. fflSM-v;-:'. 9 >>"»JU « t ■'av V cV' .y BACTERIOLOGICAL METHODS SCHNEIDER BY THE SAME AUTHOR PHARMACEUTICAL BACTERIOLOGY 80 Illustrations Octavo 246 Pages Cloth, $2.00 Post Paid "The discussion of disinfectants and of the principles of disinfection and sterihzation and of the practical application of these principles in the pharmacy would alone make the book well worth while to every phar- macist." — Jnl. Amer. Pharmaceutical Ass'n. Bacteriological Methods IN Food and Drugs Laboratories WITH AN introduction to Micro-analytical Methods BY ALBERT SCHNEIDER, M. D., Ph. D. (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 P. BLAKISTON'S SON & CO. 1012 WALNUT STREET Copyright, 1915, by P. Blakiston's Son & Co. ^^^ THE SIAPLE PHESS YOEK PA nni 29 1915 ^CI,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 cooperative manner. With such cooperation 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 iriicro-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. CONTENTS I. Outline of Micro-analytical Methods in Food and Drugs Laboratories Page 1. Introduction i 2. Grouping of Substances to be Examined in Food and Drugs Laboratories . . i 3. The Work of the Micro-analyst in Relationship to that of the Chemist and Bacteriologist 3 4. Equipment for Micro-analytical Work 4 5. Organoleptic Testing 8 6. Methods Useful in the Examination of Vegetable Drugs, Spices, Etc ... 9 7. Methods Useful in the Examination of Vegetable Food Products .... 11 8. Micro-chemical Color Reaction Tests 15 9. Making Analytical Reports 17 10. The More Important Histological Elements of Plants iS II. Bacteriological Methods in Food and Drugs Laboratories 1. Introduction 23 2. Direct Bacteriological Examinations 51 3. Numerical Limits of Micro-organisms in Foods and Drugs 53 4. Quantitative Estimations by the Cultural Methods 68 5. Preparation of Standard Culture Media. General Suggestions 71 6. Preparation of Required Standard Culture Media 75 7. Technique for Making Quantitative and Qualitative Estimations by the Plating Methods 83 8. Practical Application of the Quantitative Estimations by the Plating Methods 89 9. Qualitative Determinations 90 10. Evidence of Sewage Contamination 97 11. Possible Contamination of Foods with the Tj^hoid BacUlus 102 12. Possible Contamination of Food Substances with the Cholera Bacillus . . . 112 13. Biological Water Analysis 114 14. Bacteriological Examination of Mineral Waters 118 15. The Microscopical and Bacteriological Examination of Milk 120 16. The Bacteriological Examination of Shellfish 144 17. The Bacteriological and Toxicological Examination of Meats and Meat Products 152 vii Vlll CONTENTS Page i8. The Bacteriological Examination of Eggs and Egg Products 187 19. The Bacteriological Examination of Pharmaceutical Preparations .... 197 20. The Microscopical and Bacteriological Examination of Syrups 202 21. The Microscopical and Bacteriological Examination of Fermented Foods and Beverages 210 22. Standardization of Disinfectants , . . . . 230 23. Determining the Puritj' and Quality of Sera, Bacterins and Related Products 265 24. Special Biological and Toxicological Tests 268 I OUTLINE OF MICRO-ANALYTICAL METHODS IN FOOD AND DRUGS LABORATORIES I. Introduction The value of the compound microscope in the examination of foods and drugs is as yet not generally recognized. EflEiciency 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: 2 MICRO-ANALYTICAL METHODS 1. Vegetable drugs, crude and powdered, pharmacopoeial and other simple and compound medicinal powders. 2. Spices and condiments, whole, ground and powdered. Prepared spices and condiments. 3. Coffee, tea, cocoa, chocolate, confections, candies. 4. Tobacco and preparations made from tobacco, as snuff, smoking tobacco, cigars, etc. 5. Chemicals, minerals, solutions of chemicals, etc. 6. Tablets, pills, powders. 7. Meats of all kinds, raw, cooked, canned, sausage meats, etc. 8. Dairying products, as milk, cream, cheese, butter, ice-cream, ice cream fillers, etc. 9. Insect powders, dusting powders, cosmetics. 10. Cattle and poultry powders. 11. Unknown powders, wholly or partly of vegetable origin. 12. Starches, dextrins, sausage meat binders (starches). 13. Vegetable foods, as jams and jellies; fresh, pickled, cooked, 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. r8. 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 e.xaminations are of equal 4 MICRO-ANALYTICAL METHODS importance — assaj-able 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 40 per cent, of silica. An example like this also indicates why the micro-analyst should make the sand and ash determinations. The percentage estimates based upon microscopical examination may vary within 25 to 50 per cent, when the amounts of the admixtures are small or slight. For example, the actual amount of arrow-root starch in the so-called arrowroot biscuit is 2.5 per cent. The micro-analyst's estimates may range from a trace or small amount to 5 per cent. When the quantities of admixtures are large, from 30 to 90 per cent., the estimations may approximate within 10 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. b. Oculars, Nos. 2 and 4. c. Objectives, Nos. 3, 5 and 7. d. 1/12 in. oU-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 tools 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 Fig. I. — 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 analj'zer takes the place of the ocular. — {Baiisch &" Lomb Co.) quite different when it comes to the examination of powdered vegetable drugs, compound vegetable powders and vegetable products of unknown composition, A thorough knowledge of. 8 MICRO-ANALYTICAL METHODS and a wide familiarity with, cell-forms, tissue elements and formed cell contents is an absolute essential in order that accurately re- liable and conclusive results may be obtained and serious con- fusion may be avoided. Differences in the reports of findings by micro-analysts are in part due to the personal equation, in part due to variable methods and differences of judgment in estimat- ing the quantity of tissue elements present and also in part due to a lack of extensive and intensive experience. 5. Organoleptic Testing The organoleptic tests are indeed valuable adjuncts to the micro- scopical work. There are, however, some differences of opinion regarding the interpretation and valuation which ai e 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 fairl)-^ 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 lo 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 i 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. LigninTest. — 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. lO MICRO-ANALYTICAL METHODS 5. Beaker Sand Test. — Pour a definite amount of the powdered spice or vegetable drug into a beaker, add water, stir until the sand is washed away from the vegetable particles and settles to the bottom of the beaker. Let a stream of water run into beaker so as to wash out the vegetable matter. The final washing and decanting must be done carefully so as not to lose the sand. Salt brine may be used instead of water, should the vegetable matter have a comparatively high specific gravity. Dry the sand and weigh to obtain the percentage of sand present. 6. Ash Determination. — According to the regulation method. The percentage of the acid-insoluble residue should also be de- termined. It should be borne in mind that the ash determination gives only approximate results as far as the presence of clay and dirt is concerned, since the organic matter of dirt is combustible. The ash percentage varies greatly in vegetable drugs, especially in herbs and leaves. The sand percentage is comparatively high in those herbs and leaves having abundant trichomes, especially if the drug plants (or herbaceous spices) bearing such trichomes are grown in dry sandy soil. Dirt (and sand) percentage is apt to be high in roots and rhizomes, particularly when rootlets are abundant and when the gathering, garbling and cleaning is carelessly done. There are a number of chemical tests giving color reactions which can be done conveniently by the micro-analyst, as the boric acid reaction with curcuma, the H2SO4 color reaction with some barks, capsicum, guaiac, resin, cubeb, etc.; the H2SO4 plus for- maldehyde color reaction with morphine; the ferric chloride color reaction with salicylic acid, etc. These tests should be used when, in the judgment of the analyst, they may serve to give better information regarding the identity, purity and quality of the drug. SPECIAL TESTS II 7. Methods Useful in the Examination of Vegetable Food Products 1. Sublimation Test for Benzoic Acid.^ — Place a drop or two of the suspected liquid or semi-liquid food substance into a deep watch crystal of i 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.oi 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 i : looo. 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. Curcimia Thread Test for Boric Acid.^Boil 5 grams of powdered curcuma in 10 cc. of alcohol. To the evaporated alco- holic extract add a little soda and several cc. of 50 per cent, alcohol. In this place paper (bast fiber), cotton or linen threads and bring to a brisk boil for a few moments. Remove threads and dry between blotting paper, lay them in a very weak solu- tion of sulphuric acid and rinse in water. When dry the threads should be a golden yellow. ^ 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 lo 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 lo to 13 per cent, solution of sodium carbonate, followed by a droplet of the suspected solution. In the presence of boric acid, the thread is colored blue, which coloration remains for a longer or shorter period and then changes to gray and violet. The test is a very delicate one and is not hindered by the presence of sodium chloride, magnesium sulphate and aluminium sulphate. Strong solutions of phosphoric acid, silicic acid, calcium chlorite and magnesium chlorite, interfere with the reaction more or less. 4. Formaldehyde Test. — Concentrated hydrochloric acid added to weak solutions of formaldehyde (i : 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 I3 limited to the non-cellular portions of such substances as jams and jellies, indicating the use of fruit juices obtained from unripe fruits. 7. Microscopical Examination of Bacteria and Metals by Direct Sunlight.^ — Very minute quantities of certain minerals as iron, copper, mercury, and a few others, can be detected in liquids and semiliquids (in the form of metallic hydroxides) when examined (on slide mounts) by means of direct sunlight. All transmissible light must be cut off. Direct sunlight can also be used in making bacterial counts in liquids, using the Thoma-Zeiss hemacytometer (Turck ruling). The bacteria are readily recognizable on the dark background, standing out far more clearly than in the usual examination by transmitted light, because of the more pronounced color contrasts. 8. Micro-gluten Test.- — Mount a bit of the flour in water on a slide, being careful not to use too much water. Cover with cover glass and move cover glass to and fro a few times on the mounted material. The gluten separates into stringy fragments which may readily be seen under the low power of the compound microscope. The use of a weak solution of carbol-fuchsin, sofranin, or other stain, will bring out the gluten particles more clearly. 9. Hand Gluten Test. — Moisten wheat flour with water, making it into a dough. Knead constantly and carefully under a slow ^ 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 0.003/x, however, solid particles (as of metallic colloids) of not more than Q.003/X in diameter show no structure, they appear rather as points of light. The limits of vision with the ordinary microscope are, for air (white light) about 0.30/1, for homogeneous immersion (white light) about 0.25/u, and for homogeneous immersion when rays of shorter wave length than white light (as the blue spectrum) are used, are about o.i5;u. 14 MICRO-ANALYTICAL METHODS stream of water, washing out all of the starch. The gluten sepa- rates out as a tenacious 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. lo. 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 lo grams of the substance in 200 cc. of distilled water and centrifugalize (while still hot) for half an hour. Decant off the supernatant liquid and examine the residue microscopically. If agar has been added, characteristic agar diatoms (mostly Arachnodiscus ehren- 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 1 5 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 metalHc 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 IS 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 i 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 o.ooS 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 1 6 MICRO-ANALYTICAL METHODS acid, ammonia and ammonium sulphide by bringing a drop of the reagent in contact with the thread upon the slide. (See also Biological Test for Arsenic in Part II.) b. Zinc Test. — Dip cotton fibers into the suspected solution, allow the moisture to evaporate, and then dip the threads into a solution of gold chloride. A violet coloration develops which remains in the presence of acids but vanishes in the presence of chlorine water, indicating the presence of zinc chlorite. The reaction is appreciable in the presence of 0.003 jug of zinc chlorite, whereas in the form' of the sulphite, 0.1 ng 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. /. 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 (i : 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- SPECIAL TESTS 17 mine give the copper ferroc3'anide 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 combination valency Reaction Limit (mg. X io«) Comparative sensitiveness Bo"'. As'". Sb'". Sn"., Au'" Pt"" Cu". Hg'. Hg". Pb".. Bi'". Cd". Fe". Co".. Ni".. Curcuma thread o . i Sulphide thread | 10. o Sulphide thread i . o Violet color with sulphide thread 3.0 Sulphide thread — brown, purple. . i 3.0 Sulphide thread 8.0 Sulphide thread + ferrocyanides. 8.0 Sulphide thread + Ag j 5.0 NH3 vapor 8.0 Sulphide thread 5.0 Sulphide + PbCr04 ' 8.0 Sulphide + chromate + Bi 8.0 (NH3SH) vapor 6.0 (NH3SH) — blue i 8.0 NH3SH or nitroso — beta — naph- thol I 0.3 NH3SH , 0.3 m 33,000 in 2,500 m 40,000 m 20,000 m 22,000 m 6,000 m 4,000 in 22,000 in 25,000 in 20,000 in 13,000 in 9,000 in 9,000 in 3,500 in 100,000 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 ^ 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 i gram of the substance in solution is still appreciable. The actual limit, determined experimentally, is indicated in terms of milligrams, that is 0.00 1 mg., represented by fig. Expressing the com- parative sensitiveness (CS) in a formula we have CS = Hg limit X molecular weights amount limit combination valency or to give the example for boron, we have CS o.ooooi 0.00000006 X _ = 33,000. l8 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 No (I. S., laboratory or other serial number). Label Sample received. Sample examined Condition of wrappings and seals Organoleptic tests Consistency of feel Color Odor Taste Adjunct tests Sand (beaker test) Per cent. Ash Per cent. Acid-insoluble ash Per cent. Special tests Microscopical findings. Conclusions. . Analyst. ANALYTICAL REPORTS I9 Form No. II (No., label, dates, condition of seal and organoleptic tests, as for form I.) Adjunct tests. Sublimation tests for Benzoic acid Salicylic acid Boric acid (curcuma thread) Iodine reaction Intracellular Extracellular Special tests Microscopical findings. General Cytometric counts. Dead yeast cells per cc. Living yeast cells per cc. Bacteria per cc. Mold (hyphal fragments and hj^phal clusters) . . . per cc. Mold spores per cc. Conclusions . Analyst. 20 ' MICEO-ANALYTICAL METHODS Form No. Ill Bacteriological Examination (No., label, dates, condition of seals as for form I.) I. Direct count. (Thoma-Zeiss hemacytometer with Turck ruling.) 1. Bacilli per cc 2. Cocci per cc II. Plate and tube cultures. (Lactose-litmus-agar.) 1. Temperature differential test. a. (20° C.) colonies per cc b. (38° C.) colonies per cc 2. Color differential test. a. Pink or yellow colonies per cc b. Not pink or yellow colonies per cc 3. Gelatin liquefying colonies per cc 4 . Indol reaction (±) 5. Neutral red reduction ( + ) 6. Gas (hydrogen) formula 7. Gram stain behavior ( + ) 8. Presumptive colon bacillus test ( + ). a. Amounts used b. Number of tests c. Rating III. Special tests IV. Conclusions . Analyst. ANALYTICAL REPORTS 21 We may give an example of a report as follows: Form No. II Lab. No. 462. Label: Pure currant jelly. Made by Smith, Jones b'Co., Nan- tucket, Wis. Sample received August 5, 1914. Sample examined August 5, 1914. Condition of seals: Good, unbroken sample. Organoleptic tests: Not conclusive. Consistency or feel: Poorly jellied. Color: Normal for eurr ant jelly. Odor: Faint, somewhat disagreeable. Taste: Not characteristic, bitterish, quite acid. Adjunct tests. Sublimation tests for Benzoic acid: Negative. Salicylic acid: Very marked. Boric acid (curcuma thread): Negative. Iodine reaction: Very marked. Intracellular: Negative. Extracellular: Positive, very marked. Special tests: Salicylic acid color reaction, with ferric chloride, very marked. Microscopical examination. General. Some apple tissue {window cells and pulp cells) and currant tissue (selerenchyma) present. Added wheat starch about 5 per cent. Cytometric counts. Dead yeast cells, 80,000,000 per cc. Living yeast cells, none per cc. Bacteria, 600,000,000 per cc. Mold (hyphal fragments and clusters), 84,000 per cc. Mold spores, 5,000,000 per cc. Smut spores, none per cc. Conclusions: Misbranded. Adulterated with apple and with wheat starch and made from fermented and decomposed ma- terial, preserved with salicylic acid. Not fit for human con- sumption because of the quantity of yeast, mold and bacteria present. John Doe, xA.nalyst. 2 2 MICRO-ANALYTICAL METHODS The great advantage of the micro-analytical work as compared with chemical work lies in the fact that small amounts of the substances are used for analysis, the equipment is comparatively inexpensive and the results are quickly attained. From twenty to forty and even sixty samples of simple spices can be examined in one day, from five to twelve samples of powdered vegetable drugs, cocoas, chocolates, flours, meals, etc., and perhaps an equal number of jams, jellies, etc. Because of the very close relationship between the micro- scopical and bacteriological work, as already explained, certain essentially micro-analytical methods will be given under bac- teriological methods, more especially in Chapter 2 of Part II which deals with the direct bacterial counts, and also under milk analysis, water analysis and meat analysis. DESCRIPTION OF PLATE I Fig. I. — Types of Pollen Grains. — i. Saffron flower. 2. Flax. 3. Pink. 4. Pumpkin and squash. 5. Cloves. Mature pollen grain. 6. Cloves. Immature pollen grain. 7. Onagraceae. Circea lutitiana (Enchanter's Nightshade). 8. Scutellaria. 9. Mallow. Distended by moisture. 10. Mallow. Normal form. II. Albuco. 12. Lobelia inflata. 13. Compositae, showing one mature and two immature pollen grains. 14. Hibiscus. 15. Pine pollen. 16. Santonica. 17. Mentha species. 18. Hyoscyamus niger. Fig. 2. — Potato Starch. — The granules are large and the markings (hili, lamel- lations) are distinct. The cross bands under the polarizer are very distinct. Potato starch, mounted in water, makes a good test object for judging the resolving power of objectives. Dried and ground potatoes and potato parings are sometimes used for adulterating purposes. Fig. 3. — -Starches. — i. Sago starch from Cycas revoluta (Cycadaceae). The commercial article known as sago is usually in the form of small granules (pearl sago). There are many false sagos made from other than Cycad or Palm starch. Much of this false sago is made from corn starch. 2. Canna starch from several species of Canna. The markings are very distinct, the hili being at the larger end as a rule. Also called arrowroot {tons le mois arrow- root). 3. Cassava or tapioca starch from the tuberous roots of Manihot utillissima and other species of Manihot. Simple and compound granules; the granules are largely separated in the processing, thus giving the appearance of simple granules. Their compound origin is, however, recognizable by the contact facets. 4. Maranta starch (Arrowroot starch) from Maranta arundinacca (Marantaceas). The granules have many of the structural characteristics of potato starch. 5. Yam starch from several species of Dioscorea (Dioscoreaceae). Fig. 4.— Dextrinized Starch. — The process of baking and cooking causes the starch granules to undergo marked structural changes. They become much enlarged, the outline becomes quite indistinct and the hili and lamellations are distorted and correspondingly indistinct, i. Normal wheat starch granules. 2. Normal rye starch granules. 3. Dextrinized wheat and rye granules. 4. Normal and dextrinized corn starch. 5. Normal and dextrinized bean starch. 6. Normal and dextrinized ginger starch. Plate I Fig. I. Fig. 2. 2^ #^^ 0