aa DAS PIN ey Re ant eR, ily al Pr he (Pn Ded tee Fetes Beet ee anit ckee ie Nee 9 eek art es aa! S aavet ye? Ae RO Beas [ “soya tam fy West eye “ - . : Sone hacker castes. Wace ae ee 4s eye Mags ASG IN he oo a t- meee Bm aay Kies Nacnh ty Borge ec en nih Scena Ve A SA BS Dobe Nene nee 4 Tey ne SAN BRAS tee tig treme Re Rekie! Tn We evta T W Sons eet heehee bee bs sant me * mow Mer MR AL, a eon ee ee ae Avite Seay tib, Nase Fei LEON rte Veen Perry te 6 hens MTN Sat oes wh n rly : a ut is a J "” 7 sy Var Weal da, M Say BACTERIOLOGICAL METHODS SC TENGE DEE as 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 ALBBR te SCHNEIDER, Me D:,°P ood. ‘(CoLUMBIA 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 Espa! ON’ Ss: SONSEICO. 1012*WALNUT STREET COPYRIGHT, 1915, BY P. BLaKxiston’s Son & Co. tf -_ A) i VA) & TR OP eee / e THE MAPLE PEPFS'S YORE PA ary 29 1915 ©c1.a414306 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 coéperation 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, CALIFORNIA. a CONTENTS I. OUTLINE OF MICRO-ANALYTICAL METHODS IN Foop AND Drucs LABORATORIES PAGE t. Introduction . oO Or nn . Grouping of Eiatances: is ie nined’ in Hodd aa Beues Labetatanes . The Work of the Micro-analyst in Eee to that of the Chemist and Bacteriologist . . Equipment for Micro- pinaigGel Mion . Organoleptic Testing : . Methods Useful in the Riana atin a Veaetable aye Snaee Ete . . Methods Useful in the Examination of Vegetable Food Products . Micro-chemical Color Reaction Tests . Making Analytical Reports . . The More Important Histological Plenteats ea Plants Il. BACTERIOLOGICAL METHODS IN Foop AND DrucGs LABORATORIES . Introduction : . Direct Bacteriological eenaianens ‘ : ; . Numerical Limits of Micro-organisms in F bode ia ips : . Quantitative Estimations by the Cultural Methods : . Preparation of Standard Culture Media. General Suggestions . Preparation of Required Standard Culture Media . . Technique for Making Quantitative and Qualitative BSpmanions Dy fhe Plating Methods . Practical Application of the ‘Guanenanre, stations he the Pigtne Methods. . . Qualitative ieicroteantions. , . Evidence of Sewage Contamination . Possible Contamination of Foods with the Pyphoid Bacdias. . Possible Contamination of Food Substances with the Cholera Bacillus. . Biological Water Analysis Ears . Bacteriological Examination of Stance Water : ; . The Microscopical and Bacteriological Examination of Milk . The Bacteriological Examination of Shellfish . . The Bacteriological and Toxicological Examination of Meats cad Ment ag Products . : Vii . 102 Pak: SATTA . 118 . 120 144 Vili CONTENTS 18. IQ. 20. ai. 22. 23. 24. PAGE The Bacteriological Examination of Eggs and Egg Products Bt: ae The Bacteriological Examination of Pharmaceutical Preparations . 5 uty The Microscopical and Bacteriological Examination of Syrups . 202 The Microscopical and Bacteriological Examination of Fermented Woeds and Beverages Se ert 2Le Standardization of Tite tate” aye. : 230 Determining the Purity and Quality of se Baccara i Related Prodiet 265 Special Biological and Toxicological Tests . 268 I 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: I 2 MICRO-ANALYTICAL METHODS 1. Vegetable drugs, crude and powdered, pharmacopeeial and other simple and compound medicinal powders. F 2. Spices and condiments, whole, ground and powdered. Pecpared spices and _ condiments. 3. Coffee, tea, cocoa, chocolate, confections, candies. 4. Tobacco and preparations made from tobacco, as snuff, smoking tobacco, cigars, etc. Chemicals, minerals, solutions of chemicals, etc. Tablets, pills, powders. Meats of all kinds, raw, cooked, canned, sausage meats, etc. Dairying products, as milk, cream, cheese, butter, ice-cream, ice cream fillers, Pel ae n et etc. 9. Insect powders, dusting powders, cosmetics. to. Cattle and poultry powders. tr. 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, canned and preserved. 14. Flours and meals. 15. 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. 21. 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. 5. 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 1s 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 15 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 = rin a RHEL Ta 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 judgmerit 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 ate 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 to 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 rt per cent. or more of conium fruits a distinct mouse odor is developed in time (10 min. to 4% 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 finai washing and decanting must be done carefully so as not to lose thé 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 H2SQO, 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 De 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 1o 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 to 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 10 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, whicb 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/50 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.— M oisten wheat flour with water, making it into a dough. Knead constantly and carefully under a slow 1 The 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 0.003u, however, solid particles (as of metallic colloids) of not more than 0.0034 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 ©.30u, 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.15y. T4 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. to. 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 10 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 Avrachnodiscus ehren- bergii 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 1s 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 IT.) 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 ug 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. j. 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- pie aah . <8 SPECIAL TESTS L7 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:! Elements in | oe combination Reaction Oe pu See Romer a Curcumea thread... ..... 2... Ont I in 33,000 Es 5 ai Sulpaidethread..5 5) .2.04 bo 10.0 1 in 2,500 2 eaareeae pmtpmane thread. |. 2.054.) an | ie) I in 40,000 Sin Sle ee Violet color with sulphide thread 3.0 I in 20,000 Au’”’........ | Sulphide thread—brown, purple.. | ZO I in 22,000 Re, | Sulphide thread................ 8.0 rt in 6,000 (COA oS ae Sulphide thread + eee | 8.0 I in 4,000 i\at SO Ae Sulphide thread + Ag.. ea Be) I in 22,000 HEP oc, | NH; vapor.. ye “aaa Cm) 8.0 I in 25,000 Pipe er SS. | Sulphide firead © Lae tee keer ee 1 in 20,000 aes Sulphide 4-- PbCrOu....0. 0... .5. 8.0 I in 13,000 [Ba ee a Sulphide ++ chromate + Bi....... | 8.0 I in 9,000 ee a QNER SH vapor. . o.oo 0P 65 sss 6.0 I in 9,000 Meets at} (NE gSH) — blue? 322.002... | 8.0 1 in 3,500 Geese. 7 NESE) Or Base pera Hae (ELC) 56 8a ai RP BS I in 100,000 ner NG SE ETS. oe ole 0.3 I 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.cor mg., represented by wg. Expressing the com- parative sensitiveness (CS) in a formula we have cs = 8 limit x molecular weights ~ amount limit “* combination valency or to give the example for boron, we have 0.00001 CS = =e == 000. 0.00000006 x® 33: 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 Sample Tecelved .| Rewrite sce) seek ee eee Ce eee Few. Cheese, California; 80,000,000 GW ro ccioaesa conte Cee eee Entirely __ per- meated. Cider cera 50,000 to 50,0000. fa :jie Stove Ce lhe os oe ee 500,000 30,000,000 Cider vinegar.... T,000,000 P@W.. cs .cca es wi bee ae ook eid eke eee ee Currant jelly3 oz. | PeW..cc.%.< ake ofl eossetsten slave evap ieee retire ele eke cle eae Fruits, canned. <-|\Few:........«. ROW iae-. 56 cho 0 %.nl|ere oe where e aeie il renee ee Herring; pickled.:|8,;o00;000;000) }|/.../ci cas sacs ce oll selves meee ove | eke eee JAMS reese REWeae eee Re Wereecinncies Rew; scene Few Jellesg75.2 5 Henier Hewesr sec ecar FEW... 3.056 os |es scenes + eee eee Meat, sausage.... T,O00,000 TOF). 2 20%s aysisc ee -o/5.2/) «1 osaycceieens cists Ole nee nea 150,000,000 Milk, ordinary... 25,000 tO) 3 ese t's cise We | Sie ees ee 2,000,000 Milk, certified... TiQ00' CO: | v.35 bie’ dae ose wisi eevee ore See Sen ee 15,000 MulksSoum ane 2,000,000;000) £0 sieashs co cis wee seal letereseuoys, Sietedieo i] Cece eee 7,000,000,000 Plum preserve....| Few........... BGWis.4 seston Che HE Wrssais Bett. | Few Plomrelish. 3:4 2 100,000,000 BE Wisi sete apace 2,000,000 | Some Water, drinking 800 TOS) Sask tel nay eee ioe | + 4 ote ye ae (San F.). | 32,000,000 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 1 The counts recorded in Tables I, II, and III were made by the direct method using the hemacyt ometer. 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. gy DIRECT EXAMINATION TaBLE II 59 Number of Organisms per Cc. Name of Substance | Hyphe Bacteria Yeasts Spores ES El od eee een 3,750,000 Hew pati.t Some. Apple jam.......| 500,000 AGACOD aes tute oe eerste ees easels Apple jam....... 400,000 9,250,000 SOMe ey: Some. Apricot jam...... BELOOOM) Nine Suchet nsire INonesAh. -e None. Blackberry with | 40,000 1,728,000 Numerous. .| Very abundant. apple. CORT ea eae LOOO; OOO” W ler a eerse reere cee conteh heer oa eer ae Abundant. CHI eee AAO, OCO;COON S| aee rican ceca: 5,000,000 | Very abundant. Pete De aaa ayew)|) 500,000,000. |). 47 0).05 .awestael 27,500,000 | Very abundant. MEISUDe. nee. || 800,000,000. |. So aee eae ae 20,000,000 | Entirely _ per- meated. @atsup..........| 200,000,000 12,000,000 | 1,500,000 | Very abundant. RBALSUD = 0c cr. 5 sc ss FyOOO, 000m | Vili cate sieis eee He Weisciiecie | erace: FEAESUP 2. oes: = 80,000,000) 9) ||... «ttt els 5,000,000 | Very abundant. BU AUSID or 2 oo xceeyers AOO}OOO;000. «|e wana sane 7,500,000 | Very abundant. Cherry jam with | 5,000,000 1,000,000 200,000 | Very abundant. apple. | PRTGEAIIE SDI ope ai |\atee te Sly ercinis: ces ove BOjJOGO, O00. eI: ctoraser mote «' oallrqa nsijubeog oS cr renee RATE ANA Ft oia | cieicledieys's se oe Sse: ABOGOW, Visor ae a <\c.. | ce%.c onal ean ate OU Cho eee | 1,000,000 TOO COG Pisum e's ais| ese 37+ tates eae sree Loganberry jam...) Few...........| (POenslsio) isaaoncosuoee Some. Loganberry jam... 2,500,000 SF EG, 000% Nashik «6 «isi Quite abundant eee IAN |: fies cos. RUMEN okey sissy Aid aa eile oe Salata a ee lade , Pui jam... 5... ~ 500,000 500,000 10,000 | Some. peach jam... . «| 1,250,000 Wonelle, Alam nee os SellsormedDnn on Oc oc Bea WHEIIY, JAM. 2). 2... esses 4,500,000 INWeeoanb or Some. Serawberry jami..|.............: 500,000 750,000 | Abundant. MmOMATOES 2. ../..- | 12,000,000 | doveeabeacles of = 400,000" | Very abundant. Momatoes.....:. |2,000,000,000 PAPO GRIGG aan 4,000,000 | Very abundant. MIN ALOMpPASte:-.-|2,000;000,000 i! 422... Naenoee 1,000,000 | Very abundant. wromato paste.. ..|2,000,000,000.- | ....sis.ssee ees 1,200,000 | Very abundant. Moemato paste::.../1;400,000,000 |... 6.0. eo sk 5,000,000 | Very abundant. MamatoO paste. ....14,000,000,000. |. ..ck. eee 6,000,000 | Very abundant. sBordalo pastes:.|1,000,000,000" | ./..2) es Seiad ls 1,000,000 | Quite abundant. Tomato paste... .|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, Hyphe Bacteria Yeasts Spores — , Tomato pulp!....) Less than Less than Practically none (Helelefoicto tiem huhts Aciakettatice, hot 500,000 (1-3 per cent. of fields). Tomato pulp; .°|1,900,660;000" «| .0.204 92020. 37,000,000 | Entirely _ per- meated (100 | | | per cent.). Imitation jams...) Few.:....+<....| © 30;000;000 | Kewanee Few. 4 TasBLeE III 7 Maximum No. of Organisms per Cc. | Name of Substance = = a < | Hyphez?2 Bacteria Yeasts | Spores | Apple butter..... 5,000 to | 1,000,000 40|a0 xoct cy sles | ) nO St 1,000,000 10,000,000 | BEEMES So). oa-he | SEW sea ee TM 500,000 500,000 | 15 per cent. Catsupmeensl ee TO;O0O{O00 toll Hew. y-ne Ge ae 500,000 | 18 per cent. 50,000,000 Ciderc: ais 500,000 to 500,000 to | ov e'c.sh a | SO 2,000,000 5,000,000 BP TULUS. olen eee BOWS. ortetee oe) 50,000 to| 500,000 to 10 to 12 percent. 500,000 ~—_—I,000,000 ans: Weaetee & 1,000,000 1,000,000 to 500,000 Io per cent. 10,000,000 | Jeties... Sse ew 1,000,000 Few? sca: I to § per cent. Marmalade’: 2s. 2). one 58 232s. da ae eons ade |b = ee Tomato pastes...) 500,000,000 Few 2,000,000 | 20 to 25 per cent. Vinegars (fruit). . 5,000,000 Pewee. lee eee * Both samples were from large factories and represent the extremes in the factory conditions. The first sample is from a factory where the conditions are what they should be, the second from a factory where the conditions are just 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. Asa rule abun- dant spores indicate the presence of abundant hyphal tissue, and vice versa. * The organism in orange marmalade, under ordinary conditions of manufacture, are negligible in amount. | 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.) III, 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: of Man TREMATODA CoO RAW N- TO SCALE x 1600 Heterophyes heterophyes ee ee. aa esr emooten 60,000 per cc. POLES ses arecets seeteleree 2 afasese sae Sere a * 15,000 Per Cc. Py ple Ob mane. ic. ss see 1s oon eee 460 per cc. ' The water was at the time decidedly greenish in tint with a dis- agreeable odor, due to the numerous alge present. Water show- ing such a high and varied biological count shows surtace seepage and indicates sewage contamination and is not fit for drinking purposes, yet the biologist for the water company declared it good and harmless. The only interpretation that can be put upon a count such as the above is that the water supply is dangerously contaminated. Diatoms and desmids feed upon dead and decay- ing vegetable matter. Oscillarias occur in wet soils rich in humus. Paramecia feed upon decaying organic matter. The molds like- wise are proof of the decay of organic matter, animal and vegetable. In all cases of evidence of surface seepage, sewage contamina- tion may be suspected and all sewage contaminated drinking waters are a menace to the public health. In no case should the examination of concentrated (1000 cc. reduced to ro cc.) and centrifugalized sediment be omitted, as this will perhaps reveal contaminations which might be overlooked in the direct examination. Nor must the presumptive colon bacillus test be omitted when there is the least indication that sewage con- tamination exists. In case of slight but suspicious contaminations BIOLOGICAL WATER ANALYSIS EL7 the colon bacillus test should be supplemented by the plate count and the examination of the centrifugalized sediment. Although the bacteriological examination of water supplies is the work of the sanitarians, the food bacteriologists are frequently called upon to pass judgment on the potability of water supplies. There is no definite numerical standard for drinking waters. In the United States the presence of the colon bacillus is almost wholly the basis for condemnation, it being assumed that if bacteria are present in great numbers the colon bacillus is also generally present. This is, however, very frequently not the case. Distilled water may contain numerous bacteria without any colon organisms. Stag- nant waters may contain bacteria in great numbers without colon bacilli. It is not practicable to adopt an arbitrary numerical limit as has been suggested by various investigators. Miquel (1891) suggested the following standards: TOU ACECH Ay METICGMOLVESS: cc g eae cares sy. Excessively pure To TOOuMACLenla elt CG: a: tac. nana sree Very pure ToO-Looo bacteria per COsn se ae ee Pure TOOO—10;,O0O"DAGterla PCr CG m. -2.0 sskak aioe ee Mediocre 10,000-100,000 bacteria per CG.......2---..-.- Impure 100,000 and more bacteria per cc............. Very impure German sanitarians generally recognize a limit of 50 to 300 for drinking water. Dr. Sternberg of the Public Health Service (1892) suggested that less than 100 bacteria per cc. indicated a deep source of the water supply and uncontaminated by surface drainage and that a water supply with 500 bacteria per cc. was open to sus- picion and that 1ooo and over is presumptive indication of sewage contamination or of surface drainage. It is quite evident that there is very little excuse for the use of city and other communal drinking water supplies with a count higher than 5—10,000 per cc., and it is suggested that this be made the numerical limit for drink- ing water in the absence of or irrespective of the presence of the colon bacillus. The general routine for making the tests for the presence of the 118 BACTERIOLOGICAL METHODS colon bacillus has already been explained. It is suggested that — I cc., 0.10 cc. and o.or cc. quantities of the water be run into fermentation tubes with lactose-bile medium, making five sets of these tube cultures, and incubate at 37° C. for 48 hr., noting pos- sible gas formation. Gas formation indicates sewage contamina- tion. If the gas is formed quickly, in 6 to 12 hr., the contamination is probably recent, if more slowly, 24 to 36 hr., the contamination is probably older. Gas in the 0.01 cc. quantities or less, indicates very high sewage contamination, gas in the 0.01 to 0.10 cc. quan- tities indicates serious contamination, and condemnation of the water supply for drinking purposes may be based on the presence of gas formation in two out of three tubes containing 0.10 cc. quantities, or three out of five of the x cc. quantities, also tak- ing into consideration the rate of gas formation and the numerical plate count as well as the findings based on the direct microscop- ical examination. In brief, condemnation of water supplies in- tended for drinking purposes must be based upon the judgment of a competent sanitarian, one who comprehends the significance of the findings in relation to the source of the water supply and the sources of the contaminations. It is not practicable to lay down hard and fast rules. Each case must be considered by itself. In one in- stance the gas formation may develop in 0.3 cc. quantities (three out of five tubes containing 0.10 cc. quantities) or even in 0.10 cc. quantities and yet the water may be considered potable, as might be the case in deep well water into which street and road dust is carried, or which might contain surface drainage from field or garden. Again the water may be quite unfit for drinking pur- poses with colon bacilli in ro cc. or in 100 cc. quantities, as might be the case in wells or springs highly contaminated with old or much weathered sewage contamination. 14. Bacteriological Examination of Mineral Waters The bacteriological analysis of bottled waters is very important because it is an efficient means of ascertaining the conditions at the MINERAL WATERS 11g bottling establishments. A general opinion prevails that mineral waters are free from germs, due to the germ-destroying properties of the mineral salts present. This is not the case. Many mineral waters from contaminated sources or from unsanitary bottling establishments contain bacteria in large numbers, 300,000,000 per cc. and more. Even a medicinal water composed of concentrated ocean water (Magpotine) gave a count of 10,000 bacteria per cc. The Bureau of Chemistry has found mineral waters contaminated with sewage. Often the contamination is traceable to the inade- quate cleansing and sterilizing of used bottles and to the dirty hands of those employed in the factory. The bacteriological examination of mineral waters consists in making the presumptive colon bacillus test and in making bacte- rial counts by the plating method. It is, however, also desirable to make direct microscopical examinations, including quantitative cytometric counts of concentrates (1 liter quantities reduced to ro cc.) and of centrifugalized samples, as already explained. This will give information regarding factory conditions which could not be ascertained by the usual plating methods. In the case of bottled mineral waters, the securing, handling and shipping of samples is a very simple matter as no extra precautions and care are necessary. In the case of water from mineral springs or artificial waters in bulk, the securing of samples for examination must be done carefully to guard against outside contamination. Containers for samples must be clean and sterile and as soon as the sample is taken the container must be closed with a sterilized cork ‘or other suitable stopper, sealed and taken to the laboratory by the shortest route for immediate examination. If the samples are to be transported long distances or if for any other reason, the examinations must be postponed for from 6 hr. to several days, the sample must be kept on ice during the entire period. Mineral waters are or should be quite free from bacteria and other contaminating organisms. As yet no standards have been adopted as to the maximum number of bacteria and other organ- I20 BACTERIOLOGICAL METHODS isms permissible. The only quality test made by the Bureau of Chemistry is for the colon bacillus. 15. The Microscopical and Bacteriological Examination of Milk It is not practicable to enter into a discussion of the dairying industry or the multitudinous factors which cause modification of the quality of cow’s milk. These are matters which concern the food bacteriologist but little. Bovine diseases, inclusive of tuber- culosis, must be left to the veterinarian and the making of dairy products concern the manufacturer primarily. By this it is, how- ever, not intended to imply that the food bacteriologist need not have intimate knowledge of cattle diseases and of dairying meth- ods. Not only should he be well informed regarding these things but he should be qualified to examine cattle for diseases, tubercu- losis in particular, and should be prepared to examine and report upon the sanitary conditions, equipment and the moderness of dai- rying establishments. However, the chief efforts of the food bac- teriologist are devoted to the examination of the milk and dairying products as they appear upon the market. For the present purpose it will suffice to give a mere outline of the methods of examining and testing milk microscopically and bacteriologically. The report of the analysis should comprise the following. Securing the sample. Sealing the sample. Keeping sample on ice until ready for examination. Examining the sample. Direct examination. Determining the fat content by the microscopical method. Quantitative determination of Bacteria. Epithelial cells. Blood corpuscles. Pus cells and leucocytes. Plate cultures. Presumptive colon bacillus test. Numerical count. MILK I2I Milk may be described as a uniform suspension of fat globules in an aqueous solution of milk-sugar and casein. The fat globules represent the so-called butter fat of the milk. They are fairly uni- form in size, very uniformly distributed and under ordinary con- ditions do not tend to coalesce or clump. Pasteurization and boiling the milk does cause some of the globules to unite or rather to form aggregates but even in such cases it is possible to recognize the individual globules. On mounting a droplet of diluted milk (1-150 to 1-200) on the hemacytometer it will be found that the fat globules soon rise to Fic. 37.—Milk fat globules. Larger field as they appear under the low power of the compound microscope (X 80), globules in the corner circle as they appear under the high power (X 500).—(Huwunter, after S. M. Babcock.) the top while the heavier particles, such as bacteria and body cells, sink to the bottom of the cell, thus separating these elements auto- matically, and making the counting of globules and bacteria pos- sible in the same mount by simply focusing sharply upon the fat globules or upon the bacteria as may be desired. Some difficulty in making the counts is caused by the fact that the oil globules are out of focus when the rulings are in focus, making a constant shift- ing of focus from fat globule to lines and vice versa from lines to fat globule, necessary. Not only is this annoying but it makes accu- rate counting difficult. This difficulty can be overcome by com- 22 BACTERIOLOGICAL METHODS bining the use of an eye-piece micrometer scale with’ that of the hemacytometer, and it is suggested that such a combination be used, not only for milk examination, but also for making many of the cytometric counts of food products. A practical method for determining the fat content of milk by means of the compound microscope was worked out in the bacterio- logical laboratory of the California College of Pharmacy. The pro- cedure is as follows: Make dilutions of the milk from 1-150 to 1~200, using distilled water or normal salt solution (0.6 per cent.) and count the fat globules by means of the hemacytometer or the special counter above suggested. Numerous counts made have shown that 578,100,000 fat globules in 1 cc. of milk corresponds to rt per cent. of butter fat. This number was obtained by comparing the fat globule count with the fat determination by the standard _ chemical method (combined with the use of the centrifugal machine). The following are a few comparisons as they were ob- tained in the laboratories of the California College of Pharmacy. 1,383,000,000 fat globules per cc. corresponded to 2.30 per cent. of butter fat. 933,000,000 fat globules per cc. corresponded to 1.60 per cent. of butter fat. 566,000,000 fat globules per cc. corresponded to 1.10 per cent. of butter fat. 470,000,000 fat globules per cc. corresponded to 0.80 per cent. of butter fat. Dividing the sum total of the several counts of fat globules made by the sum total of the corresponding percentages of butter fat, gives 578,100,000 the average number of globules in 1 cc. of milk corresponding to 1 per cent. of butter fat. From this it will be seen that in round numbers, 2,000,000,000 fat globules per cc. represent a fair quality of milk, that is, milk having somewhat over 3.50 per cent. of butter fat. According to comparative tests made, the microscopical method is fully as accurate and reliable as the chemical methods. The microscopical method is not recom- mended for routine procedure in dairying establishments but it is certainly a most valuable adjunct to the food laboratory methods. It could at all times be employed’as a substitute for the chemical fat determination if for any reason the latter method BACTERIOLOGICAL STANDARDS FOR MILK 123 is not applicable. Thus, it can be ascertained microscopically whether or not water has been added to the milk or if it is full milk, half milk or skimmed milk. The bacteriological standardization of milk has received much attention within recent years and all civilized countries have adopted certain numerical limits of bacteria permissible in whole- some milk. Unfortunately, however, there is very little uniformity regarding these numerical limits in different countries or in differ- ent parts of the same country. In some cities and communities Fic. 38.—Milk fat globules very highly magnified (X 1000). A group of lactic acid bacteria at the left.—(Hunter.) there are two standards, a summer or low (numerical limit higher) standard and a winter or high (numerical limit lower) standard. The terms summer and winter are, however, misleading in certain areas of the United States and, for regulatory purposes, it would be better to base the standards on a temperature differential, irre- spective of season, combining this with a sliding scale of bacterial count. Under such a plan the Southern States, including the immediate Pacific Coast region, would be under a single standard, namely, the lower or so-called summer standard. The rest of the United States would have both standards. 124 BACTERIOLOGICAL METHODS The following is a tentative standard based upon the tempera- ture differential as above suggested. Number of Bacteria per Cc. Standards , ry Ordinary Certified Inspected Cream (Fresh Milk Milk Milk or Unripened)! Temp. from lowest to 60° F.| 30,000 to 3,000 to 12,000 to 30,000 to Winter standard 50,000 8,000 15,000 5,000,000 Temp. from 60°F. to highest.) 50,000 to 8,000 to I5,000 to | 5,000,000 to © Summer standard 100,000 15,000 30,000 150,000,000 It is not practicable to fix a numerical bacterial limit for creams. Tests made show that the count varies within wide limits, even in cream from milk which has been kept under the most favorable sanitary conditions and surroundings. Fresh creams, that is, the cream removed from the milk as soon as formed, usually within 24 hr. after the milk is drawn, contains comparatively fewer bac- teria than the cream which has been set aside to ripen. The ripening process is far from objectionable, in fact it is encouraged and regulated in the well-conducted dairying establishments in order to develop the desirable butter flavor. Most of these flavoring lactic acid bacteria are removed in the process of butter making, being drawn away and worked out with the buttermilk, only comparatively few remaining in the butter itself. Taking milk samples is not unlike water sampling. Milk should be examined not later than 6 hr. after being drawn. If it cannot be examined within that time it must be kept on ice but in no case should the examination be made later than 12 hr. after the milk was drawn. Body cell counts should not be omitted and proper judgment should be exercised in interpreting the findings. Body cell counts 1 Ripened cream contains numerous lactic acid bacilli, 300,000,000 per cc., and even more. BODY CELLS IN MILK 125 give most valuable information regarding the health condition of the cows and will serve to indicate the danger point as to the usability of the milk. It is not practicable to give exact numerical limits at the present time. Further investigation is necessary to this end. However, the following suggestions will be of great value to the analyst in arriving at a better estimate of the quality of the milk under examination. Epithelial cells few (1000 per cc.), of no significance. ; Epithelial cells many (5,000,000 per cc. and more), indicates some irritation or seri- ous inflammatory condition of udder or in milk ducts. Epithelial cells many with some pus cells, danger. The diseased animal should be found and removed from the herd. Pus cells few, indicates some slight abscess formation which should be treated if possible. Pus cells many (5,000,000 per cc. or more) indicates danger. The diseased animal should be removed from the herd. Blood corpuscles few, no special significance. Probably due to some slight injury resulting in capillary hemorrhage. Blood corpuscles many. Indicates some mechanical injury which requires attention. For practical purposes it is not advisable to attempt to dis- tinguish between leucocytes and pus corpuscles. Numerous leuco- cytes indicate some serious inflammatory condition while numer- ous pus cells indicates abscess formation perhaps following a more severe inflammation. Various methods have been submitted for making the body cell counts. That by Prescott and Breed is perhaps the simplest and also the most practical. It is carried out as follows. Spread o.or cc. of the milk on a glass slide’ over an area of 1 sq. cm., evaporating the milk to dryness using moderate heat. Next dissolve out the butter fat by means of xylol, fix with alcohol, again dry, and stain with methylene blue. Decolorize partially with alcohol and examine under the compound microscope. The body cells in the entire area of the mount are counted and the 1 The ruled slide elsewhere described (D, Fig. 5) will be found very useful for counting body cells in definite quantities of the milk. 126 BACTERIOLOGICAL METHODS entire number found multiplied by 1oo gives the number of body cells per cc. of the milk. Prescott and Breed have examined numerous milk samples and declare that the average number of body cells is 1,500,000 per cc. and that a count as low as 100,000 per cc. is uncommon. Little can be said regarding the microscopical and bacteriolog- ical examination of butter, cheese, cream and other factory products. Fic. 39.—Unglazed porcelain filters. Chamberland system; A, without pressure; B, fitted to main water supply; C, section of a porous porcelain filter. There are no bacterial standards and the laboratory work is very largely limited to the detection of adulterants such as excess of salt, of water and the presence of lard and oleomargarine in but- ter, fillers in cream and in ice cream, etc. The following simple tests will be found useful in the labora- tory: 1. Spoon Test for Oleomargarine and Renovated Butter.—Melt a small piece of the suspected butter in a tablespoon or small dish, using a’small flame. Stir the melting substance with a small piece of wood such as a tooth-pick or match. Ata MILK BACTERIA 127 brisk boil, oleomargarine and renovated butter will sputter very briskly and noisily without foaming. Genuine butter boils less noisily and with abundant foam formation. 2. Fat Cohesion Test.—Fill a medium beaker about half full of sweet milk (pref- erably skimmed) and heat to within near the boiling point. Add about 5 grams of the sample and stir until completely melted. Remove from the fire and place beaker in ice water. When the fat begins to congeal, stir with a small piece of stick. Fat or oleomargarine will collect in one mass or lump at the end of the stick, whereas pure butter granulates and will not adhere to the stick. This test is rot applicable to renovated butter which behaves like unrenovated or fresh butter. As is generally known, milk is an excellent culture medium for a great variety of bacteria. For a time after the milk is drawn, bacterial development is checked by the bacterolytic properties which all fresh milk is said to possess. These lysins, however, gradually grow less and less until there is no longer any evidence of their existence. Milk bacteria may be grouped into the acid formers, digesting bacteria and those which appear to have but little effect on the appearance of the milk. The acid-forming group is a large one and includes the true lactic acid bacteria which are carried into the milk from stable dust and other dirt in and about the stables and elsewhere. The initial bacterial changes in the milk are, however, not produced by the acid formers, but rather by those bacteria which decompose proteids, to which belong the B. subtilis and its aerobic allies. Streptococcus acidi lactict ferments both proteids and lactose as does also B. coli communis and some of its allies. In a short time, however, the true lactic acid bacteria multiply in such large numbers as to crowd out or almost completely check the development of the other species. They transform the lactose into lactic acid. On longer exposure, Ozdium lactis enters from the atmosphere which fungus begins to decompose the lactic acid and some of the remaining proteids, having the effect of lowering the acidity which again encourages the renewed multiplication of the lactic acid group. This alternating preponderance of lactic acid bacteria and higher fungi continues until the proteids and the milk sugar are almost completely used up. Butyric acid 128 BACTERIOLOGICAL METHODS bacteria may enter the milk causing the very characteristic fer- mentation changes resulting in the formation and liberation of bu- tyric and propionic acids from the splitting oflactose. Butyric acid milk has a very disagreeable odor. Various bacteria cause dis- eases of milk as blue milk and ropy milk. In some American cities the routine examination of milk for B. coli is regularly adopted. The results in Baltimore have shown the presence of this bacillus in 25 per cent. of o.oo1 cc. quantities of the milk in the winter time and 75 per cent. during the summer. It would appear that three positive tests out of a total of five from 0.oo1 cc. quantities of milk, would indicate the danger point as to quality. For making plate counts of milk bacteria, lactose- litmus-agar should be used in order to differentiate between acid formers and non-acid formers. In most communities the milk streptococci are considered objectionable, as they belong to the group of pus-forming organ- isms. It is frequently found that a high streptococcus count goes with a high leucocyte count and the two are corroborative of the existence of some severe inflammatory condition of the udder or milk ducts. There is fairly conclusive evidence that the hemo- lytic milk streptococci are frequently causative of more or less severe and even fatal intestinal diseases among children, especially during the hot summer weather. It is also fairly well proven that some of the throat and mouth infections of children are traceable to the staphylococci and streptococci of milk. The problem of tuberculous milk is of lesser importance to the food bacteriologist because the health authorities of the land have this matter under jurisdiction. It is criminally unlawful to market milk from tuber- cular cows. Ravenel states that approximately 20 per cent. of the clinical cases of tuberculosis are of the bovine type and milk from tuberculous cows continues to be a very serious menace to the public health. It would be of the greatest value if some simple and practical micro-chemical laboratory test for tuberculous milk could be worked out. We would suggest this as one of the very HYDROGEN DIOXIDE MILK TEST I29 important problems to be undertaken. It is evident that the con- trol exercised by the health authorities, while it has accomplished much, is not sufficiently stringent or far-reaching to stamp out tuberculosis in cows. A milk test much used in Holland and other European countries is to ascertain the amount of gas formation in a unit of time, in a fermentation tube containing a mixture of definite quantities of milk and hydrogen dioxide. The amount of gas liberated is di- rectly proportional to the amount of organic matter (bacteria, body cells and other organic impurities) present. Tests made in the laboratories of the California College of Pharmacy and in the laboratories of the San Francisco Board of Health would indicate that the method gives uniform results and that such a method would prove a most valuable addition to the routine milk examina- tion, serving as a check and confirmation of the bacterial and body cell counts. In order that the test may yield uniform results in all laboratories, a uniform method of procedure must be adopted. The following tentative method is suggested. A standard ro percent. volume (of available oxygen) solution of hydrogen dioxideshould beused. The peroxide should be standard- ized to the specified quality. For determining the valuation of the peroxide we would recommend the Planés colorimetric test, made asfollows. Dilute the dioxide to be tested with nine parts distilled. water. To 5cc. of this solution (1-10) add 3 cc. of a 10 per cent solution of potassium iodide and 1 cc. of 8 per cent. sulphuric acid, in a standard test-tube. The color produced is matched against a n/to iodine solution in a second standard test-tube. 1.8 cc. of the standard solution is equivalent to 1 cc. of oxygen. Into graduated fermentation tubes with slender arms, having a capacity of 25 cc., run 1o cc. of milk and ro cc. of the standard hydrogen dioxide, mix well in the bulb and at once run into the arm, excluding all air from the upper end of tube. Set aside in the incubator for 1 hr. at a temperature of 20° C. and record the amount of gas formed at the end of this period. 130 BACTERIOLOGICAL METHODS A Fic. 40.—Streptococcus (Staphylococcus) pyogenes and S. aureus. There are three principal species of Streptococci (S. pyogenes albus, S. pyogenes aureus and S. pyogenes citreus), similar in form and appearance, concerned in pus formation, as in wound infection. These organisms are very widely distributed in soil and air. Note the chain form arrangement of the cocciin A. B is asmear preparation.— (Stitt (A) and Pittfield (B).) TEST FOR WATERED MILK La The following quick and simple test is recommended to dis- tinguish between raw and boiled milk! REAGENT Methylene blue (alcoholic)... \vsa0Dee 02k Sed ose been 5CE. Formaldehyde: (ao per cent:). 01) oe sais as eee s/s 3 Sen yee Wittens (distilled) e.8 oc seat re ent te irs Mine oink fatesct ako 1QO CC. Add 1 cc. of this reagent to 20 cc. of the milk and heat for ro min. at 40°-45° C. Raw milk is decolorized while boiled milk retains the blue coloration. This test should in all cases be checked by the microscopical examination. Boiling the milk causes the fat globules to unite and adhere more or less, a characteristic which is also noticeable in pasteurized milk. The flavor and odor of boiled milk is in itself quite characteristic. Knapp recommends the following test for determining the addition of water to milk. 10 cc. of the suspected milk are run into a test-tube and curdled by adding one drop of rennet and placing the tube in the water bath for about 2 min. at a temperature of 35-40 C. The whole is then poured upon a very fine wire sieve and the liquid allowed to drain off into a tube graduate, pressing the curd with a glass rod so as to remove the liquid as completely as possible. The amount of liquid remaining in the curd is fairly constant in the tests and therefore : sore ¢ C Fic. 41.—Gelatin practically negligible for comparative purposes. culture of Staphylo- If the amount of liquid drained off exceeds 8 Ra Pe Ne cc., water has been added. This test should be checked by the chemical butter fat tests and also by the microscop- ical method for determining the fat content, as already explained. Among the micro-organisms which cause the coagulation of milk and which are often found in sour milk, particularly in old sour Io 132 BACTERIOLOGICAL METHODS milk, is the Streptococcus lacticus of Kruse. The Bacillus (lactis) aérogenes which is very closely similar to Bacillus coli, also sours milk and is likely to be present at the beginning of the fermenta- tion. The common pus streptococci and staphylococci are often found in milk in large numbers, traceable to dirt and filth and to diseased udders and less commonly to the hands of the milkers. The colon bacillus when present is traceable to stable dust and manure and to the unclean hands of the milkers. The following are some of the organisms which cause diseases of milk: 1. Bacillus cyanogenes—Blue milk. 2. Bacillus prodigiosus—Red milk. 3. Bacillus erythrogenes—Red milk. 4. Bacillus synxanthus—Yellow milk. 5. Lorula amara—Bitter milk. 6. Streptococcus hollandicus—Ropy milk. Naturally the bacilli normally present in the milk which is stored for cream formation are also present in the cream after the skimming and cause the so-called ripening of the cream. In order that the ripening process may proceed in a desirable manner, the objectionable butyric acid formers must be excluded. The butyric acid formers are more generally associated with filth, hence, a careful compliance with sanitary rules and regulations in the dairying establishment will generally encourage the invasion and development of the desirable lactic acid organisms to the exclusion of the undesirable microbes, though this is by no means always the case. Occasionally, even with the most scrupulous adherence to sanitation, cream will not ripen properly and these occasional failures have prompted the more progressive dairymen to in- oculate the milk and creamwith pure cultures of the desirable cream ripening bacilli. Others use natural cream starters, that is, small quantities of old cream which has ripened with a desirable flavor. Cream should not show colon bacilli in less than 0.10 cc. quanti- _ties and fresh unripened cream should not contain more than 5,000,- TUBERCLE BACILLI IN MILK 133 ooo bacteria per cc. Ripened cream should not contain more than 150,000,000 bacteria per cc. and most of which bacteria should be of the lactic acid group. Pathogenic bacteria which may be pres- ent in milk may also be present in the cream. Tubercle bacilli, diphtheria bacilli and typhoid bacilli are the most likely to occur. In the case of doubtful cream, the colon bacillus test should not be omitted and in the case of suspected contamination with patho- genic organisms, the cream, as well as the milk from the same source, should be examined, resorting to the usual animal inoculation tests. - The tests for the presence of tubercle bac- illi in milk, cream, meats, etc., comprises the microscopic examination of stained (Ziehl- Neelsen method of staining) sediments or con- centrates as may be required, and animal in- oculations. For the animal inoculation test, fen prrieare 4 guinea-pigs are used. Centrifugalize (in a Stained with carbol- : : fuchsin and methy- powerful machine) about 250 cc. of the milk Jeneblue—(Pitifield.) in order to throw down the tubercle bacilli (with the other inclusions), and from this make the desired cover-slip preparations and inoculate (in the region of the left knee-joint of hind leg) the remainder of the sediment into three healthy guinea-pigs. Place the inoculated guinea-pigs in individual cages and keep them under observation for from 2 to 4 weeks. The reasons why several pigs should be inoculated are as follows. Some of the pigs may be killed by bacteria other than the tu- bercle bacilli and it is always desirable to duplicate the tests. At the end of the second week, one of the guinea-pigs should be dissected and the glands of the sublumbar region as well as the glands of the superficial tissues and of the popliteal region exam- ined. If tubercular infection has taken place, these glands will be found much enlarged containing foci of tubercle bacilli. The enlarged glands are dissected and suitable cover-glass prepara- tions made therefrom. If the evidence of tubercular infection Fic. 43.—Tubercle ba- cillus slant culture on glyc- erin-agar, several months old.— (Stitt, after Curtis.) 134 BACTERIOLOGICAL METHODS is not conclusive, the other’ two inocu- lated guinea-pigs should be kept 2 weeks longer then dissected and examined like the first. Occasionally there is abscess formation at the point of inoculation but this need not necessarily interfere with the tubercular development in the glands and in the deeper tissues. It is frequently possible to isolate the bacillus of tuberculosis (from sputum, glandular tissues, meat pulp, centrifugal- ized sediments of milk, cream, etc.) by special manipulation and the use of special culture media. The following method is suggested. Spread two or three drops of the material (concentrate, sediment, crushed, suspected tuberculous meat ex- tract, etc.) evenly over the surface of two or three glass slips and place the smear preparations in the drying oven at 100° C. for 15 min., however, not before the ma- terial on the slips is well dried at the room temperature. ‘Tubercle bacilli are quite resistant to dry heat and will withstand the temperature of 100° C. for from 30 min. to thr. The exposurt to that tem- perature for 15 min. will kill most of the bacteria associated with the tubercle germs and will in fact kill some of these. At the end of 15 min. take the glass slips from the drying oven and by means of a small sterile spatula or scalpel, scrape the dried suspected material over the surface F CULTURING THE TUBERCLE BACILLI OF MILK £35 of the special medium in Petri dishes. The medium used (Hesse’s agar) is made as follows: Imitrose taonmunscaAScimAtG) 4 aol. o. ak ans cgea s 5 grams, SITALH EEN OLIOR <2. sn. Selah ptait os nit cola ce ea tae 30 grams. Glycerm=.2......" Dede a Re eEae Che RH Minnie hak ee Re 30 grams. EN EEUD ia OA ala a Past NS ohcoey aa RR Meee Rec Seechl Sc sts e Oit Sbs rine 10 grams, Na2CO,(crystalline) solution (28.6 per cent.)...... 5 ec, WRSEIMC MR VIRLEE atte ote. ates ove Ss.g cre Seperated 1000 cc, Mix ingredients. Heat until agar is dissolved. Filter through cotton. Pour into Petri dishes. Sterilize fractionally. After inoculating two or three Petri dishes in the manner indicated, incubate at 37.5° C. in a moisture-saturated atmosphere for several days. If tubercle bacilli are present young colonies will appear which may be identified with a low power by the resem- blance to broken wavy lines. Instead of making the glass-slip smears as above suggested, good results may be obtained through the use of the cotton throat swabs such as are used by physicians for taking throat cultures in diphtheria cases. Dip or roll the cotton ends of three swabs in the suspected tuberculous material, suspend in air until perfectly dry and then place in drying over (100° C.) for 15 min., then rub the cotton over the surface of the special culture medium in Petri dishes. Make some six or seven parallel streaks over the surface of the medium. Incubate and examineas before. Should the glass- slip or cotton-swab preparations be placed in the drying oven before air drying them, all or nearly all of the tubercle bacilli would be killed in the drying oven. Several investigators have recommended a direct method of examination for ascertaining the presence of tubercle bacilli in milk, and:in other materials, through the use of agents which will completely dissolve all bacterial bodies excepting the acid-fast group of organisms to which the tubercle bacillus belongs. For this purpose antiformin (really a mixture of chlorinated sodium hypochlorite and Labarraque’s solution) has been highly recom- 136 BACTERIOLOGICAL METHODS mended. This proprietary article is a strongly alkaline-solution of sodium hypochlorite. In each cc. it contains approximately 5.68 grams of sodium hypochlorite, sodium hydroxide 7.8 grams and sodium carbonate 0.32 grams. ‘The available chlorine amounts to about 5.68 grams. It dissolves all organic matter, such as that con- ms Fic. 44.—Bacillus tuberculosis in the sputum of a consumptive; stained by Ziehl method (X 2100).—(After Kossel.) tained in sputum and feces, excepting the tubercle bacilli. It is in itself an active antiseptic having a phenol coefficient of 3. For bacteriological work, a 50 per cent. solution of the anti- formin will be found satisfactory. Mix equal parts of the anti- formin solution (50 per cent.) and milk or sputum or other mate- ANTIFORMIN TUBERCLE CULTURES E37 rial supposed to contain the tubercle bacilli, in a suitable glass container and bring to a boil over the Bunsen burner. When the material is cool, add 1.5 cc. of a mixture of chloroform and alcohol (chloroform one part and alcohol nine parts) to each ro cc. of the material and shake vigorously. The tubercle bacilli absorb some of the chloroform and become heavier than the rest of the organic matter. Next centrifugalize at a high speed for 15 min. which separates the material into three layers; the antiformin at the top, the sediment in the middle, and the chloroform with the tubercle bacilli at the bottom. Pipette off the layer of chloroform and ex- amine for tubercle bacilli by resorting to the usual staining methods. The smear preparations can be made to stick to the cover or slide by mixing with serum or egg albumen solution. This method may also be tried in the examination of creams, cheese, buttermilk and butter. The strength of the antiformin solution should be graded according to the amount or percentage of organic matter to be dis- solved, taking the strength required for sputum work as the high- est. For milk work the 15 per cent. solution will be satisfactory. For cheese a 50 per cent. solution should be used, likewise for feces. Stitt recommends the following antiformin method for cultur- ing the tubercle bacilli. Mix 20 cc. of sputum, 65 cc.’of sterile water and 15 cc. of antiformin. Stir with a glass rod. After a period ranging from 30 min. to 2 hr., the mixture should be homogeneous. Centrifugalize for 15 min. or longer, decant, and wash. the sediment twice in sterile normal salt solution and smear out the well-washed sediment over serum or glycerin egg albumen ornutrose slants. Itmust beremembered that the tubercle bacillus - will not grow in sunlight and that the colonies form on the surface of the culture media only. Stitt also states that it is not wise to use the antiformin in solutions stronger than is necessary to dissolve the organic matter and bacteria other than the tubercle bacillus. For example for sputum, it is suggested that 20 or 25 per cent. of antiformin be 138 BACTERIOLOGICAL METHODS used. If stronger solutions are used, many of the tubercle bacilli are also disintegrated or considerably changed in form and in the behavior with the acid-fast stains. In addition to the routine examination of ice creams for the presence of fillers and ingredients which do not properly belong to ice creams, the food bacteriologist will have occasion to make bacteriological and toxicological tests. According to Vaughan, the toxic changes in ice cream are due to the presence of a poison designated tyrotoxicon, presumably identical with the toxin occa- sionally found in milk and cheese. Ice-cream poisoning depends upon the development of the toxin-forming bacteria in the milk and cream before it isfrozen. It is notat all likely that ice cream made from clean wholesome cream and milk will contain toxins, provided it is kept well frozen and is not stored too long. There is good evidence that slightly infected ice cream which is kept for several days and longer, may show sufficient toxic bacterial de- velopment to produce symptoms of poisoning. The virulency of the toxins produced by the bacteria appears to increase with the lowering of the temperature. The danger from ice cream is directly proportional to the un- sanitary conditions of the milk and cream used and ice-cream poisoning is far more likely to manifest itself during the hot summer weather. All suspicious ice creams should be examined bacteri- ally, making numerical plate cultures and also the presumptive co- lon bacillus test and tests for streptococci and staphylococci. The toxicological test as recommended for meats is, however, far more important and should not be omitted. Ice cream should not con- tain more than 1,000,000 bacteria per cc. and should not develop colon bacilli in less than 0.10 cc. quantities by the standard pre- sumptive colon bacillus test. Of the more common ice-cream fillers we may mention starch and tragacanth. Vegetable mucilages other than tragacanth may be suspected. Gelatin is also used. Eggs are frequently added. A filler to which a small amount of rennet had been added has BUTTER AND CHEESE 139 been extensively advertised as an ice-cream producer which did - not require the use of cream or of ice. Occasionally it may become necessary to examine sour milk and buttermilk for the presence of toxins and objectionable bac- teria and other undesirable organisms. Because of the careless and more or less promiscuous handling of buttermilk before it reaches the consumer, it is especially liable to the invasion of foreign organisms. The routine examination of buttermilk is largely limited to a direct microscopical inspection. Mold spores and yeast cells should be sparingly present and the predominating bacilli should be small’ (lactic acid formers) and of irregular and rather indefinite outline. Mold and cocci should be very sparingly present. To examine butter for the presence of bacteria (direct micro- scopical method) and other contaminations, place 1 gram of the butter in to cc. of ether and shake until all of the butter fat is dissolved. Pour the solution into the special centrifugal tube and centrifugalize for 5 min. Wash the contents of the 1 cc. end tube into 1o cc. of ether and again shake and centrifugalize. Pour off the ether and add 2 cc. of a 2 per cent. sodic hydrate solution and shake until the casein is dissolved. The sodic hydrate solution emulsifies the small amount of fat present. Examine the emulsion for bacteria, counting the bacteria and body cells by means of the hemacytometer. Butter and cheese made from the milk of animals suffering from foot-and-mouth disease have transmitted this disease to humans. The bovine type of tuberculosis has resulted from the consumption of milk, cream and butter. Tubercle bacilli have been found in the more quickly ripened cheeses. Tubercle bacilli do, however, not survive long in soured cream or milk, perhaps not over 2 or 3 days. The following are some of the more important organisms con- cerned in the ripening of cheese. 1 The Bacillus bulgarius is comparatively large (1 X 6y). I40 BACTERIOLOGICAL METHODS 1. Lactic acid bacteria—These are the chief agents concerned in the ripening of Cheddar, American and Edam cheese. Pure cultures of the Bacillus acidilactici are often used as a starter. In the manufacture of the Edam cheese, slimy whey is used as a starter (Streptococcus hollandicus). 2. Penicillium glaucum the common green mold is the principle organism con- cerned in the ripening of Roquefort, Gorganzola and Brie cheeses. In some coun- tries the green mold is scraped from molded bread and added to the curd. 3. A great variety of other bacteria, yeasts and mold are concerned in the devel- opment of the more specific flavors and aromas. Further investigation is necessary to ascertain the special function performed by each and the mutualistic relationship that may exist between them. 4. Gas generating bacteria are concerned in the formation of holes in the interior of the ripening cheese. These gas formers also modify the aroma or flavor of the cheese and in some instances constitute the chief ripening agents. Spoiling of cheese is not uncommon, due to the invasion of a variety of undesirable organisms. The cheese “hopper” or “skipper” found in and upon old and overripened cheese and in cheeses which have not been properly screened, is the larva of the black two-winged fly Piophila casei. The insect deposits its eggs in the surface cracks and crevices of the cheese upon which the developing larva feeds. The name skipper or hopper is derived from the fact that the larve are capable of projecting themselves some distance by coiling and suddenly uncoiling. This fly is a common pest in the dairying establishments. A less common but even more annoying pest is the larva of the “bacon beetle.”’ Cheeses which are comparatively hard and smooth externally are not so likely to be infested by the skipper or bacon beetle larva as are the cheeses which are rough externally. It is customary to wipe the cheese in order to remove the para- sites. If the cheeses which are stored for ripening are properly screened, the fly and beetle cannot get access to them to deposit the eggs. A small mite (Trioglyphis siro) also occurs on cheese upon which it feeds. Inadequately screened cheeses also permit flies and other pests to deposit possible infections, thus typhoid contamination and also pus streptococci and staphylococci may be found upon this food substance. DISEASES OF CHEESE I41I Bitter cheese is due to a variety of bacteria, as Tyrothrix geniculatus (the bitter soft cheese bacillus), Micrococcus casei amari (bitter cheese coccus), Weigmann’s bitter milk bacillus, Conn’s bitter milk coccus, and others. Red coloration of cheese may be caused by yeasts (Saccharomyces ruber) or by cocci. Black cheese Fic. 45.—Oidium lactis. a, 6, Dichotomous branching of growing hyphe; c, d, g, simple chains of oidia breaking through substratum at dotted line x—y, dotted por- tions submerged; e, f, chains of oidia from a branching outgrowth of a submerged cell; 4, branching chain of oidia; k, 1, m,n, 0, p, s, types of germination of oidia under varying conditions; ¢, diagram of a portion of a colony showing habit of Oidium ag as seen in culture media.—(From Bull. 82, Bur. Animal Industry, U. S. Dept. Agr. may be due to the presence of iron in milk, perhaps traceable to the action of slightly soured milk in rusty buckets. Some yeasts and molds may produce dark to black decomposition changes. Blue cheese is the result of the action of a bacillus. Putrid cheese is the result of the invasion of saprophytic bacteria and other micro- I42 BACTERIOLOGICAL METHODS organisms. Cheese poisoning is not uncommon, due to the pres- ence of bacteria which give rise to toxins (tyro-toxicon). In a general way it may be stated that cheese diseases are due to filthy and unsanitary conditions in the dairying establishment re- SO00I000N) nO Se, 0000007. =) SS SS na 0000089 9908 s O00800809 SS & Foor fF o~ ee, —=S GP ‘ee, “A Fic. 46.—Penicillium glaucum showing the characteristic spore formation. This fungus is a true saprophyte, the common green mold, occurring on a great variety of organic substances. sulting in infected milk, or to filthy and unsanitary conditions in the cheese factory, or the infection may be traceable to the im- proper and careless storing and handling of the cheese. Ripened cheese being in itself a decomposition product resulting from the invasion of certain desirable micro-organisms usually entering CANNED AND CONDENSED MILK 143 from the air, it is but reasonable to expect irregularities in the final result unless the invasion of undesirable micro-organisms, which are also present in the air, is carefully guarded against. Condensed milk is prepared by concentrating full or skimmed milk. It may be sweetened by adding cane sugar (40 per cent.). While condensed milk contains relatively fewer bacteria than does ordinary milk, due to the process of manufacture, yet none is entirely sterile. The number of bacteria usually present ranges from about 500 or even less to as high as 250,000 per cc. Colon bacilli, dysentery bacilli and streptococci are generally absent. Tubercle bacilli have been found. The method for examining condensed milk is much as for ordinary milk, with suitable modifications in making the dilutions. Canned condensed milk occasionally spoils, due to the development of bacteria and yeast organisms. Yeast organisms are not likely to appear unless the milk is sweetened with sugar. Spoiling may be- come apparent through the ‘“‘swelling”’ of Fic. 47.—Penicillium - : : : of Camembert and Roque- the can. Organoleptic testing is occasion- fart cheese. This mold ally a guide to the condition or quality of §tows at a very low tem- ‘ Z ~ perature. It is closely the milk. A numerical bacterial limit similar to, if not identical, should be adopted for condensed milk. If econ eae ee j more than 1,000,000 bacteria per cc. are present it is not suitable for human consumption. Tubercle bacilli should be absent. According to the limited reports on the subject we may assume that the process of condensing the milk kills all pathogenic bacteria which may be present, including even the more resistant tubercle bacilli. The contaminating bacteria may pro- duce toxins and in marked bacterial invasion it would be well to make inoculation tests with white mice or guinea-pigs, as for toxins in meat and in ice cream. An examination of the centrifugalized 144 BACTERIOLOGICAL METHODS sediment must not be omitted as this will convey information re- garding the sanitary conditions of the factory as well as of the dairying establishments which supplied the milk to the factory. Dried or powdered milk is prepared by spraying milk (usually skimmed) into a partial vacuum or by spraying it on a revolving drum or ona moving belt ina partial vacuum. ‘The dried material is then placed in suitable containers. The dried milk contains all of the ingredients of the milk excepting the water, the lysins and certain enzymes. The fat globules are altered physically but not chemically. Mixing dried milk with the required amounts of water makes a liquid resembling ordinary milk. The micro- scopical and bacteriological examination of dried milk is as for condensed milk. Like the condensed milk it is quite free from disease germs of all kinds but bacterial invasion is not excluded from material which has been carelessly prepared or canned. Toxins and ptomaines should be absent. The absence of moisture in powdered milk prevents the ready growth of micro-organisms and it may be kept in good condition for a period of 5 or 6 months and even longer, in dry sterile containers stored in a cool dry place. Attempts have been made to commercialize frozen milk but so far without success. It is rather difficult to handle frozen milk and the article furthermore loses the milk flavor on thawing. 16. The Bacteriological Examination of Shellfish The term shellfish includes oysters, mussels and clams. Only those species and varieties which serve as food for man are of interest to the food bacteriologist. Since it has been conclusively proven that shellfish, oysters in particular, have been responsible for typhoid epidemics, much attention has been. given to the bacteriology of this class of food. In tracing such epidemics it was discovered that the causative oysters had been floated or grown in heavily polluted waters. In several instances contamination of SHELLFISH 145 the water supply washing the oyster beds, was traceable to the discharges from typhoid fever patients. All shellfish are easily adaptable to filthy habits and surround- ings. They appear to thrive in proportion to the amount of or- ganic contamination of the water supply constituting the food beds. It must, however, not be supposed that sewage and other highly objectionable (to man) contamination is normal to the life of the shellfish. We know that the domestic hog is fond of the highly contaminated refuse materials from the kitchen known as swill but we also know that hogs thrive better on sanitary food. Thus the filth feeding oyster grows equally well, if not better, in clean sea water, that is, water free from sewage contamination and decayed animal matter. The danger from shellfish (to man) is due to the fact that these animals are often from highly contaminated water supplies and that they are generally eaten raw or only partially cooked. The possible diseases traceable to the eating of shellfish are Asiatic cholera (in countries where this disease prevails), typhoid fever and a variety of less severe intestinal diseases such as dysentery, colitis and intestinal ulcerations. The work of the food bacteri- ologist is, however, not the finding of the specific germs causing an epidemic, but rather an endeavor to ascertain the danger point in the quality of the food as represented by the positive colon bacillus tests. The prime object of the pure food laws is the maintenance of health rather'than finding the cause of disease. This most important fact is sometimes not understood as is clearly indicated by a supreme court decision permitting the bleaching of flour. It is the intent of the pure food law to clearly mark the danger points in our food supplies so that the consumer may maintain his physical well-being through the avoidance of such dangers. He who advises against the heeding of the proper and timely warn- ings set up by those entrusted with this duty, either through ignorance or indifference, is a menace to the public welfare. The danger sign, “‘avoid bleached flour” and not the actual physical I 46 BACTERIOLOGICAL METHODS disturbances which results from the eating of such flour, is the proper warning. The presence of a limited number of colon bacilli in foods and drinks is the danger mark and not the actual occur- rence of cholera, of typhoid, of dysentery, due to the eating of more highly contaminated foods. The danger signal must be within the zone of safety and not beyond it. The methods for the bacteriological examination of shellfish are but modifications of the methods used in the examination of water supplies. As in the case of drinking water, the chief index to the pollution of shellfishis the colon bacillus test. The examina- tion of the water source above the oyster beds very frequently gives inferential information as to the possibility of the contami- nation of the shellfish which obtain their food supply from beds flooded by such waters. The following is the method for the bacteriological examination of shellfish adopted by the American Health Association at the 1912 meeting. 1. Selection of Sample.—Twelve oysters of average size of the lot to be examined, having deep bowls, short lips and shell tightly closed, are picked out by hand or by means of a sterilized long- handled spoon and prepared for immediate transportation to the laboratory. 2. Making a Record of the Sample.—This record should cover the following points. The exact location of the bed from which the sample was taken. The depth of the water at the time the oysters were gathered. Weather conditions, direction and velocity of wind, state of tide, day and hour when the stock was taken from the water, the conditions under which the stock had been kept since removal from the water and up to the time when the sample was taken, presence of abnormal odors, temperature of stock, and the day and hour of taking the sample. 3. Transportation of the Sample.—The sample oysters are to be packed in a suitable metal or pasteboard container of the size and shape convenient for shipping. The important points to bear in mind are: the prevention of the mixing of the oyster liquors of SHELLFISH 147 the different samples and avoiding the mixing of the oysters with the ice water of the packing ice. The samples must in all cases be placed on ice or packed in ice if they cannot be examined inside of 36 hr. or if the outside temperature is above 50° F. It is, however, not necessary to place the oysters in absolutely tight containers provided the above conditions are maintained. 4. Laboratory Procedure.—Record the date of receiving the sample, condition of seals, of the sample oysters and the tem- perature of the interior of the container at the time of opening. The bacteriological examination should in all cases be started as soon as possible after the receipt of the sample. Before beginning operations the hands must be thoroughly scrubbed and all vessels to be used must be sterilized. The shell of the oyster may be opened by means of a sterilized oyster knife or by drilling a hole through the shell near the hinge. The drill must be sterilized and the area of the shell to be operated upon must be cleaned, flamed before drilling and flamed at least once more during the drilling process. The simplest and quickest method for opening the oyster shells is that employed by Stiles of the Bureau of Chemistry. By means of a pair of sterilized wire nippers crush and break off enough of the two valves so as to make the use of the oyster knife easy. Before opening the oysters see that they are thoroughly scrubbed and then rinsed in boiled (sterile) water, and each oyster is wiped quite dry and flamed before it is opened. 5. Bacterial Counts.— Bacterial counts are made of the compos- ite sample of each lot obtained by mixing the shell liquor of five oysters. Agar shall be used for the culture medium and in general the procedure shall be in accordance with the method recommended for the examination of water. The water used for making the di- lutions shall contain 1 per cent. of sodium chloride, in order to approximate the natural salinity of the oyster liquor. The agar plate cultures shall be incubated at 20° C. for 3 days and the col- onies counted in the usual manner. It 148 BACTERIOLOGICAL METHODS 6. Determining Bacteria of the Colon Bacillus Group.—Meas- ured quantities of the shell liquor of each of five oysters selected from the dozen shall be placed in fermentation tubes containing lactose-peptone-bile. The measured quantities shall be 1 cc., 0.10 cc., and o.o1 cc., or such other quantities or corresponding dilutions as may be desired. The fermentation tube inoculations thus prepared shall be incubated for 3 days at a temperature of 37° C., and the presence of gas noted daily. From ro to 85 per cent. of gas during this period shall be considered a positive test indi- cating a presumption of the presence of at least one bacterium of the colon bacillus group in the quantity of the water used in the test. But no final colon bacillus rating shall be made unless con- firmatory tests for the presence of organisms of the colon bacillus group shall have been obtained from the tube of highest or next highest dilution from each oyster showing the presence of gas. These confirmatory tests shall be begun immediately upon noting the formation of gas and shall be carried out in conformity with the procedure recommended by the Committee on Standard Methods of Water Analysis. 7. Statement of Results.—The results of the bacterial counts shall be expressed as the number of bacteria per cc. The results of the colon bacillus test shall be expressed either in the form of an arbitrary numerical system or in estimated number of colon bacilli per cc. of the sample. It is suggested that the arbitrary numerical method proposed by the American Health Association be given the preference. The following are the rating valuations according to this method. Colon bacillus in r.00 cc. but not in 0.10 cc., a value of 1 Colon bacillus in 0.10 ce. but not ino.or cc., a value of 10 Colon bacillus in o.o1 cc. but not in 0.oo1 cc., a value of 100, etc. The sum of these values for five oysters gives the total value of the sample examined and this figure indicates the rating for Bacillus coli. According to this system the highest (best) rating THE RATING OF SHELLFISH 149 is indicated by o and the lowest (worst) by 500, represented in tabular form by the following possible results of two analyses: EXAMPLE A Numerical Oysters I.00 cc. 0.10 cc, 0.01 cc. Aralae (oy {ea(oy (oh fo) OOO F OS 00000 00000 a BW DNDN H Total rating for B. coli = ° EXAMPLE B Numerical Oysters I.00 cc. 0.10 cc. 0.01 ce. Value +++++ ++4+++ +++++ 3 Total rating for B. coli = 500 The (+) mark means that gas formation in the lactose bile tubes took place, indicating contamination with the colon bacillus. The (0) mark indicates that no gas formation took place in the lactose bile tubes. The results above indicated are, however, not generally ob- tained in practice. The important question is at what rating shall the shellfish be pronounced unfit for human use, or rather what rating shall be the danger signal as to the quality of this food? There seems to be no uniformity of opinion as regards this point. Thus far, the Bureau of Chemistry has barred oysters from inter- state shipment which gave three positive tests out of five in 0.10 cc. quantities of oyster liquor, which standard is also adopted by I50 BACTERIOLOGICAL METHODS the Rhode Island Shellfish Commission. This standard may be graphically represented as follows (Example C): EXAMPLE C Oysters I.00ce 0.10 cc ODTee Numerical y i ; : ; : Value | I + ais ° Io 2 AF Ar (eo) Io 3 ar =F ° Io 4 + fo) fo) a 5 aR ° fe) iE Total rating for B. coli = 32 It sometimes happens in laboratory practice that the smaller quantities of shell water from a number of oysters show positive results, whereas larger amounts of liquor from an equal number of oysters show negative results. In such cases it is customary to give the next lower numerical value to the positive results in the high dilutions, and such positive results shall be considered as being transferred to a lower dilution giving negative results in another oyster. ‘This recession of assigned values shall, however, not be carried beyond the point where the number of such reces- sions is greater than the number of instances where other oysters in the series of five failed to give positive results. This may be illustrated as follows (Examples D and E): EXAMPLE D Oysters | T.00 cc. 0.10 cc. | 0.01 cc. Numerical | Value I + + fe) IO 2 + +- ° \IO 3 + ++ fe) ‘To 4 a fo) ° To (not 1) 5 -f- + + 0 (not roo) | Salar | Total rating for B. coli = lea | THE RATING OF SHELLFISH I51 EXAMPLE E Oysters I.00 cc. 0.10 cc. 0.01 cc. a ie: 0 “ a ro (not 100) 2 ae “I he ro (not 100) 3 fF fe) fe) I 4 ° | fo) fe) I (not o) 5 ° ° ° 1 (not o) Total rating for B. coli = 23 The bacteriological examination of oysters from opened or shucked stock very naturally must be somewhat modified from the method as outlined for oysters in the shell. The stock in the con- tainer from which the sample is to be taken must be thoroughly mixed. The containers (wide-mouthed glass jars) must be steril- ized and should have a capacity of 1 quart. By means of a suit- able sterilized ladle (may be flamed with alcohol on the spot), half fill the containers with the oysters and seal containers in such manner as to exclude all outside contamination. Unless the exami- nation can be made within 3 hr. after taking the sample, said sample must be placed on ice. It is very desirable to make the bacteriological examination shortly after the sample is taken. The laboratory technique is much as for oysters in the shell, though it must be borne in mind that dilutions higher than o.o1 cc. are usually required. The results of the bacteriological examination of the opened or shucked stock shall be expressed in the same way as that specified for oysters in the shell, except that in the calcula- tion for B. coli rating the values for the results of the positive fermentation tests, after confirmation, shall be recorded for each of the inoculations of each and every dilution. All tests are to be made in triplicate, that is, three fermentation tubes are to be inoculated for each dilution used. Clams, mussels and other shellfish are to be examined in the same manner as oysters, in so far as this is possible. In opening I52 BACTERIOLOGICAL METHODS soft-shelled clams it will be found that if two incisions are made through the mantle the shell water may be poured out without opening the shell. It is stated that hard-shell clams may be opened by striking the shell over the dorsal muscle with a hammer. An opening is formed which will permit the insertion of a knife with which to cut the muscle. In case any one shellfish does not contain enough shell water to make a test, the water from several individuals may be mixed. The examination of shellfish for sewage pollution is of the utmost importance, as dangerously contaminated oysters are very com- mon. In fact it would be advisable to discontinue the oyster as an afticle of diet. At its very best it is a filthy article. It is unquestionably a dangerous article of food, in this regard compar- able to the mushrooms in the vegetable kingdom. However, there is not the least likelihood that the oyster will be left from our dining tables as long as there are any available. It is therefore most desirable that the supervising of this food on the part of those who are entrusted with the safeguarding of the health of the people should be carefully and consistently done. Some authorities (English) recommend that the liquor and oysters be mixed, the latter finely chopped, for the purpose of making the colon bacillus test. There appears to be no gain from this procedure and the method cannot be recommended. 17. The Bacteriological and Toxicological Examination of Meat and Meat Products Remarkable as it may seem, food bacteriologists have given but little attention to the examination of meats and meat products, despite the fact that fatal poisoning from eating infected meats is very common. Intoxications ranging from mild to very severe, resulting from the ingestion of more or less highly contaminated meats are of daily occurrence in every community. At each in- stance of a death or deaths resulting from the eating of bad meat, MEAT BACTERIA 153 the health authorities get busy and almost invariably find the true source of the trouble, and there the matter usually rests. No ra- tional attempt is made to prevent a repetition of the occurrence. Meats of all kinds when left exposed to the air soon show signs of decomposition. The aerobic forms of bacteria are first to de- velop, causing the decomposition of proteids and sugars. Inas- much as sugar is usually present in small amounts only, the sugar decomposers are soon crowded out by the proteid-splitting forms. The small amount of acid formed by the sugar decomposers is neutralized by the ammonia which is formed during proteid de- composition. The aerobes very naturally act on the outside of the meat particles, using up the oxygen in the air on and within the immediate surface tissues of the meat. This reduction in oxygen gradually permits the anaerobes to get a start, especially B. per- fringens and B. bifermentens sporogenes. These use up proteids as well as sugar, and the complete removal of sugar encourages the more active development of pure aerobes which act upon proteids only. The following tabulation from the work by Ellis Organisms Action on Products Formed Proteus vulgaris.......2..0. Gluten and fibrin |Phenol, indol, amines, fatty acids. ZG OLEUS BULB OTUS Moje oteocix oisen os (Cason -t4: Ge neate Albumoses, peptones and amino- | acids. Streptococcus longus......... | Piri: .s,90a5-o ane Tyrosin, leucin, amines and fatty | acids. B. colt communis........... CAS CME eras kor giark om Albumoses. B. colt communis........... PReptone.s serra ‘Ammonia and indol. B. coli communis...... Ae Mixture of eggs and Skatol, phenol, leucin and oxy- meat ; acids. Micrococcus pyogenes........ Gluten ‘Phenol, indol, amines and fatty acids. Aerobic peptonizing lactic|Casein............ Leucin, tyrosin, fatty acids, aro- ACIOMDACLETI AL eee atic << matic fatty acids and trypto- | phan. B. subtilis and B. prodigiosus |Albumoses......... Leucin, tyrosin and tryptophan. Cholera Vibrio..; 4... .2 63 - PAG Maren «cate Leucin, tyrosin, indol, amines and fatty acids. 154 BACTERIOLOGICAL METHODS indicates the activities of putrefactive bacteria in culture, which correspond with the putrefactive changes produced in nature: The rotting bacteria produce the familiar changes in meat usually designated as spoiling, rotting and tainting, and such meat is universally recognized as unfit for food because of the deleterious effects following the ingestion of such meats. Tainted meats may appear entirely normal to the naked eye and slight decay of the inner tissues may not be appreciable to the sense of smell. The decomposition changes resulting in the liberation of indol, skatol and related substances having disagreeable odors usually begin near the bones and joints, and such decomposition may not become apparent until the bony structure is exposed by cutting or until the odors are dissipated more actively by boiling. This odor is very persistent; boiling for several hours will not cause it to disappear entirely. It must not be supposed that meats free from bad odors are necessarily free from ptomaines and toxins. For example, perfectly fresh meat may absorb these poisonous sub- stances when placed in contact with badly tainted meats and, again, some toxin-forming bacteria do not produce odoriferous gases. The bacteriology and toxicology of canned meats and soup stocks containing meat has not received the attention that it should. It is these substances which are so largely responsible for the mul- titudinous lesser intestinal disturbances following their use as food. The present methods of canning meats should be thoroughly in- vestigated and ways and means devised to improve them in _ accord with modern advance in the manufacture of food products. It is generally believed that the eating of canned meats and soups is fraught with danger to life and health, and this is not far from the truth. The proper canning of meats requires infinitely more care than the canning of vegetable substances. The careful super- vision of the marketing of meats and meat products is vastly more important than the supervision of vegetable foods. It is compara- tively rare for toxins and ptomaines to be formed in vegetable substances, whereas this is the rule in the decomposition of meats. MEAT BACTERIA 155 Furthermore, meats decompose much more readily than vegetable substances, which makes it necessary to observe greater care in the preparation of this class of food for the market. Some meats decompose much more readily than others. Meats of higher animals resist decomposition longer than do meats of lower ani- mals. Fish meats decompose quickly when exposed to the air. The story is current among fishermen that certain kinds of fish Fic. 48.—Bacillus welchii, also known as B. aerogenes capsulatus and B. phleg- mones emphysematose in smear preparation. This is the common “gas bacillus,” be- cause of the abundant gas formation in the tissues invaded and in culture media. It is a plump large nonmotile, anaerobic, capsulated, Gram positive, spore-bearing bacillus and is very widely distributed in nature.—(Williams.) begin to decompose before they can be removed from the hook. This is of course exaggeration, but the statements made indicate in a way the comparative resisting power of different kinds of meat to rotting bacteria. It is highly probable that the difference in the resisting power to decomposition is due in part at least to the presence of bacteriolysins. Little is known regarding the changes which take place in cold storage meats. Cold storage 156 BACTERIOLOGICAL METHODS Fic. 49.—Typical cultural characteristics of Bacillus aero- genes capsulatus (B. welchii) in agar. Culture 48 hr. old. The agar mass is separated by the gas which is formed.—(MacN eal.) does check the growth of all kinds of bacteria and of higher fungi, but not in the same ratio. For example, the freezing temperature inhibits the de- velopment of the usual rotting bac- teria very effectually, whereas many of the toxin formers multiply slowly, in time forming enough of the poison to produce marked symptoms of poisoning when meat thus affected is eaten. Little is known of the changes which take place in incompletely ster- ilized canned meats, and no attempt has so far been made to ascertain the degree of decomposition which usually takes place in the meats before they are placed in the cans and sterilized. This is a matter of the utmost impor- tance and should receive the immediate attention of the food bacteriologists. What shall be the routine method in the examination of meats? It is quite evident that the methods which are applicable in the examination of vegetable substances are not suitable in the examination of meats. We hereby suggest the following outline of methods applicable in the food labora- tory: 1. Direct microscopical examination of meats. a. Bacteria on surface of meats. b. Mold and spores present, as in moldy bacon, pork, etc. c. Presence of bladder worms, larve of parasites, etc. EXAMINATION OF MEATS 157 d. Trichine in pork. e. Cereal fillers and starches in sausage meats. Tragacanth fillers. f. Coloring substances and preservatives in sausage meats. 2. Plate cultures. (Lactose-litmus-agar and gelatin media.) a. Numerical counts of bacteria. b. Number of gas formers and acid formers. c. B. botulinus in pork meats. 3. Toxicological tests. a. Inoculation tests (guinea-pigs) to prove the absence or presence of ptomaines or toxins. b. Tests for tuberculous and other diseased meats. 4. Determining the source of the meat. a. By the precsipitin test. b. Sugar test for horse meat. c. Microscopical identification based on differences in the size and structure of the muscular fibers and the differences in the size and form of the fat crystals derived from different animals. . Of the above tests the numerical bacterial count and the toxico- logical tests are of the greatest importance and should be carried out in the examination of suspected raw meats, sausage meats and of canned meats and soup stocks. There certainly should be a limit to the number of bacteria in all raw meats, whether ground into sausage or not, as this would be the means of regulating the sanitary requirements in the proper handling of meats. The only practical method for determining the quality of canned meats is to make inoculation tests on guinea-pigs or white mice, using filtered aqueous extracts of the suspected meat products. If ptomaines or toxins are present the tests will show it. It would be very desirable to work out a micro-chemical test for determining the presence of toxins and ptomaines in meats. As above indicated, there are some very important differences between toxins and ptomaines. The former are destroyed by the boiling temperature, whereas the latter are not. For example, the thorough cooking of sausage meats prevents botulism but it does not prevent the ill effects resulting from the eating of meats with ptomaine poison. Dried and smoked meats should be examined for the presence of bacteria and molds. Dried fish in particular is very fre- I 58 BACTERIOLOGICAL METHODS quently highly contaminated with molds. It is very evident that the present method of pickling fish of all kinds must be changed. The method of pickling herring, for example, in wooden vats or casks must be abandoned, as the containers are wholly unsuitable from a sanitary standpoint. The liquor from canned fish (in tin cans) is frequently very highly contaminated with bacteria in spite of the high salt content. The gelatin of the market requires careful examination, as much of the sheet variety is not infrequently FIG. 50. FIG. 51. Fic. 50.—Bacillus botulinus from a sugar-gelatin culture.—(Pittfield, after Kolle and Wassermann.) Fic. 51.—Bacillus enteritidis. Under this name is included a number of organ- isms of the Gaertner group which play a very important part in meat decomposition and meat poisoning. It is also known as the dysentery group. The organisms are actively motile, non-sporogenous, aerobic, non-liquefying and Gram negative — (Jordan after Kolle and Wassermann.) : entirely permeated by mold and bacteria, rendering it not only unfit for food for man but also unsuitable for bacteriological work. The entire subject of meat poisoning is as yet not very well understood. Dr. Savage states that the bacteria concerned in meat poisoning may be classed under three groups: (a) the Gaertner group of bacilli, (b) aerobic bacilli not belonging to the Gaertner group, such as B. proteus and B. coli, and (c) Bacillus botulinus. In the majority of cases of outbreaks of fatal food poisoning, some EXAMINATION OF MEATS 159 form of the Gaertner group of bacilli has been the infecting organ- ism. The Gaertner bacilli are large coli-typhoid types which occupy a position intermediate between the chemically active colon group and the chemically inert typhoid group, and includes B. enteritidis, B. typhi murium, B. suipestifer and B. paratyphosus B. Sausages and ham are the commonest sources of botulism. From the anaerobic character of the bacillus it follows that poison- ing is rarely due to the eating of fresh sausage and pork. Invasion of meats by B. botulinus can take place only when the necessary anaerobic conditions exist, as for example when a ham is stored at the bottom of the pickling vat and entirely covered by the pickling solution, and in the interior of insufficiently cooked sausages and in stored masses of sausage meats. The use of the compound microscope in the examination of meats and meat preparations is stillinits infancy. The work done shows very clearly that with more experience very valuable in- formation can be obtained from the microscopical examination regarding the quality of meats of all kinds. It is highly probable that the microscope will show diagnostic differences in the muscu- lature of different animals, thus making it possible to determine the source of the meat. Considerable attention has already been given to the micro- scopical study of fat crystals derived from the fats of different species of animals. So-called rancid fats or fats which have aged considerably, even though they may not yet give evidence of ran- cidity to the unaided senses, will show more or less abundant crystalline structure, arranged in clusters, which may be readily seen under the low power of the compound microscope. ‘These crystals are not apparent in fresh fats, but are generally more or less abundantly present in canned meats and soup stocks containing animal products, in meat extracts and in other meat derivatives having fat admixtures. The presence of crystal clusters indicates fat decomposition and these are therefore an indication of the quality of the meat product containing them. The degree of ran- 160 BACTERIOLOGICAL METHODS cidity or, to state it more accurately, the quality of the product dependent upon age is in direct proportion to the quantity of crys- talline clusters present. It would appear that the quantity of crystals present is not proportional to the amount of bacterial contamination and decomposition. The indications are that it is possible to determine the source of the fat from the color, size and arrangement of the fat crystal aggregates. For example, the crystal clusters of lard are smaller than those of the fat of the do- mesticfowl. The fat crystal aggregates of the hen are compara- tively large and the individual crystals are long and slender. The inexperienced analyst is apt to mistake the crystal clusters for mold colonies (Leptothrix). This mistake can very readily be avoided by applying heat which causes the prompt melting of the fat crystals whereas the mold hyphe are not greatly disturbed or changed. The differential characteristics which would be con- cerned in the microscopical examination of fat crystals may be given as follows: 1. Differences in the size of the aggregates. 2. Differences in the length of the individual crystals. 3. Differences in the diameter of the individual crystals. 4. Differences in the form of the ends of the individual crystal. Ends may be rounded or pointed. 5. Differences in color. These will in all probability pertain to different races or families of the animal kingdom. For example, lard crystals are colorless whereas those of the domestic fowl are yellowish. The use of certain chemicals will aid in the microscopical find- ings. For example, sulphuric acid produces characteristic color reactions with certain fats. If two drops of concentrated sul- phuric acid are added to twenty drops of goose fat, a greenish-yel- low color is produced which changes to reddish brown on stirring. Under the same conditions cod-liver oil turns a violet color whereas turtle oil turns brown. Castor oil turns yellowish to yellowish brown and finally wine red with a very distinct zone. A similar reaction is observed with neats foot oil. Raw linseed oil turns a deep reddish brown to very dark brown. Lard oil shows a distinct FAT CRYSTALS 161 brown zone which deepens to wine red. The reaction for sperm oil is much as for cod-liver oil. These color reactions with sul- phuric acid are perhaps of little value in the detection of fat adul- terations and admixtures but they will prove helpful aids in the examination of these substances as to identity. Pure concen- trated acid should be used. It must also be kept in mind that the fat impurities which may be present modify the color reactions. Pure samples of fats should be kept on hand for purposes of making check and comparative tests. The Bureau of Animal Industry has suggested a method for distinguishing between fats and oils derived from the animal and the vegetable kingdoms based upon differences in the appearance of the crystals (phytosterol and cholesterol). In addition to the study of fat crystals which are formed spon- taneously in more or less decomposed and aged meat products as above set forth, certain methods for testing fat crystals isolated in the pure state by chemical methods are now generally carried out in meat inspection and food laboratories. These tests com- bine the use of the compound microscope and should therefore be carried out by the micro-analyst or bacteriologist and for that reason are hereby included. R. H. Kerr of the Biochemic Divi- sion of the Bureau of Animal Industry has worked out a method for detecting vegetable fats in mixtures of animal and vegetable fats and vice versa, the method will also serve to demonstrate the presence of animal fats in supposedly pure vegetable fats. The method is a slight modification of several methods which have been in use for some time and which are described in various text-books, and it is hereby given in full as it appears in Circular No. 212 (May 10, 1913) of the Bureau of Animal Industry. THE DETECTION OF PHYTOSTEROL IN MIXTURES OF ANIMAL AND VEGETABLE FATs Sample.—The amount of sample used depends on the amount of material avail- able. From 200 to 300 grams is the amount usually taken. The test is seldom 162 BACTERIOLOGICAL METHODS attempted if less than 100 grams are available, and an amount greater than 500 grams is never taken. Extraction with Alcohol.—The sample is melted and poured into a flat-bottomed flask of 1-liter capacity which is closed with a rubber stopper perforated with three holes. This flask is set on the top of the steam bath and connected to a reflux condenser and to a 700 cc. round-bottomed flask containing 500 cc. of 95 per cent. alcohol. A glass tube which is adjusted so that its lower end is about one-fourth of an inch above the surface of the fat and whose upper end is bent at a right angle and closed by means of a short piece of rubber tubing and a pinchcock fills the third hole in the stopper. The distilling flask is set down in the steam so that the alcohol boils briskly. The outlet tube reaches down to the bottom of the flask containing the sample so that the alcohol vapor as it distills over bubbles up through the fat and keeps it in a state of vigorous agitation. The alcohol vapor is condensed in the reflux condenser and returned to the flask containing the fat. The distillation is con- tinued until all of the alcohol has collected in the flask containing the fat. The dis- tilling flask is now disconnected. The alcohol in the flask immediately ceases to boil and soon separates from the fat. The empty distilling flask is next connected to the bent tube by a piece of glass tubing of sufficient length, the pinchcock opened, and the alcohol layer siphoned off into the distilling flask. This is then connected as before and the distillation continued until the alcohol has again collected in the first flask. It is then siphoned into the distilling flask as before, and a third extraction made. After the third extraction the alcohol layer is again siphoned off into the distilling flask and the fat is discarded. The alcohol now contains practically all of the choles- terol and phytosterol originally present in the fat. Saponification and Extraction with Ether.—The alcohol in the distilling flask is next concentrated by boiling to about 250 cc., and 20 cc. of a concentrated potassium- hydrate solution (100 grams KOH dissolved in too cc. water) added to the boiling liquid. It is boiled for ro min. to insure complete saponification of all the fat and is then removed from the steam bath and allowed to cool almost to room temperature. After it has cooled sufficiently it is poured into a large separatory funnel containing 500 cc. of warm ether and shaken to insure thorough mixing. The mixture may be clear, but is more often opalescent. There is now poured in 500 cc. of distilled water, and the funnel is rotated gently. Shaking must be avoided, as it leads to the forma- tion of extremely stubborn emulsions, but the water should be mixed with the alcohol- ether-soap solution. Separation takes place at once and is clear and sharp. The soap solution is drawn off and the ether layer washed with 300 cc. of distilled water, shaking being still avoided. After this washing it is washed repeatedly with small quantities of water until all soap is removed. The ether layer is then transferred to a flask and the ether distilled off. Distillation is stopped when the contents of the flask have been reduced to about 25 cc., and the concentrated ether solution contain- ing the cholesterol, phytosterol, and all other unsaponifiable matter is transferred to a tall 50 cc. beaker. The evaporation is continued until all ether is driven off and the residue is perfectly dry. If desired, a tared beaker may be used and the weight of the unsaponifiable matter determined at this point. sale FAT CRYSTALS 163 Preparation of the Acetates.—A small amount (3 to 5 cc.) of acetic anhydrid is added to the dry residue in the beaker and heated to boiling over a free flame, the beaker being covered with a watch glass during the process. After a brief boiling—a few seconds is sufficient—the flame is removed and the beaker transferred to the steam bath and left there until the acetic anhydrid is driven off. Purification of the Acetates.—Thirty-five cc. of hot 80 per cent. alcohol are added to the acetylated residue in the beaker and heated to boiling with vigorous stirring. The liquid is then filtered quickly through a folded filter and the in- soluble residue washed well with boiling 80 per cent. alcohol. The acetates of chol- esterol and phytosterol are dissolved, while the greater portion of the impurities present are not dissolved by the alcohol and remain on the filter. Paraffin and paraf- rele ey I ps a Fic. 52.—Phytosterol crystals. Fic. 53.—Cholesterol crystals. (aes) fin oil, if present, are likewise separated by this treatment. The combined filtrate and washings are next cooled to a temperature of 10° to 12° C. and allowed to stand at that temperature for 2 to 3 hr. During this time the acetates of cholesterol and phytosterol crystallize from the solution. They are removed by filtra- tion, washed with cold 80 per cent. alcohol, and then dissolved on the filter with a stream of hot absolute alcohol from a wash bottle, as little alcohol as possible being used. The alcoholic solution of the acetates is caught in a small glass evaporating dish, two or three drops of distilled water being added to the solution and heat applied if it is not perfectly clear. The dish is then set out ona desk in the laboratory and the alcohol allowed to evaporate spontaneously. The contents are stirred occasionally and the deposit of crystals which forms around the edges of the liquid and on the sides of the dish rubbed down into the solution with the stirring rod. As soon as a good -deposit of crystals has formed they are removed by filtering through a hardened 12 164 BACTERIOLOGICAL METHODS filter, washed twice with cold 90 per cent. alcohol, and dried by suction. After drying by suction they are dried at 100° C, for half an hour and the melting point determined. Determination of the Melting Point.—A tube of about 1 mm. diam., sealed at one end and having a slight flare at the other, is filled to a depth of about 5 mm. with the dried crystals, which are packed somewhat firmly in the lower end by tapping on a hard surface. This is attached to the bulb of a suitable thermometer and the melt- ing point determined. A thermometer graduated from 95° to 200° C. in one-fifth degrees is used in this laboratory. The determination is made in an Anschutz apparatus, the outer bulb being filled with concentrated sulphuric acid and the inner tube with glycerin. The apparatus is so adjusted that no correction of the observed temperature is required. The melting point of the first crop of crystals usually gives definite information as to the presence or absence of phytosterol, but the conclusion indicated is confirmed by recrystallizing from absolute alcohol and again determining the melting point. If the crystals are pure cholesterol acetate, the melting point of the second crop should agree closely with that of the first. If phytosterol acetate is present, however, a higher melting point should be noted, as phytosterol acetate is less soluble than cholesterol acetate. THE EMERY METHOD FOR THE DETECTION OF BEEF FAT IN LARD James A. Emery of the Biochemic Division of the Bureau of Animal Industry recommends the following method! for detect- ing beef fat in lard. It is given here because of its value in isolat- ing the crystals of fats for microscopical examination. Technique of Method.—Five grams of the warm filtered fat is weighed (on a bal- ance sensitive to 0.1 gram) ina glass-stoppered graduated cylinder of 25 cc. capacity, 150 tor75 mm. in height, with an internal diameter of about 18 mm., and warm ether is added until the 25 cc. graduation is reached. The glass stopper is securely re- placed and the cylinder is shaken vigorously until complete solution of the fat takes place. The cylinder with its contents is then allowed to stand in a suitable place where a constant temperature, at which it is desired to have the crystallization pro- ceed, may be maintained. (An apparatus described by Rogers proved efficient for the maintenance of this constant temperature.)? After 18 hr. the cylinder is re- 1 Circular 132, May 23, 1908. Bureau of Animal Industry, U. S. Dept. of Agriculture. * It is necessary to observe great caution in the use of this form of apparatus, as the sparking of the thermo-regulator is a source of danger if the solutions are care- lessly handled. A better form for this work would be one in which the temperature is controlled by a circulating hot-water system heated by a small lamp outside of the box, the regulation of which could be adjusted by using one of the many forms of gas regulators on the market. FAT CRYSTALS 165 moved and the supernatant ether solution carefully decanted from the crystallized glycerids, which are usually found in a firm mass at the bottom of the vessel. Cold ether is then added in three portions of 5 cc. each froma small wash bottle, care being taken not to break up the deposit while washing and decanting the first two portions. The third portion is, however, actively agitated in the cylinder with a sharp rotary motion and by a quick movement transferred, with the crystals, toa small filter paper. The crystals are then washed with successive small portions of the cold ether, with the use of the wash bottle, until to to 15 cc. has been used, dependent on the amount of crystals. Then by means of a slight exhaust the small amount of remaining ether is rapidly re- moved. The paper with its contents is then transferred to a suitable place, where it should be spread out and any large lumps of the glyc- erids broken up by gentle pressure. When dry the mass is thoroughly comminuted and the melting point of the crystals determined. As the difference between the melting points of the glycerids obtained in this manner from beef fat and lard is not very great, being only about 3.5 degrees, and as the writer has men- tioned a standard melting-point temperature for the glycerids of pure lard obtained under certain conditions, a description of the apparatus used in determining the melting points, together with its manipulation, is essential and may be of some assistance. Determination of the Melting Point—A Fic. 54.—Beef fat crystals. large test tube approximately 150 by 2 5 mm., ae Lee et ae ae fae othe containing water (free from air) into which the compound microscope; }, crys- bulb of a thermometer! with the melting-point tals highly magnified. tube attached is immersed, is placed in a beaker of water and so adjusted that the surface of the liquid contained in the two vessels is at the same level. The water in the beaker should be heated rapidly to about 55° C. and that temperature maintained until the thermometer carrying the melt- ing-point tube registers between 50° and 55° C., then heat is again applied and the temperature of the outer bath carried somewhat rapidly to 67° C., when the lamp is removed. The melting point of the crystals is regarded as that point when the fused substance becomes perfectly clear and transparent. The use of a dark background placed about 4 in. from the apparatus will prove of advantage. The melting-point tube should be of about 1 mm. internal diam., sealed at one end and with a slight flare at the other extremity, in order that the loading may be expedited. The amount of the substance taken for each determination should be 1 The thermometer used was one graduated in one-fifth degrees and extending from 0° to 100° C. 166 BACTERIOLOGICAL METHODS approximately the same and should occupy a space about 9 mm. in length, being somewhat firmly packed in the lower end of the tube by tapping it sharply on a hard surface. The water in the outer bath should be agitated frequently during the determination. Possible Sources of Error.—In applying the foregoing method too great care cannot be exercised with the preparation of the sample. The presence of water, the incomplete solution of the fat in the ether, or the presence of small particles of extraneous matter may interfere with the process of crystallization, frequently caus- Fic. 55.—Lard crystals. a, Clus- Fic. 56.—Duck fat crystals. a, ters of crystals as seen under the low- Clusters of crystals as seen under the power of the compound microscope; }, low power of the compound microscope; crystals highly magnified. b, crystals highly magnified. ing it to proceed too rapidly and resulting in the formation of a large mass of small fluffy crystals instead of the compact mass of larger crystals desired. These fine crystals render the preliminary washing by decantation with ether difficult, and they also persistently hold the unsaturated glycerids in larger amount than is desirable. The temperature at which the crystallization should be allowed to proceed should not be less than 15° C. nor more than 20° C., with the best results obtainable in the neigh- borhood of an average between the two. Although larger crystals are formed at the higher temperature (20° C.), only lards of high grade afford crystalline deposits in working quantity, and in many cases where lards of inferior grades are tested the amount of solid glycerids entering into their composition is so reduced as not to yield any deposit at all. HORSE MEAT 167 Pure fresh butter shows no crystalline structure. Salted butter will of course show the characteristic salt crystals. Melted butter which is allowed to cool slowly shows a marked crystalline structure under polarized light, even under the low powers of the compound microscope, but this is not a diagnostic character inasmuch as other fats show a similar behavior with polarized light. Horse meat has been used as food for man for many ages and is at the present time a regularly marketed food article in many countries. During the siege of Paris (1870) when food became very scarce, experiments with the meats of various animals were made, as that of rats, mice, cats, dogs, mules and horses. Horse meat especially met with general favor and since that time has become quite common in the French meat markets. It is stated that it is a frequent substitute for beef in our restaurants (the cheaper eating places in our larger cities). Horse meat differs from beef in that it is somwhat coarser grained, darker in color and that it contains a higher percentage of glycogen. As a rule the meats from cattle contain little or no glycogen, although it is stated that fresh meat from well-nourished cattle may contain as much gly- cogen as does the meat of the horse. It must also be borne in mind that the meat from dogs, cats, starved calves and fetuses | contains considerable glycogen. Should such meats be added to sausages the admixture might be recognized by the color, the meat of fetuses and starved calves being much lighter than that of the horse or of mature cattle. In time the glycogen of horse meat is changed into grape sugar and will respond to the Fehling’s solution reaction for sugar. For this purpose use a cold aqueous extract of the suspected meat. In the case of fresh horse meat the following tests are recommended. The Braéutigam and Edelmann test for the presence of horse meat is made as follows: Grind or chop (finely) 50 grams of the meat and boil for 1 hr. in 200 cc. of water. Add 1.5 grams (3 per cent. by weight of the meat) of caustic potash and heat over 168 BACTERIOLOGICAL METHODS water bath until the muscle fibers are disintegrated. Boil down to 50 grams and filter. When cool add an equal part of dilute nitric acid (10 per cent.) to precipitate the albuminoids, and again filter. Pour the filtrate into a test-tube and carefully pour iodine water down the inside of the tube. If horse meat is present a burgundy red zone appears at the point of contact of the two solutions. The width and intensity of the colored zone is in direct proportion to the amount of horse meat present. Peel If starch is present (as in sausages and sausage meats seasoned with starch-bearing spices or mixed with starch fillers), this must be precipitated from the boiled meat extract and removed by filtra- tion. To the extract add two or three times the volume of con- centrated acetic acid and let stand for 2 or 3 hr., and then filter through two or three thicknesses of filter paper. Test the filtrate with the iodine water as above suggested. However, before making the glycogen test the test for starch should be applied, for if it responds to this test the precipitation of starch must be repeated. Because of the dilution with the three or more volumes of acetic acid (to precipitate the starch) negative re- sults may be obtained in cases where horse meat is present. It is therefore advisable to precipitate the glycogen by means of alcohol, using from ten to twelve times the volume of the acidu- lated meat filtrate. The cloudy alcoholic suspension is run through a small filter and the precipitated glycogen on and in the filter paper is washed out by means of hot acidulated (acetic acid) water, and this filtrate is then tested with the iodine water. This test is positive in the presence of 5 per cent. quantities of horse meat. The wine-red color reaction is temporary only and it must be kept in mind that dextrin interferes with the reaction. Because of the fact that meats other than that derived from the horse may contain glycogen, it is sometimes necessary to supplement the above color reaction with the biological test or the precipitin test which has come into use within recent years. The general routine for making the test is as follows: Inject PRECIPITATION TEST 169 (subcutaneously or intravenously) rabbits with ro cc. of filtered defibrinated horse blood (or serum) every other day five or six times. At the end of this time draw blood from the rabbit, allow it to clot kept on ice, remove the serum and filter, where- upon the reagent is ready for use. Express and extract (in saline solution) the juice from the meat suspected to contain horse meat, filter and keep onice until wanted foruse. To the filtrate thus prepared add a few drops of the equinized rabbit serum. If cloudiness and slight whitish precipitate forms it constitutes a positive test, proving conclusively that the suspected meat is horse meat or contains horse meat. Only raw fresh meat re- sponds to this test. Heating destroys the action of the reagent. Inoculating rabbits with the defibrinated and filtered blood serum of various animals, as of hog, domestic fowl, deer, dog, bear, etc., and testing in the manner outlined in the following method by Dr. Karl F. Meyer of the State University of Cali- fornia, the meat of the responding animal may be identified. THE PRECIPITIN TEST FOR THE DETECTION OF HORSE AND DEER MEAT AND FOR MEAT ADULTERATIONS IN GENERAL The method can be used for fresh, dried, frozen, pickled, raw and smoked, but not for boiled, meat. The meat may not be heated above 60°70° C. for the biologic test. For the tests are needed: a. Specific antisera (anti-horse or deer precipitin serum; pre- cipitin). b. Aqueous extract of the meat to be identified (precipitinogen). 1. Antisera.—The sera must be specific and highly active against the meat protein to be determined. Rabbits are in- jected subcutaneously, intravenously or intraperitoneally with serum, defibrinated blood or extract of the fat free meat. The best results are obtained by inoculating fresh serum intravenously. The sera for injection can readily be obtained from abattoirs 170 BACTERIOLOGICAL METHODS or from serum institutes or laboratories. Horse serum is not as toxic to rabbits as are some other sera. Meat extracts should always be filtered to avoid infection of the animals to be im- munized, but extensive sloughing is likely to occur with any method of immunization and the mortality rate is high. The blood or serum used as antigen can be preserved by the addition of chloroform (1—2 per cent.), or by drying. On account of the individual differences existing in rabbits in regard to the development of precipitins, it is advisable to treat at least six animals at the same time. ‘The injections of 2-3 cc. of horse or deer serum are made at intervals of 5 days. Ten days after the last injection the blood is tested for pre- cipitins. The further treatment of the animals differs individu- ally, depending on the precipitin contents of the rabbits. Ani- mals which show a high precipitin reaction are given subsequent inoculations subcutaneously or intraperitoneally, to avoid ana- phylactic death which frequently results from intravenous in- oculations. Some rabbits fail to produce precipitins, whatever the method used. Fornet and Miiller’' recommend the intraperitoneal injection of 5, 10 and 15 cc., respectively, of protein material on the rst, 2d and 3d day, respectively. The test for antibodies is carried out on the 12th day. Gay and Fitzgerald' inject on three consecutive days 1 cc. of the antigen, bleed, and test the serum on the roth day. Both methods frequently give very good results. The precipitin content of an immune serum is occasionally titrated during the process of immunization by withdrawing a few cubic centimeters of blood from an ear vein. The hair over the marginal vein is removed and the skin rubbed with alcohol. A fine pipette is introduced into the vein and the blood collected by capillary attrac- tion or by suction. It is, however, advisable for the beginner to cut the vein transversely and to collect the blood in a centrifugal tube. The hemorrhage is stopped by covering the wound with 1 University of California publications, Pathology, Vol. II, 75, tor2. PRECIPITATION TEST 171 cotton soaked in liq. ferri sesquichloridi (ferric chloride) or by placing a small hemostat for 14 to 1 hr. on the incision. The serum which has separated from the clot is centrifugalized and the titer is determined as follows: Preliminary Titration—Into each of a series of six test-tubes place 2.0 cc. of the following dilutions of serum (horse or deer) antigen, mixed with 0.85 per cent. saline 1:100, I:500, 1:1000, I:5000, 1:10,000 and 1:20,000. To each cubic centimeters of the dilution 0.1 cc. of antiserum is added. The solution of 1:1000 should become turbid instantaneously or within 1 to 2 min., the other dilutions in from 3 to 5 min. The serum should have a titer of 1:20,000; that means the serum should cause a turbidity in a dilution (of horse serum or extract of meat) of 1: 20,000 in less than 5 min. The antiserum is either introduced by allowing it to run down the side of the tube (no shaking is permissible), or it is stratified on the diluted horse serum. In the first case the turbidity appears from the bottom, in the second case in form of a grayish ring; both reactions are positive. The coloration is best seen against a dark background. The pipettes and test- tubes must be perfectly clean and sterile. The equipment de- signed by Uhlenhuth is very satisfactory. The test-tubes are long and narrow, to cm..by 0.3 cm., and are suspended in beveled holes of the test-tube rack. Pipettes of 1 cc. capacity graduated. into Woo cc., and 5 and 10 cc. pipettes graduated into 49 cc. will be found satisfactory. Preservation of Serum.—In case the titer of the serum is satisfactory, the rabbit is bled to death (aseptically) from the carotids. For full details on technique, consult the text-books on Immunity. The centrifuged serum should be perfectly clear and sterile and should not be opalescent. Kept cool and in the dark (ice chest) it will remain potent for months, even years. To avoid opalescence the animal should be bled only after a period of fasting. On account of autoprecipitation, it will lose some of its potency. The precipitate formed can be removed by cen- 172 BACTERIOLOGICAL METHODS trifugalizing or by filtration, but the titer must again be tested. Preservatives such as carbolic acid, etc., should not be added to the sera. Sterile sera are obtained by filtration through Berke- feld filters. Drying of the sera on filter paper is the best method known for preserving them (Jacobsthal und z. Eisler). 2. The Preparation of the Meat Extract——To make the bio- logic test for horse or deer meat, remove from the deeper parts of the specimen, by means of a flamed or boiled knife and through a fresh opening, a piece of muscle of about 30 grams weight. It should contain as little fat as possible. On a sterilized tile (best covered with unused writing paper) chop the meat carefully. The finely minced meat is placed in a sterilized too cc. Erlenmeyer flask and spread out with a sterile glass rod and covered with 50 cc. sterile saline solution. Salted meat is washed for tro min. in a large flask with distilled water, renewing’ the water several times, without shaking the flask. The mixture of saline and meat is kept for about 6 hr. at room temperature, or over night in the refrigerator. To obtain a clear solution the flask should not be shaken. Since the presence of fat interferes with the reaction, it is advisable to remove it by means of ether and chloroform. To make the extraction, take 75-100 grams of the minced meat, place in a large Erlenmeyer flask and cover with equal parts of ether and chloroform. After 24 hr. the ether and chloroform are poured off, the meat is washed once or twice with saline solution and then extracted, as stated above. To determine whether a sufficient quantity of protein sub- stances has passed into solution, place 2 cc. of the extract in a test-tube and shake vigorously. If a fine foam develops and persists for some time, the extraction may be said to be sufficiently complete. The protein solution must be perfectly clear and must therefore be filtered. With extracts from fresh meat this is usually accomplished by filtering through a firm filter paper previously moistened with saline solution. If it is not crystal PRECIPITATION TEST 73 clear, and especially if the meat to be examined was fat or salt, it is filtered through a sterile Berkefeld or through a layer of infusorial earth stratified in a Biichner funnel. The filtrate is suitable for the test when a foam is developed by shaking and when it contains about 1 part of protein in 300 parts of salt solution. ‘To determine this, 2 cc. of the clear filtrate are placed in a test-tube and heated, and a drop of dilute nitric acid (sp. gr. 1.153) is added; if a marked cloudiness and a flocculent precipitate forms, the extract is too highly concentrated and must be diluted with normal salt solution until the heat and acid test causes only a diffuse, opalescent cloudiness which settles to the bottom of the tube after 5 min. as a slight precipitate. Before proceeding with the test, the reaction of the meat ex- tract should be tested with litmus paper and if it is found to be acid it should be neutralized very carefully with o.1 per cent. sodium hydroxide or magnesium oxide solution. Only slightly acid or alkaline solutions should be used. For the extraction of the meat, spigot, tap or distilled water should not be used. Fresh meat frequently produces a sufficiently strong protein solution in rt hr. In boiled, preserved and decomposed meat, the ex- traction proceeds very slowly (24 hr.) and the solutions are difficult to clarify. Technique of the Test—If, for example, the object is to determine whether a piece of meat is horse flesh or, if sausage, contains the meat of this animal, the test is conducted as follows: Tube 1.—2 cc. of unknown extract (1: 300) + o.1 cc. of anti-horse serum. Tube 2.—2 cc. of unknown extract (1: 300) ++ 0.1 cc. of normal rabbit serum. Tube 3.—2 cc. of horse flesh extract (1: 300) 4- 0.1 cc. of anti-horse serum. Tube 4.—2 cc. of pork extract (1: 300) + 0.1 cc. of anti-horse serum. Tube 5.—2 cc. of beef extract (1: 300) + 0.1 cc. of anti-horse serum. Tube 6.—2 cc. of saline solution + o.1 cc. of anti-horse serum. The immune serum is added to each tube very carefully and run down the sides of the tube, or stratified. The tubes must not be shaken. The tubes are kept at room temperature. The 174 BACTERIOLOGICAL METHODS test must not be made with a mixture of the sera of different rabbits. Inter pretation of the Results —If in tubes 1 and 3 a misty cloudi- ness should appear within 5 min., and if a definite precipitate forms within 30 min., the other tubes remaining perfectly clear, the extract is very probably one of horse flesh or the flesh of some other single-toed animal. Precipitates which develop more slowly cannot be considered as positive. The protein of horses and donkeys cannot be differentiated by this test. In a similar man- ner, tests may be made for the meat of deer, dogs or any other animals, if the respective immune sera are used with the extract. Frc. 57.—Types of syringes: 1, Roux’s bacteriologic syringe; 2, Koch syringe; 3, Meyer’s bacteriologic syringe. The Meyer syringe is the simplest and best for general purposes.—(McF arland.) Heterologous precipitates, which occur when antisera are added to concentrated foreign protein solutions, rarely are disturbing factors of the tests when the above technique is used. The elective absorption (according to Kister and Weichardt) with the foreign protein is occasionally necessary for scientific tests. The organoleptic tests are not always conclusive as to the quality of the meat. It is a well-known fact that the stinking or putrefactive odors are generally wholly absent in even highly decayed salted and brine-pickled fish and meats and in heavily seasoned sausage meats and in smoked meats. On the other MEAT BACTERIA 175 hand, it is advisable to reject or condemn all meats which emit offensive odors, provided such odors are not normal to the meat. Under normal offensive odors may be mentioned the fishy odor of meats from animals which feed upon fish, mussels and other aquatic animals; the sex odor which is often marked in the meats from older males; the various vegetable odors due to feeding, such as the turnip odor and taste in beef, fenugreek odor, etc., etc. Distinctively putrefactive odors in meats are a very reliable indication of their unfitness for consumption. Marked changes in consistency (sloppy, smeary and porous meats) and in color (grayish, yellowish, greenish) usually indicate advanced stages of decomposition. Some authorities have recommended that the presence of free ammonia should be the test for putrefactive changes in meats and should serve as the basis for condemnation procedures, but others point out the fact that toxins are formed even before there is any appreciable formation of ammonia. The safest guide to the quality of meats is undoubtedly the bacterio- logical test. As to the question on what bacteriological findings shall the quality estimates of meat be based, it is suggested that judgment be based upon the number of bacteria present and generally irrespective of kind. If exposed and comminuted meats do not contain more than 1,000,000 bacteria per gram, they may be presumed to be reasonably wholesome. The exceptions to this numerical limit are the finding of pathogenic and toxin-forming bacteria. The conclusive proof of the mere presence in meats of bacteria which are pathogenic to man is sufficient to condemn such meats. It is reasonable to assume that most bacterial in- vasions of meats are of the putrefactive kind and hence objection- able, and it is therefore fair and just to all concerned to fix a nu- merical limit at which such foods are still reasonably wholesome, as suggested. There are, however, those notable exceptions where meat contains toxins and ptomaines in quantities sufficient to produce serious and even fatal poisoning without bacteria being present, as when fresh meat has been in contact with decomposed 176 BACTERIOLOGICAL METHODS ' and toxin-bearing meats from which it has taken up the poisons by absorption. It is therefore desirable and often necessary to supplement the bacterial count by the toxicity test. The numerical limit above suggested (1,000,000 per gram of the meat substance) pertains to bacteria found upon the ex- terior of the meat bulk or in the outside cells and tissues of the meat bulk or meat particles. Proper care must therefore be observed in taking samples and in preparing the sample Miah for plating. In the case of i of Ay WE bulk meats such as whcle WB eS slaughtered animals, hams, NN, ag bacon, etc., pieces as nearly cubical as possible (about 1 gram each) are removed with a sharp sterilized scalpel, the outer surface of the meat forming one face of the cube. This is to be weighed and WW LOGGER. ulped in a sterile mortar Ave WELZ rit an equal amount of rs sterile normal salt solution Fic. 58.—TIllustrating the method of making an intravenous injection into a and this pulped material is rabbit. The ear is manipulated to induce then made up to the desired hyperemia and the surface vein is com- pressed near the base of the ear, to facilitate dilutions for plating, using the inserting of the syringe needle.— . (UcFarland.) normal salt solution. Gela- tin media should be used for culturing and incubation should be done at 20° C. for a period of 3 days and the counts made. In the case of sausage meats and comminuted meats generally, take 1 gram quantities, pulp thoroughly and mix thoroughly with the required amount of nor- mal saline and plate. In the case of soups and soup stocks hay- ing a meat or meat derivative base, take 1 cc. quantities, from the thoroughly mixed sample, dilute and plate. SSA kK LEZ TOXINS IN MEAT 177 Weinzirl and Newton describe a method of determining the bacterial content of meat, in which the meat is ground in a mortar with sterile sand and normal salt solution to obtain an emul- sion for inoculation into the culture media, and report the appli- cation of this method to the determination of the bacterial con- tent of a number of samples of market Hamburger steak. The result showed that the standard of 1,000,000 bacteria per gram advocated as a maximum limit for the salable product is much too low, as nearly all the samples examined would be condemned on this basis, though showing no taint or other evidences of putrefaction. The authors propose a limit of 10,000,000 bacteria per gram. For making toxicity tests of meats, broths, sausage meats, soup stocks and other meat products, the following general method is recommended. In case of solids such as meats (raw, . smoked, cooked, canned or pickled), sausages, sausage meats, etc., 1o grams of a well-mixed average sample are well pulped in 10 cc. of boiled distilled water. Let stand for 20 min. with frequent stirring. Express and filter the extract through a clay bougie. The toxins being soluble will be found in the filtrate. Inject 2 cc. of the clear filtrate into the subdermal connective tissue or intraperitoneally into guinea-pigs or white mice, using three animals for each test. If one or more of the animals thus inoculated die within 48 hr., or if they show marked symptoms of intoxication without dying, the meat is unfit for consumption. In the case of soups, broths, soup stocks, chop suey and other meat products which contain liquid, the procedure is much simpler. Take suitable quantities of the thoroughly mixed sample and filter, first through filter paper and finally through the clay bougie, as for the meat extract already described, and inject 2 cc. quantities as already explained. The toxicity tests should in all cases be supplemented by the plate count. Botulism or sausage poisoning is due to a toxin (botulin) formed by the Bacillus botulinus (Lat., botulus, a sausage), a 178 BACTERIOLOGICAL METHODS large anaerobic sporogenous saprophyte especially common in sausages and sausage meats, particularly in liver sausages, blood sausages, jelly sausages, in hams, in liver pate, canned meats, etc., etc. The bacillus, inclusive of the spores and the highly virulent toxins which it forms, are destroyed by boiling and thorough cooking. The digestive ferments do not destroy the toxin. The usual smoking of hams and sausages does not de- stroy the toxin or the bacillus. The bacillus is killed by strong brines, but this does not also destroy the toxin. ‘The oval spores are quite readily killed by heat and chemicals. Heating to 80° C. for t hr. kills them. Ichthyotoxism (fish poisoning) and mytilotoxism (shellfish poisoning) are closely akin to botulism and are in all probability caused by the same bacillus or perhaps a varietal form of B. botulinus. The occurrence of the Bacillus . botulinus is, however, not limited to pork and sausage meats. Well-authenticated cases are on record of the occurrence of this bacillus in canned vegetables and in domestically prepared string — beans served without previous heating. There is no doubt that the heat employed in the canning process destroys the toxin formed, but the temperature may not always be high enough to kill all of the bacilli and their spores even though the spores are not very resistant to heat (80° C.). Bacillus botulinus does not multiply in the living organism. It grows readily in slightly alkaline media at a temperature of 18° to 25° C. At higher tem- peratures (35° to 37° C.) it grows only sparingly and without the formation of toxin. Cultures give out an odor of butyric acid. In pickled, canned and otherwise prepared and preserved meats, and mixtures of meat and vegetables (chop suey, pork and beans, etc.), the processes of bacterial development are greatly modified. The use of deodorants, of preservatives and color- ing agents mask or obscure many of the decomposition changes in meats. Very frequently the only cause for suspicion is an unusually heightened color or a lack of the normal meat flavor. MEAT BACTERIA 179 Sausage meats are found on the market so highly colored as to produce a red ink with the water in which they are boiled. The meat dealer tries to deceive the housewife by stating that the red color is derived from the rich red blood of the meat itself, whereas the red coloring matter of the blood is decomposed by the boiling and the boiled meat extract is only slightly colored. Very frequently pickled pigs’ feet appear on the market which look quite normal, the only suspicious character being an unusual pallor of the surface with a smeary consistency and a lack in the flavor. On microscopical examination it will be found that the surface of the meat is covered or coated with yeast cells, mold hyphz and mold spores and bacteria. The American method of making sausage and sausage meats from carelessly and pro- miscuously handled meat trimmings which accumulate during the day’s work in the retail meat markets, is accountable for the high contamination with bacteria and other organisms (10,000,000 to 100,000,000 per gram). Such sausage meats are also very frequently colored to reduce the pallor due to the use of ex- cessive amounts of fatty tissue trimmings, thus leading the cus- tomer to believe that there is a considerable amount of muscular (red. meat) tissue present. The coloring also serves to hide the beginnings of decomposition changes in the meat. Preservatives are added to check and mask the decomposition changes which have begun to manifest themselves. It is unlawful to add coloring substances to sausage meats, but it is permissible to color sausage casings. Numerous chemical tests for ascertaining the existence of putrefactive changes in meats have been recommended. The Ebers test appears to have met with considerable favor and is made as follows: Into a test-tube pour about 3 cc. of a mixture composed of 1 part of pure hydrochloric acid, 1 part ether and 3 parts alcohol. This tube may be closed with a perforated rubber stopper carrying a glass rod which is pushed through the opening of the stopper so that the end almost touches the 13 180 BACTERIOLOGICAL METHODS liquid in the tube. Dip the free end of the tube into the meat pulp, meat extract or meat broth and, after shaking the tube in order to fill it with the acid vapors, insert the rod, closing the tube with the rubber stopper. If the juice or the meat particle is from decayed meat, a grayish smoky vapor appears at the end of the glass rod, which settles to the surface of the liquid. There must be no free ammonia in the room while making the test. The test is not applicable to pickled meats. ‘Dhisigteas should be made supplementary to the microscopical, bacterio- logical and toxicological exami- nations already explained. In place of the test-tube or reagent glass above recom- mended, the small perfume sample bottles with glass rod stoppers may be used in mak- ing the test. Fic. 59.—Bacillus tetani as seen ina Sausage meat binders or scraping from a wound. Some of the filers are very readily detected organisms show spore formation while others do not. The pale globules are by means of the compound eae eS en microscope. Corn starch and wheat starch fillers are most commonly employed, the object in adding them being to increase the water content of the sausage meat. Some brands of sausage contain corn meal and other cereal products. Egg albumen and tragacanth fillers are used occasionally, and it is said that it is possible to increase the water content of the meats by 30 per cent. with only 3 per cent. of the tragacanth filler. The increase in water content through the use of the starch fillers is about 5 to 10 per cent. In examining meats for starch fillers or added cereal it must not be forgotten that some of the spices used contain starch (pepper, allspice). CEREAL IN SAUSAGE MEAT I8I Graham, of the laboratory division of the Bureau of Animal Industry, has recommended a method for determining the per- centage of starch added to sausages and sausage meats. A small pellet of a thoroughly mixed sample of the meat preparation is well pulped and teased out. Make the usual slide mount, using just enough of the prepared material to fill the space be- tween slide and cover, using some pressure. Count the number of starch granules in the areas (squares) of the ocular scale and compare with the known number of similar starch granules in I, 2, 3 and 4 per cent. mixtures of the same starch. Rarely does the amount of starch filler added exceed 3 or 4 per cent. Mr. Graham states that the method gives results accurate within 10 per cent., which is sufficiently accurate for all practical purposes. It is suggested that the special spore and mold counter de- scribed elsewhere (A or B, Fig. 5) be used with the ocular counting scale (Whipple’s) for making the starch determinations in sausage meats. The exact number of starch granules in mixtures con- taining 1 per cent. of starch should be carefully ascertained, follow- ing the general method recommended for finding the number of oil globules representing 1 per cent. of butter fat in milk. For determining the number of granules in 1 per cent. suspensions of the starch, it is suggested that weak solutions of gum arabic (1 per cent.) be used. The gum solution keeps the meat particles as well as the starch granules in suspension until the counting is com- pleted. Having once determined the exact number of granules in rt per cent. of the starch suspension, it is a simple matter to make comparative determinations of homologous starch in sausage meats, or in other substances, as may be required. Add 1 gram of a well-mixed sample of the sausage or sausage meat to about 2 cc. of water in a suitable dish and mix thor- oughly, in order to wash the starch from the meat particles. Next add enough of the gum arabic solution to make a total of 9 cc. of the liquid, thus making a dilution of 1-10. Mix thoroughly in order that the starch present in the meat may be uniformly 182 BACTERIOLOGICAL METHODS distributed and make the counts as for spores or yeast cells, and from the findings determine the percentage of starch which has been added. This quantitative method for determining added starch is applicable even if the starch has been dextrinized through the cooking of the sausages, provided the individual granules are still recognizable and provided also the identity of the starch is still ascertainable. Corn meal and corn starch are the more common sausage fillers used in the United States. The above method for determining the percentages of starch in mixtures could also be employed, modified to suit special cases, in the examination of compounds of flour, of meals, for ascertain- ing the percentage of starch in baking powders, in almond meal, in adulterated mustard and in other products where starch or flour is used for purposes of adulteration, and to ascertain the pro- portions in flour or meal compounds, etc. In frozen meats the red blood corpuscles are almost com- pletely decolorized and disintegrated (hemolyzed), changes which are readily observed under the compound microscope. The microscope will also prove useful in the detection of added coloring substances. The micro-sublimation test will readily demonstrate the presence of benzoic and salicylic acids in meats and meat products. The microphytic examinations of meat include the following groups of the plant kingdom: 1. Penicillium Species.—Especially common on hams, bacon and smoked meats generally. These molds are essentially aerobic saprophytes and are therefore found on the exterior of meats. 2. Aspergillus Species.—These molds are apt to occur on and in fish meats, in gelatin, in canned meats and in pickled meats. 3. Mucor Species.—These small molds are less common than the above. They may occur on pickled meats and on meats that are kept in damp places. 4. Yeasts.—Yeast cells may occur on pickled meats and, MEAT BACTERIA 183 more especially, in meats and meat products which contain starch and sugar. 5. Bacteria.—It is not necessary to enter into any extensive discussion of the different species and varieties of bacteria which may occur in and upon meats. The more important bacterial invasions of meats have already been mentioned. ‘The following is a partial list of the more important species which the food bac- teriologist may be called upon to look for in meats: a. Bacillus botulinus——-Most common in sausages, as already Fic. 60.-—B. tetani, showing flagelle. stated elsewhere. Forms highly virulent toxins and produces rancid changes. b. Bacillus tuberculosis —Will be found in meats of tuber- culous animals. c. Bacillus tetani—May occur in meat products, more es- pecially in gelatin. It is essentially anaerobic but thrives better in association with aerobes, and it produces one of the most virulent toxins known which is, however, very unstable in its chemical composition and easily destroyed. A temperature of 60° to 65° C. destroys it and it is also very quickly destroyed on exposure to air and light. The danger from the tetanus bacillus pertains to possible inoculation with the bacillus rather than the 184 BACTERIOLOGICAL METHODS ingestion of the toxins, which might be formed outside of the body and absorbed by the meat. d. Cadaver bacilli—Under this head are included a: variety Fic. 61.—Tetanus bacillus stab culture in glucose-gelatin 6 days old—(McFarland, after Fraenkel and Peiffer.) of bacteria which cause putrefactive changes in dead animals and in meats, with toxin and ptomaine formation, and to which reference has already been made. e. Bacillus anthracis —The anthrax bacillus may occur in all food-produc- ing animals, and its isolation from beef and other meats may become an occasional necessity in the food labo- ratory. f. Staphylococcus group—These may occur in great abundance in liv- ing animals, causing septic decomposi- tion changes in tissues and organs. g. Streptococcus group.—tLike the Staphylococci, these organisms pro- duce pyemic or septic changes in liy- ing animals. h. Numerous other bacteria may on occasion come to the attention of the food bacteriologist, as the bacillus of hog cholera, of swine plague, of swine erysipelas and others. In this connection we must not forget the possible presence in beef, and less fre- quently also in pork, sheep and horses, of the ray fungus (A ctin- omyces bovis) which is the primary cause of “lumpy jaw” in cattle and which disease is transmissible to man. Examination of meats for the presence of encysted trichine (Trichinella spiralis) is incidental rather than a routine in the TRICHIN 185 food laboratory. Even if the meat is found to contain trichine it does not warrant condemnation procedures, because these organ- isms are harmless provided the meat is properly cooked be- fore eating; however, it cannot be denied that no consumer could be persuaded to use meat thus infected. The examination of pork for the presence of encysted trichine was at one time a regular routine in the larger slaughtering houses of America be- cause of the European (largely German) boycott against Ameri- Fic. 62.—Actinomyces bovis from broth culture ( 1000).—(Williams.) can pork. In recent years the routine examination for trichine has been very generally abandoned. Trichine are not uniformly distributed in the muscular tissue of the animal. They are most abundant in the diaphragm, next in the base of the tongue, in the laryngeal, lumbar, mas- ticatory, and abdominal muscles and nearest the tendinous insertions of the bones. They are never found in adipose tis- sue. They may occur in wild hogs, in dogs and in bears and of course also in man. To examine meat for trichine, cut bits 186 BACTERIOLOGICAL METHODS from the organs of chief distribution of the parasite. From these samples cut small flat pieces and compress between two Fic. 63.—Colony of Actinomyces bovis from cow.—(Williams.) glass slips and examine under the low power of the compound microscope. As a clearing agent a solution of acetic acid (1-30) ro SUEM Ie , UAT cca it aa ety THM z Sea a eS NTO N Scie ak ou FRY UTUMAA (TANCE Leavtaiagce ts el - Ae AUDACITY Mirra Cr a2 SHANTI ae ee oy MT 7 Tt are twin i Yn oR i a LOW cae AG aes } nc tat Cy! Be, CN Reuters ey TANT . Maer iver i Hint attri ren ait i qeuenst se TU AUC attou arctan - AINA ua Fic. 64.—Encysted Trichina spiralis (Trichinella spiralis) in muscle tissue.— (Stitt, after Ziegler.) may be used. To clear sections of salted hams or other meat, use diluted potassium or sodium hydrate. Examining minced EGGS 187 meats and sausages for trichine requires greater care and persistency. Encysted trichine retain their vitality for a long period of time when kept at a low temperature, and persist even after the meat has undergone decomposition through bacterial infec- tion. The wandering embryos are harmless and the muscle trichine continue their development only in another host, as man, dog or bear. In the intestinal tract of this second host they become sexually matured, growing to a length of 0.5 to 0.75 mm., and produce young in large numbers. ‘Trichinella does not produce ova. The inexperienced analyst might mistake vinegar eels (in pickled meats), Miescher’s bodies (Sarcocystis), lime concretions, muscle degenerations and trichine-like worms (Pseudo-trichine), found in the muscles of the rat, mouse, rabbit, fowl, fish, mole and other animals, for trichine. 18. The Bacteriological Examination of Eggs and Egg Products ° Among the foods which require the attention of the bac- teriologist are eggs and egg products such as evaporated eggs, frozen eggs and dried egg albumen. Many fresh eggs are quite free from bacteria, or if bacteria are present they do not exceed negligible quantities, usually not over 500,000 per cc. Ex- tensive investigations made by Stiles (Bureau of Chemistry) show that the contamination of eggs is in proportion to age and favorable temperature. Thus during warm weather the bacterial development is quite rapid, whereas cold retards such develop- ment. Placing contaminated eggs in cold storage checks bacterial development temporarily and even causes a reduction in the number of organisms present at the time the eggs were placed in storage, but within a short time the temporary numerical reduction in bacteria is not only regained but there is a steady 188 BACTERIOLOGICAL METHODS increase in proportion to the time of storage, until a maximum development is reached. The bacterial flora of the white and of the yolk of the egg differs quantitatively as well as qualita- tively. It may happen that the yolk is badly infected while the white is in comparatively good condition. As a rule, how- ever, if the yolk is highly contaminated the white is similarly affected. In fact the first decomposition changes generally take place in the periphery of the egg albumen, the infection taking place via the exterior of the shell. Commercially, eggs are designated as fresh, stale, storage; firsts, seconds and thirds (when sorted as to size); watery and weak when the white is thin; heat eggs; leakers, checks and mashed when the shell is more or less broken; eggy, strong, musty, sour and stale as to odor; blood ring, sour rot, white rot, light rot, spot rots, moldy, black rots, etc., when more or less rotted and decomposed; green or grass eggs when the white is more or less green colored through the invasion of bacteria. These terms have no scientific importance and are of no signifi- cance to the food bacteriologist, beyond that of indicating the probable or likely condition and contamination and_ probable cause of the change or deterioration of the eggs so designated. The old-time popular methods of testing eggs by candling, by shaking to determine “looseness,” floating on brine, noting discoloration of the shell, and by the odor, have their value in practice but are far from reliable. An egg which gives off the odor of sulphuretted hydrogen is universally recognized as bad, rotten or spoiled. In Germany eggs are pronounced spoiled if the white is gelatinous in consistency (as in old eggs from which moisture has escaped) or yellowish in color (also due to age), or if the yolk is more or less adherent to the shell or is more or less mixed with the white. A fresh egg broken in the manner customary in the kitchen allows the entire contents, yolk and all, to fall out into a receptacle without rupturing the yolk. The white should be of uniform consistency, uniformly trans- EGGS 189 lucent and without marked yellowish or amber coloration. The yolk should be uniformly soft and entirely free from all lumpiness and should not be adherent to the shell. Eggs are preferably used in the comparatively fresh state, that is, within a few days or at the longest 8 days after they are laid. It is, however, not always possible or practicable to use the eggs while still fresh, and egg preservation has become a very important industry. Eggs may be preserved in brine, in liquid glass and in various chemical preservatives. They may also be preserved in oil, in lard, or coated with tallow, wax or paraffin,