrating is done with the N/20 caustic soda solution, the normal solution is added to bring the medium to the desired reaction, the stronger solution being preferred because it reduces the amount of liquid intro- duced. The Committee on Standard Methods specifies that the reaction of all standard culture media shall be + 1.0 per cent. and if it differs in reaction by more than 0.20 per cent. the medium shall be readjusted and when a reaction other than the standard is used it shall be indicated and the reasons for using a different reaction shall be fully stated.
Media are preferably made in large quantities as this will reduce to a minimum the discrepancies due to variation in the composition of the ingredients used. As soon as made and titrated, the media should be put into tubes and in other culture containers, after which media containers and all are to be sterilized according to the methods already described. To guard against the evaporation of moisture from the media, the tubes, flasks, etc., should be sealed by dipping the plugged ends into melted paraffin, or they may be capped with rubber coverings especially made for that purpose. In case media are to be used within a few days, sealing is not necessary but they should be kept in a moist place, preferably in the ice-box.
6. Preparation of Required Standard Culture Media
Culture media used in bacteriological work may be divided into those which are required for general purposes and those which have special uses. The former should by all means be prepared according to the standards suggested by the Committee of the A. H. A. If special media are used, their exact composi- tion and mode of preparation should be fully and explicitly given.
76 BACTERIOLOGICAL METHODS
Furthermore, the reasons why the special media are used should be clearly set forth, so that co-workers may judge of their special value and may try them out intelligently, should they care to do so.
As special media are adopted into general use by the majority of bacteriologists they are to be relegated into the group of general media. For example, a few years ago, lactose-litmus-agar, Endo medium, Hess’ medium, lactose-bile medium, etc., were special media. They are now in general use and they should be pre- pared according to a standard method. We hereby give the methods of preparing some of the more important media used in general bacteriological work, following the directions of the Committee of the A. H. A.
1. Nutrient Broth.—Infuse 500 grams of chopped lean meat for 24 hr. in distilled water. Shake occasionally and keep in the refrigerator. Any loss by evaporation is to be restored. Strain the infusion through cotton or through cotton flannel. Add 1 per cent. of peptone and warm over water bath or steam until the peptone is entirely dissolved. Heat for 30 min. in rice cooker or in steam sterilizer and restore any loss by evapora- tion. Titrate with normal sodic hydrate (or normal hydro- chloric acid) to a reaction of + 1.0 per cent. Boil for 2 min. over open flame, stirring constantly. Restore loss by evapora- tion. Filter through cotton (placing the cotton on cotton flannel or on perforated filter paper). Pass the medium through this filter until it comes out perfectly clear. Again titrate and record the final reaction. Pour into tubes (10 cc. in each tube) and sterilize in the manner as already directed.
This medium is much used for general cultural purposes. It is used in making the cultures of typhoid fever germs, for determining the phenol coefficient of disinfectants by the Ander- son-McClintic method of rating the germ destroying power of disinfectants. It is also used in culturing motile bacteria, etc. Various indicators may be added.
STANDARD MEDIA 17
2. Sugar Broths.—Broths to which sugars are to be added are prepared in the same manner as nutrient broth, adding 1 per cent. of dextrose, lactose, saccharose or other sugar. The sugar is to be added before sterilizing. Sterilizing in the auto- clave is to be preferred because the longer steam sterilization is apt to cause inversion of the sugar. The reaction of the sugar broths shall be neutral to phenolphthalein.
These media are much used in testing for the presence of Bacillus coli (dextrose broth). The committee states that the removal of muscle sugar by inoculating with B. coli is not nec- essary if small amounts of gas formation are to be disregarded. In the routine work of testing water for the presence of the B. colt a sufficient volume of the water to be tested is added so that the resulting mixture will be one of normal strength. The com- mittee also advises against the use of beef extracts in place of the laboratory made beef infusions.
3. Nutrient Gelatin —Make the beef infusion in the manner al- ready described. After the first filtering through cotton or cotton flannel, add to per cent. of gelatin (the per cent. being based on the weight of the beef infusion instead of volume and the weight of the gelatin to be on a basis of dry condition, and 1 per cent. of peptone) and warm over water bath with constant stirring until the peptone and gelatin are entirely dissolved. While dissolving the peptone and gelatin the temperature should not rise above 60° C. Boil for 2 min. and adjust the reaction to +1.00 per cent. Heat for 40 min. over water bath or in steam sterilizer and restore any loss by evaporation. Again adjust the reaction if necessary and boil over open flame for 5 min. with constant stirring. Restore loss, filter until clear, titrate and record this final reaction. Tube and sterilize as for beef broth and at once store in ice chest. Protect against evaporation as already explained.
4. Nutrient Agar.——Boil 15 grams (dry weight) of washed thread agar in 500 cc. of distilled water for half an hour and make
78 BACTERIOLOGICAL METHODS
up weight to 500 grams. Infuse 500 grams of lean meat in 500 cc. of distilled water for 24 hr. in ice chest. Make up loss by evaporation, strain, weigh filtered infusion and add 2 per cent. of peptone. Warm on water bath with constant stirring until all of the peptone is dissolved. To 500 grams of the meat infusion add 500 cc. of the 3 per cent. agar solution, keeping the temperature below 60° C. Boil for 1 min. and titrate to +1.0. Sterilize in steam for 4o min. and restore any loss by evaporation. Re- adjust if necessary and then boil for 5 min. with constant stirring. Restore any loss due to evaporation and filter by passing it through the filtering material (cotton and cotton flannel or perforated filter paper) at least three times. Titrate and record the final reaction. Tube, sterilize and store as for gelatin media. It must be borne in mind that agar media are never as clear as broth or gelatin media.
5. Lactose Litmus Agar.—To make this medium add r per cent. of lactose to nutrient agar just before sterilizing and make the reaction neutral to phenolphthalein.
If this medium is to be used in tubes the sterilized azolitmin (1 per cent. aqueous solution) is added just before the final mass sterilization, that is, the sterilization before pouring into the tubes.
If the medium is to be used in Petri dishes, the azolitmin is not added until ready to pour into the dishes.
The azolitmin and the lactose should be sterilized separately before adding to the agar medium, though it is permissible to mix the lactose with the agar and sterilize together, preferably in the autoclave (120° C. for 15 min.).
It would appear that the azolitmin of the market varies con- siderably and many bacteriologists prefer the pure litmus. A 1 per cent. aqueous suspension of azolitmin should dissolve readily when boiled for 5 min.
This medium is much used in bacteriological work on pre- sumptive sewage contaminations, as estimating the temperature
STANDARD MEDIA 79
differential colonies (20° C. and 37° C.), red colonies and total colonies, etc.
6. Lactose Bile—This medium is to be made in two ways: Add 1 per cent. of peptone and 1 per cent. of lactose to sterilized undiluted fresh ox gall; or add the peptone and lactose to a Io per cent. aqueous solution of freshly made dry ox gall. It is used without titrating. Old dried ox gall should not be used. Obtain it from a reliable dealer. If possible, make arrangements to get the fresh undiluted ox gall from some abattoir.
This is the standard medium for making the quantitative as well as qualitative tests for the colon group of bacilli.
7. Liver Broth.—Chop 500 grams of fresh beef liver into small pieces and place in 1ooo cc. of distilled water. Weigh infusion and container. Boil for 2 hr. in rice cooker, starting cold and stirring occasionally. Make up loss in weight and pass through wire or cloth strainer. Add 1o grams of peptone, 10 grams of dextrose and 1 gram of di-potassium phosphate (K.- HPO,). Dissolve the added ingredients by warming in rice cooker with stirring and then titrate to the neutral point (to phenol- phthalein). Boil for 30 min. in the rice cooker and for 5 min. over open flame with constant stirring to prevent the carameliza- tion of the dextrose. Make up loss due to evaporation and filter. Tube, sterilize and store as for other media.
This is a much used enriching medium which gives asg forma- tion with all of the species which ferment dextrose. It is also much used to rejuvenate pure cultures of bacteria and encourages the development of attenuated forms of bacteria.
8. Hiss Typhoid Bacillus Medium.—Two media are used. One for the isolation of the typhoid bacillus by the plating method, and the other for the differentiation of the typhoid germ from other forms in tube cultures. The former is designated as the plate medium and the second as the tube medium. They are prepared as follows:
80 BACTERIOLOGICAL METHODS
a. Plate Medium
AGAL. 4 nt asad tad haniudandy roa oiueue See es t34 wee ro grams GEA ied So ue anlnneng ghia aba eoe gees Oe 25 grams Salt. crea oh see ey, # are ites tse epee atta we hnshyisey 5 grams Liebig’s méat extracts so. xs 0mecume a4 ea pepe oe Io grams WOXTTOSOS. 2 ond Son bee TRD RR EER Aer uray sat Io grams Water (distilléd) i c.g ccuen see eSh eee seers 1000 CC.
Add gelatin when the agar is melted, dissolve the gelatin, add the other ingredients, titrate to +2.0 per cent., filter, etc., as for other media. The medium is to be clarified by adding the whites of one or two eggs, well beaten in 25 cc. of distilled water, boil for 45 min. and filter through absorbent cotton. Do not add the dextrose until after clearing.
b. Tube Medium
PBA 48 Rants i crena seed ctte dit serene ge Samedi acest 5 grams SS LAE TAN cpety ch steed noe ateh ch Alene ete FES ch SOI dha in yt tne tes PS 80 grams ALL Ec est aw tel aed ace A ee gt ney ea Ak inte at 5 grams Tiebig’s meat extracts cic es dad i neag eugene 5 grams TD GRETOS Chuo pand ec cSaaritchal a oboe vin Sra beac a abs Bag Io grams Water (distilled)...... ..... be GA ee Ney th eres atatshs 1000 Cc.
The manner of preparation is the same as for the plate medium. However, the reaction is to be +1.5 instead of +2.0 per cent. Without the dextrose and less salt and titrated to +1.0 per cent., the plate medium constitutes the ordinary nutrient agar- gelatin medium which was formerly very much used because it possessed the solidifying properties of agar combined with the nourishing properties of gelatin.
9g. Endo Medium.—This medium is much used in testing for the colon bacillus. It is variously modified by different workers and it is highly important that some standard method of pre- paring the medium should be adopted and adhered to. The following is the method of preparation and use recommended by the committee.
Add 30 grams of powdered agar to 1 liter of cold water by sifting slowly upon the surface of the water and allowing it to
STANDARD MEDIA 81
settle. Add ro grams of peptone and 5 grams of Liebig’s meat extract. Heat in rice cooker until the ingredients are entirely dissolved. Neutralize with sodium carbonate, using litmus as an indicator, and then add to cc. of a 1o per cent. solution of sodium carbonate.
Store the medium in lots of roo cc. using flasks large enough to permit the addition of the other ingredients. Sterilize for 2 hr. in streaming steam.
To use the Endo medium proceed as follows: Make a 10 per cent. aqueous solution of sodium sulphite and add 2 cc. of fuchsin solution (10 per cent. of basic fuchsin in 96 per cent. alcohol) to 10 cc. of the sulphite solution and steam this mixture for a few minutes in the steam sterilizer. Add 1 gram of chemically pure sterilized lactose to each roo cc. of the Endo medium after the medium has been liquefied and while the temperature is not above 60° C. While the medium is still liquid, add 0.5 cc. of the fuchsin-sulphite solution and then pour into the Petri plates and allow to harden in the incubator. The sulphite solution must be prepared fresh as needed.
10. Milk.—The milk to be used for cultural purposes must be pure and recently drawn: In all cases the milk of the grade or quality known as ‘‘certified milk” is to be preferred. The recently drawn milk is to be placed in the refrigerator for 12 hr., so as to permit the cream to rise to the top and any sus- pended matter to sink to the bottom. Skim the milk and siphon off all but the bottom sedimentary portion. Adjust to +1.0 per cent. Tube and sterilize.
Litmus milk is made by adding 1 per cent. of sterilized azo- litmin to the above. In using litmus milk always set aside a control tube with the inoculated tubes for purposes of color comparison.
Because of the difficulty of always getting a uniformly high quality of cow’s milk, it has been suggested that an artificial substitute be employed. Hill and his pupils recommend a
82 BACTERIOLOGICAL METHODS
medium in which prepared casein (nutrose) is the principle in- gredient. Chemically, nutrose is a caseinate of sodium and is prepared as follows: Moist casein precipitated from skimmed milk is washed with water in a solution of sodium hydroxide, evaporating the solution to dryness in vacuo, powdering the residue and washing successively with alcohol and ether and then dry- ing. It is a coarse, white, odorless and tasteless powder, forming a turbid adhesive solution with water, having an alkaline reac- tion toward litmus and an acid reaction toward phenolphthalein, It is a food product intended for the sick because of its easy di- gestion. It is made in Germany but may be secured through any of the larger American pharmaceutical houses (Victor Koechl & Co., New York City). The formula for making the artificial milk is as follows:
INUEPOS Gai s secsce cp RLS ad no AO, GA ae ne Bea 24 grams TACOS Eric d Faded end ne Phaeing sabe tandes ARdsn na ese Io grams Distilled: waters, pics /acarscrdx cr 4-eemneg rend eco k Heese 1000 CC.
Dissolve the nutrose and lactose in the water (cold) for 12 hr. with occasional thorough shaking and then filter through cotton. Tube and sterilize at 110° C. for 20 min., or in the steam sterilizer in the usual manner. No adjustment is required.
This medium contains all of the nutritive ingredients of cow’s milk with the exception of fat which is not desired for the ordinary cultural work. It is of uniform quality and is said to give far more uniform results than cow’s milk. It is furthermore more translucent than cow’s milk and shows the reactions with in- dicators much better. It would be advisable to make the artificial milk the standard substitute for cow’s milk.
11. Peptone Medium.—This is simply a 1 per cent. peptone solution in distilled water and is intended to be used for making the indol test. Beef broth from which muscle sugar has been removed by inoculating with B. colz is believed to be objection-
STANDARD MEDIA 83
able because of the toxins present and which interfere with the growth of many species of bacteria.
Other media of a more or less special character will be described or referred to under the discussion of methods. Those described above are the more important ones required in the bacteriological examination of foods and drugs.
7. Technique for Making Quantitative and Qualitative Estimations by the Plating Methods
As has been explained, the plating method is intended to de- termine the number of living bacteria present in foods and drugs and the results supplement the results of the method of making the direct counts already described. From this statement it is evident that the quantitative results by the two methods are not the same. For example, the bacterial count of a catsup by the direct method may be very high while the plating method may give negative results, due to the fact that the heat sterilization employed at the cannery killed all of the bacteria present. This also shows why it is absolutely necessary to employ both methods in order to form a correct estimate of the total contamination of the substance.
The following suggestions on laboratory technique are given with a view to the unification of methods, thereby leading to greater uniformity in comparative results.
1. Apparatus.—Test-tubes to be used for the usual cultural purposes shall be of medium weight and thickness, 15 cm. long by 1.6 cm. diam. Petri dishes shall be 10 cm. in diam. Petri dishes with porous covers are preferred. All glass ware must be scrupulously clean and may be sterilized by exposing to a dry heat of about 150° C. for a period of 1 hr., after being cleaned, wiped dry and plugged with a good grade of commercial cotton. A browning of the free ends of the cotton plugs indicates that the right degree of heating has been attained. A standard wire
7
84 BACTERIOLOGICAL METHODS
loop is made as follows: Bend the end of a No. 27 platinum wire, ro cm. long, around a piece of No. 10 wire. The free portion of the straight platinum wire inoculating needle, shall be 10 cm. long (No. 27 wire). The standard fermentation tube shall be of the following proportions. The length of the closed end of the fermentation tube (diameter about 1.5 cm.) shall be about 14 cm., and the open end shall be of bulbous form (diameter of bulb about 3.8 cm.) large enough to hold all of the liquid in the closed end. Larger and smaller fermentation tubes than the standard just described may be used for special purposes. Standard and other fermentation tubes may or may not be graduated as the special purposes may require.
2. Amounts of Media to be Tubed.—The standard amount of culture medium to be placed in each test-tube of standard size is 5 and ro cc., the media to be introduced by means of a suitable burette. Greater or lesser quantities may be used as occasion may require. Tubes containing just ro cc. of culture media are required for the plating purposes. 5 cc. quantities (of gelatin, agar and other solid media) are required for making slants.
3. Amounts of Culture Media to be Plated.—For the usual quantitative determinations by the plating method, ro cc. of the culture medium shall be poured in each standard Petri dish.
The required number of tubes each containing ro cc. of agar or gelatin culture medium are placed in the steam sterilizer until the medium is entirely liquefied and then placed ina beaker or other suitable container with lukewarm water, with thermometer. Plate the gelatin medium when the thermometer registers be- tween 25° and 30° C. The temperature of the medium must not be more than 30° C. If the temperature is less than 25° C. the gelatin will begin to coagulate and will not pour and spread properly. Agar media must be plated at a higher temperature than gelatin media, usually 40° to 42°C. The Petri dishes should be warm when the media are poured, the temperature being ap- proximately the same as that of the medium when it is poured.
TECHNIQUE 85
This will insure a more uniform spreading of the medium over the bottom of the dish.
To pour the liquefied agar or gelatin from the tubes, remove the cotton plug and flame the mouth of the tube so as to kill any bacteria or spores that may be present; raise one side of the cover just high enough to permit bringing the tube to the middle of the dish and pour contents into the dish over the material planted into the middle of the dish. Let cover of the dish sink into place and by very slight tilting of the Petri dish induce the culture medium to spread evenly over the bottom of the dish before the medium has had time to coagulate. As the medium spreads it also causes the spreading of the planted material.
Many workers use 5 cc. of the medium for plating, instead of 10 cc. asabove recommended. The smaller amount is satisfactory when 1 cc. quantities are to be planted or inoculated. However, in order to make sure that the entire area of the bottom of the dish is well covered, 10 cc. quantities should be used. The larger amount also minimizes the influences which the changes in evaporation in the media may have upon the quantitative results.
4. Method of Making the Plate Cultures.—Absolutely clean sterilized (dry heat of 150° C. for 1 hr.) Petri dishes of the standard size (10 cm. diam.) are used. 0.1 cc. quantities of the substance to be cultured, or dilutions thereof, are planted or delivered into the middle of the dish, an absolutely clean and sterile 1 cc. pipette accurately divided into tenths. The cover of the dish is to be lifted just high enough to permit placing the pipette in position, and is to be replaced just as soon as possible.
In the usual water analysis work, 1 cc. quantities are generally planted, instead of o.1 cc. quantities as above recommended. For purely quantitative results, the smaller amounts should be planted because the larger amounts may include enough of the inoculating liquid to interfere with the uniformity of results.
Formerly it was customary to mix the material to be planted with the medium in the tube before plating. This method has
86 BACTERIOLOGICAL METHODS
some very objectionable features, chief of which is that the residue remaining in the tube after pouring retained a certain percentage of the organisms, thus interfering with the accuracy of the results. It must, however, be admitted that the method has some ad- vantages, chief of which is the more uniform mixing of the bacteria with the medium and their more uniform distribution in the plate, making accurate counting of the colonies easier.
5. Making the Dilutions—Whether or not making dilutions is necessary depends upon the number of organisms present in the substance to be analyzed. The number of colonies in a Petri dish must not exceed 200 in order to make counting fairly easy and accurate. In fact with the method of direct planting, as usually recommended, which generally results in a some- what irregular distribution of the bacteria (hence also the colonies to be counted) it would be desirable to make the dilutions such that the number of colonies in each plate shall not exceed roo. If 0.1 cc. quantites are to be plated or planted, as above recom- mended, it would follow that dilution would not be necessary as long as ‘the number of bacteria per cc. does not exceed 1000.
However, since most food and drugs contain more chan that number of bacteria per cc., it becomes necessary to make dilu- tions. The standard dilutions are made by tens, as 1-10, 1-100, I~1000, and 110,000. The dilutions are made by adding 1 cc. of the substance to be analyzed to 9, 99, 999 and 9999 cc. of sterile distilled water, or other desirable sterile diluent, and shaking thoroughly. In practice it is desirable to plate three of the graded dilutions, so that the second higher dilution will in all probability yield about 100 bacteria in the o.1 cc. of the material plated. Thus with fairly pure drinking water, the plant- ings would be made from the undiluted water, the 1-10 and the 1-100 dilutions, presuming that there are about 10,000 bacteria per cc. present. In case of unusually pure drinking water, that is water in which the number of bacteria is probably not more
TECHNIQUE 87
than 5o per cc., it would be desirable to use 1 cc. quantities for plating which would give about 50 colonies in the plate.
The thorough mixing of the sample before making the dilutions is of the greatest importance, likewise the thorough mixing of each dilution before taking out the quantity to be*plated. Each
/ 2 3 4 pe ve 7 A 6 4 g
Fic. 23.—Types of growth in stab cultures. A, Non-liquefying. 1, Filiform (Bacillus colt); 2, beaded (Streptococcus pyogenes); 3, echinate (Bacterium acidi lactici); 4, villous (Bacterium murisepticum); 5, arborescent (Bacillus mycoides).
B, Gelatin liquefying. 6, Crateriform (Bacillus vulgare, 24 hr.; 7, napiform (Bacillus subtilis, 48 hr.); 8, infundibuliform (Bacillus prodigiosus); 9, saccate (Microspira finkleri); 10, stratiform (Pseudomonas fluorescens).—(M cFarland, after Frost.)
test should be made in triplicate, taking up the 1 cc. amounts for making the dilutions with three different clean sterile pipettes. It is preferable to use a new pipette for each dilution. [If it is not convenient to have on hand a sufficient number of clean sterilized pipettes, the pipette in use must be thoroughly
88 BACTERIOLOGICAL METHODS
rinsed in sterilized distilled water, using a fresh supply of dis- tilled water for each rinsing.
Draw the liquid to be plated into the pipette, place thumb over the upper end of pipette, let liquid run out until one of the o.1 cc. marks is reached, then bring the lower end of the pipette close to the surface of the liquid in the diluting tube and let just o.r cc. run out, the finer degrees of accuracy are attained by using or not using the meniscus of the liquid projecting from the lower end of the pipette after the last drop has fallen. This correcting droplet is secured by touching the lower end of the pipette lightly against the inside of the diluting tube at a point near the surface of the liquid, without, however, actually touch- ing the liquid. A similar adjustment may be made when taking up the 1 cc. amount to be diluted, only in this case the pipette is of course to be touched against the side of the tube or vessel containing the liquid of which the dilution is to be made.
Thorough mixing of the contents of the diluting tube is at- tained by vigorous shaking. Place the thumb over the open- ing of the tube, interposing a piece of sterilized rubber sheeting such as is used by dentists. Some workers mix the contents of the tube by rapidly rotating between the two hands and by tap- ping against the palm of one hand.
6. Incubation.—The regulation incubators are to be used. It is highly important that there should be ample ventilation, a matter to which amateurs and even experienced bacteriologists as a rule give little or no attention. All modern incubators are supplied with ventilating openings at the top which should be kept open most of the time. The air in the incubating chamber should be practically saturated with moisture, which may be accomplished by placing a flat dish containing water in the lower chamber.
Two standard incubating temperatures are employed, namely, 20° C. and 37° C., the first corresponding to the ordinary room temperature and the second to the body (human) temperature
TECHNIQUE 89
or blood heat. The devices to regulate the temperature should be such that the variation from the two standards given shall not be more than 2°, that is, not more than 1° in either direction. There is no standard time of incubation. For work in the study of water sanitation as carried out in Germany, England and also in the United States, gelatin plates are incubated for 2 days at a temperature of 20°C. It is suggested that the period be extended to 3 days in order to get more accurate results.
7
stents)
2! Scene
gt 7
Fic. 24.—Types of streak culture. 1, Filiform (B. coli); 2, echinulate (B. acidi lactici); 3, beaded (Sir. pyogenes); 4, effuse (B. vulgaris); 5, arborescent (Bacillus mycoides).—(McFarland after Frost.)
From 1 to 2 days is the usual time of exposure for the higher temperature (37° C).
8. Practical Application of the Quantitative Estimations by the Plating Methods
The relative importance of the quantitative bacteriological de- terminations by the method of direct counting and by the plating method has been explained. Both methods must be made standard in every food and drug laboratory. Quantitative esti- mations by the plating method should take precedence with all substances containing largely living organisms such as water supplies of all kinds, milk, raw meats, and shellfish, etc., and all substances in which infection is suspected, even though such sub-
go BACTERIOLOGICAL METHODS
stances may have been subjected to processes of sterilization during some phase of the processing or of manufacture.
In a general way the quantitative results by the plating method are to be interpreted in a manner similar to the results by the direct count. In some cases the question at issue may be relative to the presence or absence of viable bacteria in substances which presumably do not contain living organisms, such as canned foods generally. Manufacturers of canned products are of the opinion that the methods of heat sterilization employed will kill all of the bacteria which may have been present at the time of canning. This is undoubtedly true in many cases, but in other instancesitis only too evident that retarded fermentation processes continue after the cans are sealed, which accounts for the high counts in canned food products which contained only small numbers of bac- teria at the time the cans were sealed. These subdued fermenta- tion processes as a rule do not result in the formation of sufficient gas to produce ‘‘swells” and hence the article is not suspected until the container is opened when a more or less disagreeable or peculiar odor is noticeable, which is, as a rule, not sufficiently pronounced to prevent the use of the article as food.
In addition to the purely quantitative results, the plating method indicates the general qualitative character of the organ- isms present and conveys some idea as to the course of the infection or contamination as shown by the characters of the colonies de- veloped in the Petri dish or in the tubes.
9. Qualitative Determinations
The chief qualitative determinations in food and drugs labora- tories pertain to sewage contamination. The recognition and determination of pathogenic bacteria as the typhoid bacillus, the cholera bacillus, diphtheria bacillus, etc., is an incidental possibility in the food laboratory routine and not a regular part of it. Of far greater significance is the recognition of the evidence
QUALITATIVE DETERMINATIONS gI
of the presence of intestinal parasites, such as the segments of tape worm, the larve and ova of such parasites, etc., as already stated under the discussion of the direct method of making counts. The bacteriologists in food and drugs laboratories should be qualified to recognize all of the possible disease germs and the smaller carriers of disease which may be associated with food substances and they should be able to demonstrate the presence of such contamination, if necessary. The thus far recognized routine in food and drugs laboratories and in public health laboratories is limited to making the so-called presumptive colon bacillus test, as indicative of sewage contamination or contamination with fecal matter. Sewage con- tamination means primary contamination with fecal matter. The reason why the colon bacillus test has been selected as giving satis- factory evidence of sewage contamination is because this bacillus is most abundant and is constantly present in fecal matter. Any considerable number of colon bacilli in water supplies or in food substances is evidence of gross negligence and defects as to sanitary requirements.
As far as the practical work in finding evidence of the sewage contamination of food substances is concerned, there is no effort made to isolate and identify a definite bacillus recognizable as Bacillus coli. It is rather the recognition of certain cultural characteristics which have come to be recognized as being peculiar to the bacilli, known as the B. coli group, all of which are traceable to intestinal origin. Furthermore this group of bacteria is very widely distributed in the animal kingdom, being in no wise limited to the intestinal tract of man. The B. coli group of the lower animals is in all probability different from that which inhabits man and certain workers have made attempts to differentiate them by means of special cultural methods, but thus far these methods are not sufficiently perfected to be used practically in food and drugs laboratories. These statements also apply to the Streptococci group of intestinal origin. However, some of the laboratory results thus far attained would indicate that in the
g2 BACTERIOLOGICAL METHODS
near future it will be possible to differentiate between pollutions traceable to human origin and such as are traceable to cow, horse or hog manure, for example. It cannot be denied that food materials intended for human consumption which are contamin- ated to any distinctly appreciable amount with the contents of the intestinal tract of any animal, are unsanitary and hence unfit for human consumption.
In-as-much as the intestinal bacteria (bacilli and streptococci) are very abundant and very widely distributed, it is quite evident that it would be impracticable to pronounce all foods unfit for use if only one ora few intestinal organisms were found to be present in a comparatively large quantity. Human feces contains about one-third bacteria (dry weight), the majority of which belong to the colon group, and the exterior of the human body carries bacteria derived from the intestinal tract, especially the hands and the deposits under the finger nails. Flies are carriers and distributors. of intestinal bacteria. The dust of the streets and street sweepings contain large numbers of bacteria derived from the intestinal tract of the horse, etc. It would be impracticable to enter into a fuller discussion of the distribution of bacteria traceable to in- testinal origin. Suffice it to state that it is the work of the food bacteriologist to determine the presence, in articles intended as food, of those bacteria which indicate contamination with fecal matter, no matter what the source of such objectionable matter maybe. The basis for the condemnation of contaminated foods is quantitative in the comparative sense. For example, the finding of a few colon bacilli in large quantities of water or their occasional presence in small quantities of water, does not indicate that the water is unsuitable for drinking purposes. If, however, the colon bacilli appear in a large proportion of many small samples (1 cc. or less) of water it is safe to conclude that there is considerable recent sewage contamination and that such water is dangerous to health. The intestinal bacteria are in themselves not seriously pathogenic to man even when taken in considerable numbers.
QUALITATIVE DETERMINATIONS 93
The real source of danger lies in the fact that the intestinal bacteria normal to man and the lower animals, may be and frequently are associated with pathogenic bacteria, such as the typhoid bacillus and the dysentery bacillus. Our long experience with the con- sumption of sewage contaminated water supplies, has shown that, as a rule, the first danger sign of the excessive contamination was usually a marked increase in the number of cases of dysentery, generally followed by sporadic cases and epidemics of typhoid fever.
The practical results of the quantitative bacterial determi- nations of food substances combined with the qualitative tests for the colon group, has proven of the highest value and it is considered entirely feasible to continue the application of the tests and to suggest ways and means of improving the laboratory technique covering such methods. The qualitative methods thus far worked out are based upon a knowledge of the life history of the bacteria concerned and may be briefly stated as follows: Normal intestinal bacteria and such other bacteria as may develop in the intestinal tract, such as the typhoid bacillus, the cholera bacillus, the dysentery bacilli, etc., are adapted to a temperature of about 37° C. and they feed upon the food materials found in the intestinal tract and have a somewhat reduced oxygen supply. Among the substances peculiar to the intestinal tract we find bile, pancreatin, and other enzymes and a certain water percentage and the: various ingredients of food materials more or less digested and the various products elaborated by the different species and varieties of bacteria present. It is a study of the peculiarities of the intestinal bacteria which has suggested the technique for their isolation and their quantitative as well as qualitative esti- mation in food supplies, as can be seen readily from a study of the culture media and cultural methods recommended. To enter into any fuller discussion of the work done by American and European investigators on the bacteria which are normal to the intestinal tract or which may occur in the intestinal tract in
94 ; BACTERIOLOGICAL METHODS
disease, is not practicable but we desire to give the following tabulation showing the relationship between the different species of the colon-typhoid group of intestinal bacteria in their behavior with dextrose and lactose media.
BacTERIA OF THE COLON-TYPHOID GROUP
Dextrose Lactose Species Gas Formation|Acid Formation| Gas Formation|Acid Formation B, alcaligenes . sSgseeatidie WY oe | ODES none none none yo A) 2) ee eer none slight none none B, dysenteri@seaiacicccigss none distinct none none B. enteritidis.............. active strong none slight Paratyphoid group........ active strong none slight Hog cholera bacillus....... active strong none slight Bie: COM bractuact mas ay x tiers ae active strong active strong
There are numerous other distinguishing characteristics be- side those indicated in the above tabulation, as agglutinating phe- nomena and behavior with other special culture media. It is simply desired to indicate somewhat more specifically the lines of research which were necessary to determine the identity of the related species and varieties of intestinal bacteria.
The Bacillus coli was isolated as early as 1884 from the feces of a cholera patient, at which time this organism was supposed to have some causal relationship to cholera. Later it was proven that this bacillus was a normal inhabitant of the intestinal tract of man and of other animals, being regularly present in their excreta, and this discovery proved of the highest importance to sanitarians as the presence of this germ in water supplies, in milk, in mineral waters, etc., is generally regarded as evidence of sewage contamination. The colon bacillus has been found in sewage contamination, river water, in spring water, ice, milk,
QUALITATIVE DETERMINATIONS 95
cream, butter, buttermilk, sour milk tablets, mineral waters, oysters, clams, flour, oatmeal, cornmeal, cereals, frozen eggs, dried nuts and fruit, etc. B. coli is not normally present in sea water and its occurrence in salt water shellfish is evidence of sewage contamination. The occurrence and general distribution of the colon bacilli is almost in direct proportion to the density of the population. Animals of all kinds are disseminators of colon bacilli, particularly the larger animals as the horse, cattle, the dog and domestic fowls. Within and about the home, the house-fly and the stable-fly are the chief distributors of colon bacilli. We may repeat that colon bacilli are found on the skin of persons, particularly on the hands and under the finger nails. The under- clothing worn carries these bacilli and is the agent instrumental in distributing them over the exterior of the body, especially in those of uncleanly habits. Water in which the hands have been rinsed will generally yield positive colon bacillus tests. It is also apparent that the colon bacillus does not survive for a great length of time outside of its natural environment; thus sewage- contaminated waters purify themselves of colon bacilli after a time, the period varying with the temperature and the amount of organic matter present. Thus it sometimes happens that a water supply may show a high bacterial count and yet be quite free of colon bacilli. As a rule, however, water supplies and substances brought in contact with such water supplies which show a high general bacterial count, will also show a comparatively high count in colon bacilli. There may be notable exceptions to this rule. A water supply, or other liquid substance, may show a comparatively low bacterial count and yet yield numerous colon bacilli. Such an occurrence would indicate an unusual source of extensive sewage contamination.
From the foregoing it is evident that the sanitary examination of foods and drugs resolves itself into the making of quantitative bacterial counts, as already fully explained, and the presumptive colon bacillus test, with an occasional test for other specific
96 BACTERIOLOGICAL METHODS
organisms, as will be explained later. It is also evident that the isolation or the identification of the colon bacillus in a mixed contamination as all sewage-contaminated substances are, is not as simple a matter as might appear on first consideration. However, the presumptive tests for the presence of the colon bacillus in definite quantites of the food materials or liquids used with, or associated with, certain food materials, is almost uni- versally accepted as evidence of the dangerous contamination with sewage. It is, however, quite clear that health officers should not adopt hard and fast rules or standards for the con- demnation of foods because of such evidence of sewage con- tamination. Very naturally, the standard for water supplies will not apply to oysters and shellfish generally and the standard for shellfish will not be practically applicable to mineral waters, etc. With substances of which the standard or quality is quite generally based upon a numerical count, as for example milk, the presumptive colon bacillus test need not be applied, unless it is to be carried out as giving corroborative evidence of the sewage contamination.
One of the first important duties of the food and drugs bac- teriologists will be for them to get together and agree upon uni- form methods and to decide upon the kinds of bacteriological examination under the pure food and drugs act to which the quantitative as well as the qualitative (presumptive colon bacillus test) determinations are applicable, in harmony with our present knowledge of food bacteriology. The working laboratory methods adopted must be practicable and must be carried out primarily as a better protection of the physical well-being of the consumer, incidentally also safeguarding the business interests of the con- scientious manufacturers. The following suggestions are in- tended to indicate along what lines the practical qualitative work may be done and also to outline certain research work which should be carried on in order to develop the working methods to greater perfection and to add such new methods as may prove useful.
SEWAGE CONTAMINATION 97
1o. Evidence of Sewage Contamination. General Methods.
It may be assumed that the presence of any or all of the large group of colon bacilli in water or in food substances is indi- cative of sewage contamination or contamination with fecal matter. The colon bacilli are aerobic, nonsporeforming, motile, short and produce acid and gas in dex- trose and lactose media and develop best at a comparatively high tempera- ture (37° C.). A practical presumptive colon bacillus test depends upon the characteristics thus indicated and is car- ried out as follows:
1. Presumptive Colon Bacillus Test. —Add the substances to be tested (water, sewage, mineral water, shellfish liquor, washings from vegetables, etc.) in 0.01 cc., 0.10 cc., I cc., § cc. and 10 cc. quantities (or these equivalents in dilutions) into fermentation tubes hold- ing at least 40 cc. of lactose bile, incu- bate at 37° C. and look for the forma- tion of gas. If gas formation is observed Fs r pare eee ee the presence of colon bacilli may be sus- _ tion tube is especially conven- pected. If, in the case of water supplies ‘¢nt for making the gas deter- for example, the o.10 cc. tubes show gas__ lus. Other forms of fermenta-
7 ; tion tube may be used.—(Pitt- formation then it may be reasonably as- feild.) sumed that colon bacilli are present. If two out of five of such tubes give positive gas reactions, the test may be considered conclusive. To test the gas formed, fill the tubes showing gas formation with a 2-per cent. solution of sodic hydrate, hold thumb firmly over the opening of the fer- mentation tube and-mix contents by tilting back and forth carefully. The volume of gas absorbed is COz whereas the un-
98 BACTERIOLOGICAL METHODS
absorbed portion is supposedly hydrogen. The colon bacillus shows a gas formation of 14 hydrogen. The standard time of incubation is 48 hr., but if colon bacilli are abundant, gas forma- tion will be observed in the tubes carrying the larger amounts of the inoculated material at a much shorter time, occasionally within a few hours. Small numbers of attenuated colon bacilli may require 2 and 3 days before there is any gas formation noticeable. In this connection it may be mentioned that the attenuated colon bacilli indicate remote contamination, as all B. coli of recent contamination develop readily in lactose bile.
Fic. 26.—Bacillus coli. Superficial colony on a gelatin plate 2 days old (X 21). —(McFarland after Heim.)
This constitutes the usual presumptive test for the presence of sewage contamination. Some investigators, however, recommend that the test be supplemented as follows: Plant the suitable quantities or dilutions into liver broth (in test-tubes) and in- cubate at 37° C. for about 12 hr. and then transplant these cultures into the lactose bile as above explained. The liver broth en- richment medium is said to bring out the attenuated forms of colon bacilli. In routine procedures the liver broth culturing is usually omitted as the important point at issue is the determina- tion of fairly recent contamination with sewage, or of sewage
COLON BACILLUS TEST 99
contamination in large amount, and the lactose bile medium gives conclusive results regarding this.
The presumptive colon bacillus test is to be supplemented further as follows: Plate suitable dilutions of the substances to be tested for sewage contamination (0.001 cCc., 0.01 CC., 0.10 CC., 1.00 cc.) into lactose litmus agar Petri dishes, making two sets. Incubate one set of these plate cultures at 20° C. and the other at 37° C. and note the following:
1. The relative number of colonies which develop at the two temperatures. 2. The number of acid-forming colonies.
The time of incubation at the lower temperature (20° C.) should be 3 days, although fairly conclusive results may be noted at the end of the second day. The standard time of incuba- tion at the higher temperature (37° C.) is 48 hr., although certain results may be noted at the end of 24 and 36 hr. If the propor- tion of high temperature colonies is high, it is indicative of the presence of numerous bacteria derived from the intestinal tract. If the high temperature colonies approximate (numerically) the low temperature colonies, sewage contamination may be suspected. If in addition many of the high temperature colonies show pink or vermilion (on lactose-litmus agar), the sewage contamination is practically proven. Both the colon bacilli and the sewage streptococci show pink colonies on this medium, the latter being the brighter, more vermilion in coloration. This coloration is due to the formation of acid by the organisms named which reacts with the litmus. Examine the pink colonies under the micro- scope in order to determine which are the colon bacilli and which the streptococci. Asa rule, high temperature colonies should not exceed 1 : 100 as compared with the low temperature colonies. It must be kept in mind that the pink colonies may turn blue within 24 hr. due to the liberation of ammonia and amines. Red colonies indicate lactose fermentation with formation of acid, but
since bacteria other than the colon bacillus form acid (notably 8
100 BACTERIOLOGICAL METHODS
the streptococci), it is desirable to examine such colonies micro- scopically and to inoculate into other media and perhaps to test for indol formation, in order to obtain satisfactory proof as to whether or not they are colon bacilli.
Neutral red (a safranine dye) reduction was at one time con- sidered a very important check test for the colon group. Stokes, as early as 1904, recommended that neutral red be added to lactose broth in the fermentation tubes which contain the required dilu-
Fic. 27.—B. coli showing flagellz stained by the van Ermengen method (X 1000).—(MacNeal, from McFarland after Migula.)
tions of the liquids to be examined. 30 to So per cent. gas forma- tion in the closed arms of the tubes and the change of the neutral red to canary yellow, is said to be characteristic for the colon group. It would appear that the majority of bacteriologists are inclined to omit the neutral red test as being of little value.
The production of indol in peptone broth or solutions is another colon bacillus test much used in the United States. Boehmes’ modification of the Ehrlich method is now generally employed, made as follows: Two solutions are required.
COLON BACILLUS TEST IOI
Sotution No. I
Para-dimethyl-amido-benzaldehyde................. 4 parts Absolute aleohol.s.. ¢sacas iaaca he eae amare ca nS Bee 380 parts Concentrated HCl is scsciec esuscneae sacs meee wxmee ueu 80 parts
Sotution No. II Sat. sol. of potassium persulphate. The indol test is performed as follows: Add 5 cc. of solution I to 10 cc. of a broth culture and then add 5 cc. of solution II, the
Fic. 28.—Bacillus of typhoid fever, stained by Loeffler’s method to show flagella (X 1000).—(Williams.)
whole being then shaken. A red color indicates indol. Some of the leading bacteriologists consider this a very valuable test.
The so-called hog cholera group or the Gaertner group of bacilli is important from the standpoint of the food bacteriologist. The Gaertner group occupy a position intermediate between the chemically active coli group and the chemically inert typhoid group and includes the following important species or rather strains —the Bacillus enteritidis strain which includes many of the bacteria isolated in cases of food poisoning and some of the B. typhi murium
102 BACTERIOLOGICAL METHODS
varieties, as B. psiltacosis, and B. suipestifer and B. paratyphosus B. They differ from the typhoid group by gas formation in dextrose, and from the colon group by the production of an alkaline reaction in milk. They are concerned in the development of intestinal disturbances such as dysentery and diarrhea. No practical routine working method for the isolation of the Gaertner group has as yet been recommended. Some of the more important cultural characteristics are indicated in the table of Bacteria of the Colon-typhoid Group.
Another important group of bacteria from the standpoint of the food bacteriologist is the large group of sewage streptococci. They occur in the intestinal tracts of many animals. There are numerous strains of this group and they are somewhat less widely distributed than the colon group. The determination of sewage streptococci adds but little more than may be learned from the colon test and for this reason we shall not enter into any fuller discussion. This statement also applies to the host of other bac- teria and related organisms which are more or less constantly associated with sewage and sewage contaminations.
For all practical purposes, the presumptive colon bacillus test supplemented, as the special cases may require, with certain special tests, combined with the quantitative counts by the plating method (gelatin media) will give all the information which is necessary to judge of the quality of certain foods, drinks and medicamenta, as far as the contamination with sewage is concerned. These points will be more fully discussed under special heads.
11. Possible Contamination of Foods with the Typhoid Bacillus
Testing food substances and medicamenta for the presence of the typhoid bacillus will never become a regular routine in the food laboratory. On occasion it will become an incidental pro- cedure and must therefore receive some consideration. To under- stand the special significance and importance of this organism as a possible contaminator of foods, it is necessary to enter into
TYPHOID BACILLUS CONTAMINATION 103
a brief statement of the typhoid fever and the organism which causes this disease. The primary cause of typhoid fever is the Bacillus typhosus, which in its general morphological characteris- tics resembles the colon bacillus, differing in that it is somewhat longer and more actively motile. When introduced into the intestinal tract of man it multiplies very actively and produces the symptoms of the disease known as typhoid fever. In disease, therefore, this organism grows in the same environment as the colon bacillus, excepting that the temperature (fever temperature) is higher. After recovery from the disease, the germs may remain
Fic. 29.—Bacillus typhosus, 72-hr. gelatin culture—(Stilt, after Kolle and Wassermann.)
in the intestinal tract for long periods of time, for months and years. Furthermore, those who have never had the disease may become infected with the germs and carry them for long periods of time without developing the disease. Persons infected with the germs of typhoid fever without suffering from the disease are known as typhoid carriers, and it is self-evident that they may cause typhoid fever in those with whom they may come in contact. Numerous such carriers have been found and many sporadic cases of typhoid have been traced to such source. However, the majority of typhoid epidemics are traceable to foods and drinks contaminated
Io4 BACTERIOLOGICAL METHODS
with the intestinal secretions of typhoid patients. The subject of typhoid contamination is therefore intimately associated with the general subject of sewage contamination or contamination with human fecal matter. Very naturally, the typhoid bacillus is far less common than the colon bacillus. In a general way it may be stated that the distribution of the typhoid bacillus is as wide as the distribution of typhoid contaminated sewage. As long as we adhere to the antiquated and highly unsanitary method
Fic. 30.—B. typhosus from gelatin smear preparation stained with fuchsin (X 1000).—(MacNeal.)
of emptying our sewage into the drinking-water supplies just so long will we continue to have epidemics of typhoid fever. Numer- ous statistical records show that the mortality rate from typhoid fever in our larger cities is directly proportional to the filthiness of the drinking-water supply. House-flies are known to be carriers of typhoid and the germs have been isolated from vegetable food materials, from oysters and other shellfish, from milk, etc.
The laboratory procedure in the examination of foods and liquids for the typhoid bacillus includes the isolation and identi-
TEST FOR TYPHOID BACILLUS 105
fication of the germ. The proceedings are similar to those out- lined for the colon bacillus, excepting that in this case the quan- titative factor is not considered. The finding of a single typhoid fever germ in a mass of food materials is sufficient to condemn it. It may be assumed that where there is one typhoid bacillus there are more in the same vicinity and these may initiate an epidemic of typhoid fever.
The food bacteriologist may be called upon to examine food substances for the presence of typhoid contamination (from the feces of typhoid patients or of carriers) in instances where it is known that food has been exposed to typhoid infection or where such infection is merely suspected. The isolation from foods and the positive identification of the typhoid bacillus is by no means a simple matter. It is necessary to make use of special cultural methods, supplemented by the agglutination test, etc. The methods tried out by various bacteriologists are too numerous to even review and most of them have after a time been abandoned as unsatisfactory. The following tabulation from the work of Prescott and Wilson indicates some of the more practical laboratory procedures which have been tried with more or less success.
a. By filtration. 6. By agglutination
1. Physical concen- Schuder’s tration c. By chemical | Fischer’s | Proc- precipitation | Wilson’s ess.
Miller’s
. Hoffman and Ficker’s caffein process. . Jackson’s lactose bile. . Parietti’s carbol broth.
a 2. Enrichment..... { 0 ¢ a. Elsner’s gelatin medium. b ¢
Examination of water for typhoid
bacilli . Endo’s medium.
. Loeffler’s malachite green medium. d. Drigalski-Conradi agar.
e. Hiss’s medium. f. Hesse’s medium.
a. Morphological and cultural charac- 4. Identification.... teristics. b, Agglutination,
3. Isolation........
106 BACTERIOLOGICAL METHODS
Space will not permit discussing the methods thus outlined nor is this essential for the present purpose. Those interested are referred to the work by Prescott and Winslow, Elements of Water Bacteriology (1913), which contains a fairly complete digest of the methods. Furthermore, the methods adopted must be suited to the special cases in hand. The most suitable procedure for isolating the Bacillus typhosus from drinking water would not be practicably applicable in the examination of typhoid con-
Fic. 31.—B. ltyphosus from an agar culture 6 hr. old. Highly magnified vA te ee the flagelle stained by the Loeffler method.—(McFarland after facNeal.
taminated sewage or milk, for example. For the time being there is no routine laboratory method for the isolation of the typhoid bacillus and we must content ourselves with a brief consideration of those methods which will in all probability give the best results.
It is of the highest importance that the food bacteriologist should search out typhoid contaminated foods before the occur- rence of an epidemic. In fact, if such work is not undertaken until cases of typhoid have developed, the bacteriological find-
TEST FOR TYPHOID BACILLUS 107
ings are often wholly negative, because of the long incubation period (14 days), so that the bacilli may all have disappeared from the sewage or water between the time of the infection and the manifestation of the symptoms of the disease. Under con- ditions favorable to the typhoid germs, as food supply, temperature, absence of sunlight, etc., they may survive for several months. It is generally conceded that the Bacillus typhosus is quite re- sistent and persistent. According to Ravenel, the germ survives for several months and longer in fecal matter deposited in snow which when carried into the stream supplying a city with drinking water by the early spring rains caused an outbreak of typhoid.
The highly objectionable method of using human excrement for fertilizing the soils of truck gardens, as practised by the Chinese and a others, may lead to the typhoid contamination Fie. pa of the vegetables grown in such gardens. Wash- trating the Widal ings of the soil and of the vegetables should be ee Rite examined for typhoid germs. eg ie sa
The following general method for the isola- shows clumping of tion of the typhoid bacillus is suggested, subject il either) hae to modification to suit special cases.
1. Concentration—Run from 1 to 10, and more, liters of water (as from well, cistern, stream, water tank, etc.) through a clay filter. Just before all of the water has passed through the filter, shake it up and pour into a suitable centrifugal tube (the special tube already described will answer the purpose very well) and place in incubator for 30 min. at a temperature of 37° C. The incubating is done for the purpose of increasing the motility of the typhoid bacilli.
2. Separation by Centrifugalization——Take tube from the incubator and centrifugalize for from 5 to 30 min. at a high speed. The non-motile bacteria will be thrown down first, while the
108 BACTERIOLOGICAL METHODS
highly motile Bacillus typhosus will tend to remain near the middle
and upper parts of the tube. 3. Cultural Separation on Basis of Motility——By means of a
sterile pipette take up the upper half or third of the contents of the centrifugalized tube (2) and place in the special loop tube with phenol-broth and incubate at 37° C. for 24 hr., or longer if necessary.
4. Plate Cultures.—Take up several platinum loopfuls from the loop tube (the opening opposite the inoculated end) and plant in lactose-litmus-agar (at 37° C.) and note the character of the colonies which form. Compare with the colon bacillus colonies. Examine colonies microscopically.
5. Other Cultural Tests.—Test for absence or presence of gas formation. Enrichment in liver broth may be tried, etc.
6. Agglutination Tests—Two methods may be used. The microscopical and the macroscopical. The usual routine mi- croscopical method is carried out as follows: By means of a clean sterile pipette place 0.1 cc. of the typhoid serum and 0.9 cc. of physiological salt solution (salt is necessary to bring about agglutination) in a clean sterile Syracuse watch crystal and mix thoroughly by means of a clean sterile glass rod. This gives a serum dilution of 1-10. Place one platinum loopful of a 24-hr. bouillon culture of the typhoid bacillus on a clean cover glass and add one loopful of the mixture from the Syracuse watch glass. This gives a dilution of 1-20. Two loopfuls of the culture and one of the serum mixture gives a dilution of 1-40. Three loopfuls of culture and one of serum mixture gives a dilu- tion of 1-80. Make the dilutions one at a time and place the cover glass holding them (inverted) on a vaselined hollow or concave slide and examine at once under the high power, con- tinuing the observation for 30 min. if necessary. The first change noticeable will be a gradual loss of motility, followed by a clumping of the now non-motile germs. This constitutes a positive agglutination reaction. Clumping with the lower
TYPHOID AGGLUTINATION TEST 10g
dilutions (1-20, 1-40) is not considered characteristic for the typhoid organism, since other bacteria may also produce agglutina- tion with the typhoid serum. It is, however, not likely that sera will agglutinate other than the specific one in dilutions as high as 1-80. Higher dilutions should be tried on the principle that the positiveness of the test is in proportion to the serum dilution which will produce clumping. It should also be borne in mind that the agglutination phenomena are more marked at the body temperature (37° C.) and that in the case of the typhoid serum, the paratyphoid group will also give positive results. In reporting on the agglutinating phenomena always give the dilution and the time factors. The novice must frequently be reminded that all manner of solutions of salts, acids, etc., will produce agglutination with most bacteria. We would not recommend the use of the blood-counting pipette (which accom- panies the hemacytometer) for making the dilutions and inixtures of the serum and the bacterial cultures, as is advised by some investigators, largely because of the danger of possible infection in sucking up the quantities of bacteria, and also because this method adds nothing to the value of the results.
For the so-called macroscopical method or precipitation method, as it is also called, small test-tubes are used in which the suitable dilutions of the serum (with normal salt solution) and the bacterial cultures are mixed. A positive reaction is indicated by flocculency and the deposition of a slight precipitate. Dead (formalized) typhoid cultures may be used. The method in general use in Germany is preferred, a description of which may be found in most text-books on bacteriology. Some of the American pharma- ceutical houses (Parke, Davis & Co.) market a full equipment for imaking the macroscopic agglutination test with the typhoid germ. It contains full directions for using and according to re- ports is as reliable as this test can be made for practical purposes. It need hardly be stated that in all cases it is desirable to make a control test with normal salt solution.
IIo BACTERIOLOGICAL METHODS
The following is offered by way of fuller explanation of some of the details of the method above outlined for the isolation and identification of the Bacillus typhosus. The unusually active motility of the typhoid germ has been utilized by several in- vestigators (Drigalski and Starkey) as a means for separating it from less highly motile forms. Drigalski allowed from 5 to ro liters of the suspected water to stand in tall milk cans for 1 or 2 days at the room temperature, after which he plated definite amounts taken from the surface of the container into litmus- lactose-agar. By this method he was enabled to isolate typhoid bacilli from several contaminated springs. Starkey used glass tubes bent into four loops which after being filled with phenol broth were inoculated at one end and incubated anaerobically at 37° C. for 24 hr. The more actively motile typhoid bacilli found their way to the fourth loop from which they were isolated by plating. The centrifugal method above recommended is merely an adjunct to the methods employed by Drigalski and Starkey. The non- motile bacteria are thrown down first and in a very short period of time thus being an advantage over the Drigalski method in which gravity is the separating force. It is true that in time the motile forms would also be thrown down. It is therefore im- portant not to prolong the centrifugalizing more than is necessary. In place of the four-loop Starkey tube we would suggest the use of four separate tubes; one a simple U-tube or single-loop, a W- or double-loop, a three-loop and a four-loop tube. These tubes, after being cleaned and sterilized are filled with phenol broth and in- oculated at one end at the same time. Incubate at 37° C. or even at 4o° C. and examine loopfuls taken from the ends opposite the ends inoculated as follows: The U-tube at the end of 6 hr., the double-loop tube and the three-loop tube at the end of 12 hr., the three-loop tube (reexamination) and the four-loop tube at the end of 24 hr., and the four-loop tube again at the end of 36 hr. ifnecessary. The phenol broth and the higher temperature hinders the growth of most bacteria without checking the growth of the
TEST FOR TYPHOID BACILLUS Iil
typhoid germs. These conditions will enable the highly motile
Bacillus typhosus to reach the more remote loops first where they
may be taken out by means of the platinum loop or the pipette. In place of the loop tubes above described and which can be
a b Qa b
m
Fic. 33.—Loop tubes for culturing and isolating typhoid bacilli and other motile bacteria as explained in the text. 1, Single-loop or U-tube; 2, double-loop or W-tube; 3, three-loop tube; 4, four-loop tube. The tubes are filled with phenol broth or other desirable media and inoculated at the ends marked (a). Material for subculturing and for microscopical examination is taken from the opposite end (6), at varying intervals of time.
made in the laboratory, it would be preferable to use a single tube of four or five loops provided with openings at each of the upper
turns of the loops, thus making five or six openings in all, from which the quantities to be examined and plated may be taken.
II2 BACTERIOLOGICAL METHODS
The tubes must be fastened to suitable stands or supports to pre- vent, as much as possible, the mechanical mixing of the contents after the inoculations are made. It is perhaps self-evident that concentrates or high contaminations are to be inoculated into the tubes. The tubes should be large enough to hold at least 50 to too cc. of medium and suspected water in equal parts.
12. Possible Contamination of Food Substances with the Cholera Bacillus
In the United States the contamination of foods with the cholera vibrio is far less likely than the contamination with the typhoid fever germ, yet it is a possibility to be reckoned with.
Fic. 34.—Spirillum cholere, from broth culture, stained with fuchsin ( 1000).— (Stitt, after Kolle and Wassermann.)
The cholera germ is found in the feces (but not in the urine) of patients and in the feces of carriers, in which regards it resembles the typhoid bacillus. It is less resistent than the typhoid organ- ism, disappearing rapidly from the stools, usually in 5 to 10 days. Under certain conditions (as in fresh water supplies) the infection may endure for longer periods, for several months and more. Like the typhoid germ, it shows some marked tend-
THE TEST FOR THE BACILLUS OF CHOLERA 113
encies to locate in the bile duct or gall-gladder, where it may re- main dormant for a long period of time. This observation has led to the use of bile as an enriching medium for both organisms. The cholera vibrio work in the food and drug laboratory may resolve itself into the isolation of the germ from water supplies, from vegetables and possibly from feces and from sewage, and consistsin the use of special culture media, special cultural methods, inoculation methods and agglutination tests. It is interesting to note that the method now in use for isolating the cholera vibrio from water supplies is the original Koch method, done as follows. ee Add 1 percent. each of peptone »@ a @ and salt to 100 cc. of the suspected és SLs aS % water and incubate at 38° C. 7 e' a “ens Examine microscopically at inter- 9 NM
vals of 8, 12 and 18 hr. As soon as; = a ioe as curved and comma-shaped - “ 14 organisms appear, plate on agar ee ay re
and make such additional tests “Qc
as may be necessary to prove the Fic. 35.—S. cholere showing invo- lution forms (X 1000).—(Mac Neal, presence of the cholera germ, such after Van Emengen.) as the nitroso reaction, agglutina- tion test, Pfeiffer’s phenomenon, etc. It is not practical to enter into a fuller discussion of the subject. More complete details will be found in the works on bacteriology and in bulletins and reports on bacteriology and on hygiene. For example, the U.S. Public Health Service has worked out a quick routine method for isolat- ing the cholera germ from feces, used in the U.S. Quarantine Ser- vice and at the quarantine station of New York, as reported in the Journ. of the Am. Pub. Health Association (Dec., 1911) and a condensed summary of the general methods may be found in the admirable little work by Stitt (Practical Bacteriology, Blood Work and Parasitology, 1913). Numerous special reports will be found in American and foreign bacteriological literature.
It4 BACTERIOLOGICAL METHODS
13. Biological Water Analysis
The complete biological analysis of water supplies is, as a rule, not a regular routine of the food and drugs bacteriologist, yet he should be prepared to make such analysis when occasion makes it necessary. The food bacteriologist will have to do more with the analysis of sewage contaminated water supplies and with foods and other substances which have come in contact with such contamination.
The complete biological analysis of water supplies may be out- lined as follows the fuller details of which may be found in special text-books, bacteriological journals and reports on water analysis.
Securing the sample. Bacteriological examination. Quantitative. Qualitative; the presumptive colon bacillus test. Alge; significance of. Diatoms. Desmids. Nostoc and oscillaria. Other alge. Molds and spores; significance of.
Ova and larve of higher parasites; significance of. Sand, dirt, etc.
The water supply of a city or community should be watched at all times, but perhaps more particularly in the early spring when the melting snows and the heavy rains bring in materials accumu- lated and held back during the winter months. Furthermore, the rise in temperature encourages the rapid multiplication of various organisms, such as alge and bacteria. In late summer and early fall the drinking water often becomes vitiated, through a reduction in supply, perhaps as the result of lack of rainfall. In the early spring, after the first days of warm weather, the water supply often becomes murky due to dirt washed in, green in tint due to the enormous development of alge and generally accompanied by a decidedly disagreeable odor which is traceable to the presence of
BIOLOGICAL WATER ANALYSIS IIS
blue-green alge of the Nostoc and Oscillaria groups. Various more or less futile attempts are made by the water companies to correct these conditions. In order to reduce the growth of alge the reservoirs are roofed over (the alge requiring sunlight for their development), forgetting that while one evil is thus in a measure corrected, another and perhaps greater, is encouraged by such pro- cedure, namely, the growth and development of bacteria which thrive best in the absence of sunlight. Numerous desmids, di-
Fic. 36.—S. cholere very highly magnified, showing flagellee—(MacNeal, from Kolle and Schiirmann.)
atoms and blue-green alge in drinking water, indicate the presence of dead and decaying organic matter in comparatively large amount. Diatoms are especially abundant in water supplies from old wooden tanks and wood-lined reservoirs. Nostoc and Oscillaria are especially abundant in water supplies fed from soii drainage. Bacteria are present in all soil and sewage contaminated waters. The well water of the farms may be contaminated with all manner of organisms, such as sewage organisms and disease germs, includ- 9
116 BACTERIOLOGICAL METHODS
ing the larve of Nematodes and the spores of fungi, to say nothing of