Neui ^atk g»tat0 (5OIU90 of Agriculture Kt (StOtnM Inineraity jCtbracg _ Cornell University Library QR 121.07 Dairy bacteriology. 3 1924 003 232 398 I Cornell University § Library The original of tiiis book is in tine Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924003232398 DAIRY BACTERIOLOGY DAIRY BACTERIOLOGY BY ORLA-JENSEN DR. PHIL. , PEOPBSSOE OF TECHNICAL BIOCHEMISTRY IN THE POLYTECHNIC COLLEGE, COPENHAGEN. FORMERLY DIRECTOR OP THE SWISS EXPERIMENTAL DAIRY STATION TRANSLATED FKOM THE SECOND DANISH EDITION, WITH ADDITIONS AND KEVISIONS BY P. S. ARUP B.SC. (LOND.), F.I.C. CHIEF CHEMIST TO ENGLISH MARGARINE WORKS (1919) LIMITED WITH 70 ILLUSTRATIONS PHILADELPHIA P. BLAKISTON'S SON & GO. 1012 WALNUT STREET 1921 Printed in Great Britain. TRANSLATOR'S PREFACE The success of Professor Orla-Jensen's " Dairy Bacteriology," in the original as well as in the German, Dutch, and Finnish translations, has led me to hope that an English translation might find a welcome. The author has had the unique experience of an intimate knowledge of the highly-evolved dairy industries of two countries so widely different as Denmark and Switzerland, and, as the reader wUl find, he is keenly interested in the valuable work which has been carried out in English-speaking countries, particularly in America. The translation has been based on the second Danish edition of 1916, which is the standard text-book on its subject for Danish students of dairying. As Professor Orla-Jensen has spared him- seK no pains in correcting and adding to the text in order to bring it thoroughly up-to-date, the present edition may be regarded as an entirely new one. Certain portions of purely local interest have been omitted, though the number of omissions on this account have been very few ; the scientific character of the work as a whole renders it of international interest. At the present time, when the question of pure mUk is beginning to attract the attention which it deserves in the United Kingdom, it is hoped that the work may offer some useful suggestions to those engaged on the difficult problems which the whole question involves. In spite of the modesty of the author's claims I consider that his book should also convey something to those who can read between the lines, and on this accoxmt I venture to hope that not only students, but also dairymen and aU others who have to deal with milk and dairy products, whether from the medical, veterinary or analytical side, will find something of interest in its pages. In conclusion, I wish to express my thanks to Mr. D. R. Wood, Public Analyst for the County of Somerset, for kindly reading the MS. and offering useful hints. PAUL S. AEUP. Limerpool. AUTHOR'S PREFACE In order to avoid the inclusion of superfluous matter in this work on Dairy Bacteriology, I have, on the one hand, omitted from the teaching of dairy practice everything which is not exactly of bacteriological interest, and, on the other hand, from the bacteriological side everything which is not of interest in dairy practice. It is, therefore, assumed that the reader wUl have obtained from other sources a knowledge of dairy practice, and that through the study of this book he will obtain some guidance in the bacteriological technique. This work is not a treatise, but only a text-book, for which reason one must not expect to find in it mention of every single microorganism to which is ascribed the power of affecting milk or dairy products in one way or another. I have purposely scrutinised the literature on the subject as closely as possible, and through my own investigations I have sought to form an opinion as to the actual conditions. The book is in all details built up on my own experiences, culled from twenty-five years of research work, and I, therefore, feel that I can confidently recommend it to my pupils. ORLA-JENSEN. Copenhagen. CONTENTS PAGE Translator's Preface ........ v Author's Preface ......... vii List of Illustrations ........ xi PART I.— GENERAL CHAPTER I MiCBOOEGANISMS AND FERMENTATIONS ..... I General Description of Microorganisms, their Nutrition and Growth — Fermentations — Enzymes — Variability — Methods of Culture and Examination. CHAPTER II Bacteria 26 Classification — The Lactic Acid Fermentation and the Classi- fication of the Lactic Acid Bacteria — The Propionic Acid Fermentation — The Butyric Acid Fermentation — The Putre- faction Process. CHAPTER III Yeasts and Moulds ........ 49 The Species commonly found in MUk and Dairy Products and their Action. PART II CHAPTER I Cleaning and the Procurement op Milk .... 54 Cleaning and Disinfecting — The Procurement of Milk — Clean- liness in Milking, etc. — Microorganisms in Milk, their Origin. CHAPTER II The Normal and Abnormal Microflora of Milk ... 63 The Normal Flora : Microorganisms in Milk, their Growth in and Influence on MUk under various Conditions — Abnormal Flora : Primary Milk Defects — Diseases of Cows, their Influence on Milk — Secondary Milk Defects — Infections with Disease Organisms — Abnormal Behaviour, Appearance, Taste and Smell. X CONTENTS CHAPTER III PAGE The Preservation of Milk and its Treatment for Direct Con- sumption .......... 77 Cooling — Sterilisation — Pasteurisation — Bacteria in Pasteurised Milk — Condensed Milk — Dried Milk — Preservatives — Milk for Town Supplies — Cleaning, Pasteurising and Cooling — ^Milk for Infant Feeding. CHAPTER IV The Applications of the Lactic Acid Fermentation in the Dairy Industry ........ 100 Dietetic Preparations — The Souring of Cream — Lactic Acid Cultures and Starters — Treatment of Starter Milk — Buttermilk — Separated Milk — Whey for Preserving Manure. CHAPTER V The Normal and Abnormal Microflora of Butter . . 120 The Normal Flora : Butter from Unpasteurised and Pasteurised Cream — Changes in Butter on Keeping — Fat Hydrolysis — Rancidity — The Abnormal Flora: Original Butter Defects — Secondary Defects. CHAPTER VI The Ripening Processes of the Different Cheeses . . 128 Action of Rennet and Lactic Acid in making the Curd and in Ripening — Types of Ripening Processes — Changes undergone by Constituents of Cheese in Ripening — Hard Rennet Curd Cheeses — Moulds in Cheese Ripening — Soft Rennet Curd Cheeses — Smeared and Mouldy Cheeses— Acid Curd Cheeses. CHAPTER VII Defects of Cheese ......... 151 Origin of Defects — Sponginess — ^Defects in Colour, Taste and Smell. CHAPTER Vm The Grading of Milk 157 Taste and Smell — Estimation of Dirt — Leucocyte Test — Catalase Test — Rennet Test — Acidity — Fermentation Test — Reductase Test — Combined Reductase and Fermenting Test, with special Reference to the Interpretation of Results. Index • 175 LIST OF ILLUSTRATIONS Fig. page 1. Acetic Acid Bacteria {BacteriMm aceti). (After Hansen) . . 2 2. Bieweis' YeaL&t {8accharomyces cerevisice). {Aitei Hansen) 3. Penieillium glaucum ....... i. Growing Top Yeast. (After Mitseherlich) 5. Clostridia. (After Prazmmvski.) Spore Formation in the Common Butyric Acid Bacteria .... 6. Plectridia. (After Migula.) Spore Formation in the Tetanus Bacterium ........ 7. Spore Formation in Wine Yeast. (After Hansen) . 8a. Mueor Mueedo. (After Kemer) ..... 8b. Transverse Section of a Single Sporangium. (After Brefeld) 9. Penieillium, glaucum. (After Brefeld) .... 10. Oidium,Jfactis. (After de Bary) ..... 11. Jlf Mcor racemosMs with Chlamydospores. (After Bre/eW) . 12. Zooglcea of " Leuconostoc," a Coccus which forms slimy lumps in Cane Sugar Solutions. (After Zopt) . 13. The Bacterium of " Long Milk." Capsule Stage. Milk not yet slimy ......... 14. The Bacterium of " Long Milk." Slimy Stage 15. The Bacterium of " Long MUk." Milk less slimy, but beginning to go thick owing to acid formation .... 16. Stab Cultures. A. The Anthrax Bacterium (aerobic). B. The Swine Erysipelas Bacterium (facultative anaerobic). (After Migula.) C. The Tetanus Bacillus (obligate anaerobe). (After Ball) 17. Involution Forms of Bacterium aceti produced by cultivation at 39° to 41° C. (After Hansen) .... 18. Freudenreich Flask ....... 19. Petruschhy Flask ....... 20. Incubator with Petri Dishes, Plugged Test Tubes, Apparatus for Measurement of Gas Production, etc. 21. Desiccator for Anaerobic Cultivation .... 22. Stribolt's Method for the Cultivation of Anaerobic Bacteria. (After Salomonsen) ...... 23. Jorgensen's Moist Chamber ...... 24. A. Cornefs Forceps. B. Kiihne's Forceps 25. Bacterium pyocyaneum, monotrich. (After Migula) 26. Bacterium syncyaneum, lofotrich. (After Migula) . 27. Bacterium typhosum, peritrich. (After Migula) 28. Bacterium, vulgare, peritrich. (After Migula) . 29. Various Cocci. (After Fli^gge) ..... 30. Fi6no cholerce. (After Migula) ..... 31. Various Screw-shaped Bacteria. (Aitev Flugge) 32. Actinomyces bovis. (After Bostriim) .... xa LIST OF ILLUSTRATIONS Fig. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. Thermohacterium bulgaricum. Grown in sterile milk. Stained with methylene blue. The grains are round and dark blue .......... Thermobaoterium bulgaricum. Grown in milk pasteurised by heating previously to 80° C. for half an hour. Stained with methylene blue. The grains are long-drawn and red. Capsule clearly shown ....... Thermobacterium helveUcum from Emmental Cheese. (After PAGE fermenting Plec Streptobacterium casei from Danish Dairy Cheese Streptoeoceus eremoris (Streptococcus laclicas) . Streptococcus eremoris (Starch's No. 18) . Streptococcus thermopJiilus . . ' . Streptococcus lactis ..... Betacoccus from thin juice from Nakskov Sugar Factory Streptococcus liquefaoiens (FreudenreicV s Micrococcus amari) ....... Streptococcus liquefaciens {Escherich's Streptococcus coU gracilis) Various Betaoocci in Stab Cultures in Cane-sugar Gelatine The CoU Bacterium which spoilt the MUk and Butter at Duelund Dairy in 1888. (After 0. O. Jensen) Bacterium acidi propionici (a) . . . Bacterium acidi propionici (6). Grown at 39° C. Bacillus subtilis. (After Migula.) a. Active rods before spore formation. 6. A piece of film. c. Rods with spores d. Stained to show the flagellae 49. Mycoderma cerevisice. (After Hohn) 50. MoniUa nigra. (After Burri and Staub) 51. Two Varieties of Penicillium brevicaule . 52. Rotting Swede containing numerous Pectin tridia .... 53. Milking Room at Fauerholm 54. Ourler's and StadtmUller's Milk Pails. (After Conn) 55. Cooling and Straining of Milk at Fauerholm. In the foreground Ice is being put into the bottom of Busck's Milk Pail 56. Jonas Nielsen's Steriliser 57. Heine's Cleaning Separator 58. Laval's Cleaning Separator 59. Hygienic Stopper 60. Orla- Jensen's Household Pasteurising Apparatus 61. Bacteritim hifidnim, distinctly branched. From fseces of bottle fed infant ..... 62. Kefir Grains, natural size. (After Kern) 63. Section through a Kefir Grain 64. Leucocyte Sediment from the Milk of a Cow suffering from Streptococcic Mastitis 65. Catalase Test Apparatus 66. Gelatinous Types 67. Blown Types 68. Spongy Types 69. Cheesy Types 70. Apparatus for Reductase and Fermenting Test to take 200 Samples 30 31 168 DAIRY BACTERIOLOGY PART I GENERAL Chapter I Microorganisms and Fermentations By microorganisms or microbes, we understand all organisms which are too small to be seen by the naked eye ; they remained unknown until it had become possible to construct strong magni- fying glasses or the combination of lenses which make up the microscope. Bacteria were first observed by the Dutch optician Leeuwenhoek in 1675, but no real knowledge of their nature was gained until the latter third of the last century, when Pasteur carried out his classical researches. Bacteriology is thus a comparatively new science ; nevertheless it has already revolu- tionised both medical science and the technology of the fermenta- tion industries, among which may be included the dairying industry. As far as the fermentation industries are concerned, only three groups of microorganisms come into consideration. Bacteria, Yeasts and Moulds. Bacteria and yeasts proper are unicellular, while moulds are generally multicellular. Unicellular organisms may be united in chains, but the individual cells of the chain show no. dififerentiation as regards structure or function, excepting such modifications as may be due to differences in age or nutrition. In the moulds, on the other hand, it is possible to distinguish between two groups of cells ; first, the mycelium, which is con- cerned with the nutrition of the organism, forming a tangled network in the nutrient medium, and second, the fine thread-like shoots which bear the reproductive organs and generally project out of the medium so that the spores may be carried away by air currents (see Fig. 8). DAIRY BACTERIOLOGY The size of microorganism is given in micromillimetres, or microns, designated by the Greek letter (i, being one-thousandth part of a millimetre. Bacterial cells generally measure from 0'5 to 2 ju, in thickness. Some idea of their minuteness may be formed on consideration of the fact that the space of a cubic millimetre will hold one thousand million bac- teria of average size. From Figs. 1, 2 and 3 it will be seen that the cells of yep,sts and moulds are from five to ten times as large as those of the bacteria. Fig. I. — Acetic Acid Bacteria (Bac- terium aceti). (After Hansen.) Fio. 2. — Brewers' Yeast {Saccharomyces cereviaim). (After Hansen.) Fig. 'i.—Penfcillium glaacum. All magnified 1,000 times (x 1,000). GROWTH AND REPRODUCTION Under normal conditions a bacterialcell will soon grow to its maximum size, often becoming more or less elongated in form, when a cross partition appears dividing it transversely into two MICROORGANISMS AND FERMENTATIONS separate daughter cells of equal size, which in their turn grow longer and divide, the process being repeated indefinitely. This mode of reproduction is known as fission. Yeast cells, on the other hand, reproduce by Imdding, or gemmation ; small round outgrowths appear and continue to develop until they attain the size of the mother cell (see Fig. 2). In the moulds we generally find growing points as in the higher plants ; only the outer cells are capable of reproduction, which mostly takes place by fission and only rarely by budding. Spore Formation. — ^As the higher plants form seeds, so many of the microorganisms form spores, the function of which is to preserve the species under adverse conditions. Spores are accordingly more resistant to the influence of desiccation, poisons and high temperatures than the ordinary cells. Two distinct © I ^ 1^. 1^ % , 6' Fig. 4. — Growing Top Yeast. (After Mitscherlich.) I. At 7 p.m. II. Next -" morning, 8 a.m. III. 9 a.m. IV. 10.15 a.m. V. Noon. VI. 3.30 p.m. VII. 8 p.m. VIII. 9 p.m. IX. 10 p.m. X. 11 p.m. types are found : endogenous and exogenous spores. It is com- paratively seldom that the cell becomes completely changed into an Arthrospore, absorbing reserve food material freely and thickening its walP Endogenous spores are formed inside a cell while exogenous spores arise as constrictions on the end of a cell. Bacteria only form spores of the first mentioned type, only one spore apipearing in each cell. If the spore causes the cell to bulge out locally, drumstick (Plectridium) or club-shaped {Clostridium) formations arise according as the spore lies at the end or in the middle of the cell. Germination in a direction approximately at right angles to the length of the cell is said to be lateral, and germination from the end polar. The yeasts likewise form endospores only, but several spores, up to ten in number, may be 1 In this connection it may be mentioned that, according to Preisz' investigations (" Centralblatt f. Bakt.," 1 Abt., 1918, Bd. LXXXII., p. 321), the spore proper (the refractile body) is always only condensed reserve food material ; outside this may be demonstrated a mass of protoplasm from which growth proceeds. 4 DAIRY BACTERIOLOGY formed in a cell. As a rule, two, three or four spores iare found together (Fig. 7), and. they -generally grow by budding like the ordinary yeast cells. When moulds form endospores (e.g., the various Mucors) this takes place in specially shaped cells known as sporangia, which may contain several hundred spores (Figs. 8a and 8b). As a rule, however, the moulds form exogenous spores ; these are known as Conidia when formed as constrictions on specialised spore bearing members (Conidiophores) (see Figs. 3 and 9), and as Oidia when formed on the ordinary branches of the mycelium (Fig. 10). Considered as spores, the oidia are less characteristic than the conidia ; the Chlamydospores, however, are exceptional, being unusually thick walled oidia, which may be formed anywhere in the mycehum (Fig. 11). In a chain of conidia, the outer members are generally the oldest, and the inner ones the youngest. STRUCTURE AND CHEmCAL COMPOSITION OF CELLS The main constituent of the cell is a viscous jelly-like trans- parent solution of proteins, known as protoplasm, which is the living svhstance of the cell. It is an extremely complex mixture of unstable compounds, which are largely built up of combinations ■^ (X) Fig. 5. — Clostridia. (After Praz- mowsH.) Spore Formation in the Common Butyric Acid Bacteria. FxG. 6.— Plectridia. (After Migula.) Spore Forma- tion in the Tetanus Bacterium. All X 1,000. Fig. 7.— Spore Formation in Wine Yeast. (Mtev Hansen.) of a considerable number of different amino acids. The feeble acid and basic properties of these acids are also characteristic of the proteins, a circumstance which enables the latter to combine loosely with a vast number of other substances, thus fulfilling a condition necessary for the vital processes. Oil drops and other reserve foodstuffs are often found in the protoplasm, and as a rule MICROORGANISMS AND FERMENTATIONS 5 there are one or more spaces containing cell sap (vacuoles). Young and vigorous cells are nearly filled with protoplasm ; old or starved cells have only a thin layer on the cell wall. AH cells possess a micleus which, although similar to the rest of the proto- plasm in composition, is of a still more complex nature. When reproduction takes place, the nucleus is divided between the two new cells. The protoplasm is bounded by a cell wall which grows thicker as the cell ages. It is composed not of cellulose, as in the case of Fig. 8a. — Mucor Mtuxdo. (After Kerner. Fig. 8b. — Transverse Section of a Single Sporangium. (After Bre- Jeld.) the higher plants, but of an allied carbohydrate which has been named hemiceUidose ; it becomes coated with a nitrogenous substance identical with or very similar to chitin, the chief con- stituent of the shells or exoskeletons of the Crustacea and the insects. Mucin, a substance which forms a sticky mass with water, may also be present. As these substances are typical of many of the lower forms of animal life, it may be- surmised that the bacteria form a sort of link between animal and plant life, and this theory derives some support from a consideration of their mode of life. The cell wall may sometimes swell up considerably ; in cases where DAIRY BACTERIOLOGY Fig. 9. — Penicillium glaiicum. (After Brefeld.) Pig. 10.— Oiddum laetia. (After de Bary.) Fig. 11. — Mucor race- monus with Chlamy- dospores. (After Brefeld.) MlCHOORGANlgMS AND FERMENTATIONS 7 it retains its sharp, contour, it is known as a capsule. Numbers of cells may thus form large jelly-like masses, known as zoogloea Occa.sronally the cell walls will become assimilated with the surrounding liquid, which then becomes slimy or ropy throughout (see Figs. 13, 14 and 15). Fio. 12. — Zoogloea of " Lew.onostoc" a Coccus .which forms slimy lumps in Cane Sugar Solutions. (After Zopl.) x 1,200. Fig. 13.- -The Bacterium of " Long Milk." Capsule Stage. Milk not yet slimy. X 1,000. NUTRITION The following twelve elements enter into the composition of all organisms : — Oxygen, Hydrogen, Carbon, Nitrogen, Sulphur, Phosphorus, Chlorine, Sodium, Potassium, Calcium, Magnesium and Iron. It follows that substances composed of these elements are necessary for the normal growth of microorganisms. Some of them, e.g., sulphur, calcium and iron, are only used in such minute "BAIEY BACTERIOLOGY Fig. 14.— The Bacterium of '• Long Milk." Slimy Stage, x 1,000. Fia. 1.5. — The Bacterium of " Long Milk." Milk les.s slimy, but beginning to go thick owing to acid formation, x 1,000. quantities that it is unnecessary to add them specially to the nutrient media if ordinary tap water is used. Chlorine and MCROORGANISMS AND iFERMENTATIONS 9 sodium are only required in appreciable, amounts by organisms normally living in salt liquids, i.e., marine and many pathogenic organisms. Like all other organisms, the microorganisms are able to derive the necessary sulphur, phosphorus and metallic elements from inorganic salts. These salts are found in the required proportions in plant ashes, and formerly it was the practice to add them to nutrient media in this form. If micro- organisms are cultivated in milk or the extracts of meat or plants they will generally be well supplied with the requisite inorganic nutriment. Nitrogen. — Microorganisms may be divided into two main groups according to their ability or inability to assimilate all the nitrogen they require from inorganic soiirces. A few species belonging to the former group can assimilate nitrogen from the air, and thus incidentally improve the soU for plant growth. The great majority of these, however, require their nitrogen in the form of ammonia or nitrates ; they are represented by the t3rpical water bacteria, acetic acid bacteria, and many yeasts and moulds. Belonging to the group which cannot grow in Ihe absence of proteins or the immediate decomposition products of proteins are many of the putrefactive bacteria and the true lactic acid bacteria. Carbon. — ^Microorganisms faU into two groups with respect to carbon assimilation. A few species of soil bacteria which oxidise ammonia to nitrates resemble the higher plants in being able to utilise atmospheric carbon dioxide as their sole source of carbon. Unlike the higher plants, however, they are best able to carry out this process in darkness — ^no bacteria tolerate direct sunlight. Most microorganisms reqitire organic sources of carbon. Some putrefactive bacteria can obtain all the carbon they need from proteins, thus presenting a broad analogy to the carnivorous animals. As a rule, however, microorganisms require special sources of carbon *such as carbohydrates, alcohols and organic acids, and often show marked preferences for certain substances ; thus some species will only grow in presence of certain sugars, a circumstance which is put to advantage in distinguishing closely related species from one another. Oxygen. — ^Oxygen and Hydrogen are assimilated from most of the nutrient substances and from water. Water must be regarded as the most important of the nutrient substances ; it constitutes About four-fifths of the total substance of the microorganism, besides which it is absolutely essential to life as a solvent and distributing medium for all the other nutrient substances. Like animal and plants, most microorganisms are capable of assimilating oxygen direct from the atmosphere. The analogy is only super- 10 DAIRY BACTERIOLOGY ficial, for nearly all microorganisms can live without atmospheric oxygen for shorter or longer periods, provided that other sources of energy are available, and towards some species oxygen acts as a poison. Organisms which cannot live without atmospheric oxygen are defined as aerobic, and those which can do without it as anaerobic. The latter group is subdivided into facultative and obligate anaerobes, which are respectively helped or hindered in their growth by the presence of atmospheric oxygen. As a rule y. -Ll^t Fio. 16.— Stab Cultures. A. The Anthrax Baot'enum {ae.robic). B. The Swine Erysipelas Bacterium (facultative anaerobic). (After Migula.) C. The Tetanus Bacillus {obligate anaerobe). (After Ball.) aerobic organisms will, sooner or later, form a film on the surface of the culture solution or spread over the surface of solid media, only penetrating slightly below the surface. Facultative anae- robes will grow equally well at all depths or on the surface, while obligate anaerobes only thrive below a certain depth. These relations are illustrated by the accompanying figures, The true lactic acid bacteria generally tolerate air, but thrive best in its absence ; in stab cultures, therefore, they will not spread over the surface, but penetrate the medium evenly from all points. MICROORGANISMS AND FERMENTATIONS 11 FERMENTATION PROCESSES The assimilated nutrient substances are employed, partly in building up the cells during growth and reproduction, and partly as sources of energy for these and other vital processes. All substances are not equally suited for both purposes, and distinction may be made between the nutrient substances proper and those which merely function as sources of energy. As long as active reproduction is taking place, a considerable proportion of the latter will be pressed into service as cell-building material, later on to be decomposed into simpler products, when the energy thus liberated will be utilised by the microorganisms. That portfon of the period occupied by the reproductive process during which no appreciable amounts of decomposition products are separated is known as the incubation period. The decomposition products, which bear a certain general comparison with the substances contained in the urine of animals, are known as fermentation products, and fer- mentations are to be regarded as decompositions brought about by microorganisms. Exceptions occur when the organic sub- stances undergo complete oxidation to carbon dioxide and water ; such changes are looked on as respiration processes analogous to those observed among the higher organisms. Fermentation thus implies only a partial decomposition through the agency of micro- organisms. It is evident that such a process entails a certain loss of efficiency, and consequently a larger consumption of material. Complete decomposition is only effected by aerobic microorganisms, more particularly by the moulds which, as previously mentioned, are able to form aerial shoots. Moulds only function as fermenta- tion organisms when excluded from access to air, under which cir- cumstances many of them produce alcohol, like the yeasts. It will now be understood that the most typical of the fermentation organisms must be facultative or obligate anaerobes. Fermenta- tions are also known which are simple oxidations, such as the acetic acid fermentation. Processes of this nature require a plentiful supply of air, and can, therefore, only be carried out by aerobic organisms. Fermentations and fermentation organisms are often named after their most characteristic products, e.gr., alcohol and butjrric acid fermentations, lactic acid or butyric acid bacteria. Some- times the name is derived from the substance attacked, e.g., cellulose fermentation or cellulose bacteria. The former system is the' more logical. Soil formation is the natural bacterial decomposition of plant remains into products, which, as a rule, are not stinking. Putrefaction is the decomposition of animal remains into products which are usually stinking. As 12 DAIRY BACTERIOLOGY animal tissues consist for the most part of proteins, the term putre- faction has become synonymous with the bacterial decojtnposition of proteins. Formerly, when nothing was understood of the real nature of the different fermentations, they were generally looked on as chemical changes which apparently originated spontaneously, and which might improve a natural product. In cases where the natural product was spoilt instead of being improved the process was known as putrefaction. As alcohol fermentation is the most widely-known example, it is often supposed that the evolution of gas is characteristic of fermentations in general ; but in its modem sense the term fermentation includes a number of processes, such as the lactic acid and acetic acid fermentations, in which no gas is produced. ENZYMES The manifold chemical changes due to microorganisms are carried out through the agency of certain special substances, known as enzymes. These are bodies of unknown composition, possibly more complex than the proteins, which may be separated more or less easily from the protoplasm, and which are able in small amounts to induce certain chemical changes in relatively large amounts of material. Enzyme action may be extracellular or intracellular, according as the enzyme acts outside (exoenzyme) or inside (endoenzyme) the cell in which it has been formed. To the former class belong the digestive enzymes of animals ; as the function of these is to prepare the food for assimilation, they must necessarily act outside the assimilating cells. Many micro- organisms form enzymes which are analogous to these. On -the other hand, the typical fermentation enzymes decompose or oxidise the assimilated food material inside the cell, and must ' therefore act inside the cell, so that the energy liberated in the process may be directly available to the cell. Distinction was formerly made between unorganised ferments, i.e., enzymes such as are contained in the digestive juices, and organised ferments, by which were understood the microorganisms. It was not until 1897, when Buchner succeeded in separating from yeast the enzyme which brings about alcoholic fermentation (zymase), that it became clear that all fermentations are ultimately due to enzyme action. The activity of enzymes increases with the temperature, but above certain limits the enzymes, like the proteins, become de- natured and lose their special properties. The optimum for most enzymes lies between 35° and 65° C. In aqueous solution they are generally rendered completely inactive at temperatures MICROORGANISMS AND FERMENTATIONS 13 between 65° and 85° C. In the dry state, some enzymes will stand temperatures of about 150° C. Enzymes resemble living organisms in being destroyed by heat ; on the other hand they are unaffected by many substances which act as poisons towards living protoplasm, such as toluene, ether, chloroform, ethereal oils, phenol, salicylic acid and benzoic acid. Some such substance, usually toluene, may therefore be added to an enzyme solution to keep it sterile ; boric acid is added to preparations of rennet. Most enzymes act best in feebly acid, all but neutral, solutions ; pepsin is exceptional in acting in presence of the appreciable proportions of acid contained in the gastric juice. As a rule, enzymes are named after the substances on which they act, thus maltase acting on maltose, lactase acting on lactose, urease on urea, etc. In some cases, however, previous usage has established other names. Diastase or, better, Amylase (amylum — starch) con- verts starch into dextrin and maltose ; it occurs plentifully in malt, and plays an important part in the brewing and distilling industries ; during the mashing process it converts the starch into fermentable products. It also occurs in the saliva and the pancreatic juice of , mammals ; as it only makes its appearance some time after birth, ' newly-born animals are unable to assimilate starchy foods. Invertase hydrolyses saccharose, i.e., cane or beet sugar, into dex- trose (d. glucose) and Isevulose (d. fructose), a mixture which is found in honey and many fruits. Invertase, maltase, lactase and amylase are the chief carbohydrate splitting enzymes. Enzymes which hydrolyse fats into glycerol and fatty acids are known as lipases, and those which split proteins as proteolytic enzymes. Proteins may be hydrolysed by stages, each stage yielding a simpler product, thus : proteins to metaproteins to proteoses to peptones to polypeptides to amino acids. The first decomposition product of casein is paracasein, which, according to Hammarsten, differs from natural casein in being precipitated by the small amounts of calcium salts found in solution ift normal cow's milk^. A microorganism which secretes proteolytic enzymes wiU always coagulate milk, and then gradually redissolve, i.e., peptonise, the precipitated paracasein ; unless the organism in question also belongs to the acid-producing group, the process of peptonisation will tend to produce an alkaline reaction in the milk. Peptonising organisms generally liquefy gelatine more or 1 The coagulation of casein by rennet appears to be analogous to the coagulation of other proteins by heat. See Orla Jensen, " Kemiske Under- sogelser over Maelkens Koagulering og Koaglets Oploselighed i Saltvand." Det kgl. danske Videnskabernes Selskabs Oversigter, 1914, No. 4. Chemisohe Untersuchungen tiber die Uerinnung der Milch und fiber die Loslichkeit des Ger. in Salzwasser. Zeitschr. f. Physiol. Chem., 1914, Bd. XCIII., p. 283. 14 DAIRY BACTERIOLOGY less readily, being usually referred to as liquefying organisms. The proteolytic enzymes of the gastric juice, chymosin (rennet) and pepsin, only carry the cleavage of proteins as far as the peptone stage ; the trypsin of the pancreas effects complete hydrolysis to amino acids. The erepsin of the intestinal juice is particularly thorough in its action, but only attacks proteins which have already been partially broken down by other enzymes. Casein is exceptional in being directly attacked by erepsin^. Oxidising and reducing enzymes are known as oxidases and reductases ; they are of prime importance in connection with the breathing of animals and the reduction of carbohydrates to fats. Starch's well-known reaction, by which it is possible to ascertain whether milk has been heated to over 80° C. or not, depends on the presence in milk of an oxidase which is destroyed at 80° C, and can transfer the loosely bound oxygen of hydrogen peroxide to paraphenylene diamine or certain other colourless substances giving coloured products. Paraphenylene diamine gives a violet or, in the presence of casein, a blue colour. Many vegetable products, such as potatoes, fruits or fungi, contain oxidases as well as substances which yield coloured products on oxidation ; hence the darkening in colour which takes place when they are cut into pieces and left exposed to air ; if the material has been boiled, the oxidase will have been destroyed, andLno darkening occurs. Reductases, on the other hand, are generally recognised by taking advantage of the fact that many substances, such as methylene blue, are decolorised on reduction. Catalase occupies a position intermediate between oxidases and reductases ; it decomposes hydrogen peroxide, but the oxygen so liberated will not act on paraphenylene diamine or similar oxidisable substances. Catalase only occurs in small quantities in milk, but plentifully in blood ; as constituents of the blood always pass into milk, which is drawn from diseased udders, an abnormally copious evolution of oxygen in the catalase test (see p. 159) is to be regarded as a bad sign. Certain poisons, easily destroyed by heat, known as toxins, are closely related to the enzymes. They are found in a few plants, in poisonous reptiles and other animals, and are frequently « secreted by microorganisms. The various toxins are generally the active agents in diseases due to pathogenic organisms. Healthy living tissues are highly resistant towards enzyme action," and are provided with special poisons, bactericidal substances, which repel ^ It follows that microorganisms which secrete no other enzymes than erepsin attack casein, but do not liquefy gelatine. Conversely, the author has iound that micrococci which liquefy alkaline, but not neutral or acid gelatine, do not peptonise casein. MICROORGANISMS AND FERMENTATIONS 15 bacterial invasion unless they have previously been weakened by the action of toxins. To counteract this, the higher organisms secrete substances, known as anti-toxins, which play an important part in recovery from disease and the subsequent more or less permanent period of immunity from the disease in question. VARIABILITY Microorganisms are classified together when they resemble one another in structure and habits of living and growth. Many microorganisms, however, are very liable to vary in appearance and in their biological characteristics, so that we may often have to deal with dissimilar forms which nevertheless belong to the same species. As we have really no sure guide enabling us to distinguish between essential and non-essential characteristics, it may often be difficult to determine whether we are dealing with different species or only variants of the same species. Undoubtedly, the biological properties, such as those which relate to nutrition, growth and energy, are more important in this respect than out- ward form, and, again, the means by which energy is produced are of more importance than the particular raw materials from which the energy is derived. Though it may be granted that an organism which derives its energy from an alcoholic fermentation instead of a complete oxidation to carbon dioxide and water would hardly be classified among the higher organisms, yet it is often observed that closely related organisms are able to utilise different nutrient materials. If a lactic acid organism undergoes variation, it does not follow that it will start producing alcohol or butyric acid instead of lactic acid, but it may easily lose its power to ferment a poly- saccharide, such as milk sugar, which requires a special enzyme, lactase, to convert it into fermentable material. In other words, the intracellular enzymes are much more characteristic of a given microorganism than the extracellular enzymes. It may be assumed that the chief products of a fermentation process will always be the same under similar conditions, but the by-products may vary considerably according to the condition of the organism. The ability to produce certain substances which affect the taste, smeU or colour of the medium is especially variable. The nature of the cell wall may also vary, so that an organism may appear sometimes with and sometimes without a capsule, or the cell wall may dis- integrate into a mucilaginous mass. Bacteria are particularly liable to undergo this form of degeneration — especially the lactic acid organisms — generally as the result of over-nutrition ; an analogous instance is seen in fatty degeneration in animals. Temperature is an important factor in determining the form of 16 DAIRY BACTERIOLOGY microorganisms ; on cultivation at temperatures approaching the maximum, acetic acid bacteria may be induced to undergo a striking transformation. In the case of acid-producing bacteria, the high concentrations of acid produced in the medium wiU also tend to cause the appearance of abnormal forms, known as involution forms, and in old cultures of lactic acid bacteria strange elongated, swollen or even branched cells may often be observed. Prolonged cultivation under adverse conditions is the best method of producing new varieties ; thus Emil Christian Hansen induced yeasts permanently to lose their capacity of forming spores by Fia. 17. — Involution Forms of Bacterium aceti produced bv cultivation at 39° to 41° C. {Mter Hansen.) x 1,000.' cultivating for several generations at temperatures above the maximum for sporulation. METHODS OF CULTURE It is not possible to compound a universal nutrient medium, for some substances may be essential for the well-being of certain organisms and poisonous to others. While certain water bacteria wUl only thrive^ in very dilute solutions of organic nutrient matter, yeasts and moulds thrive best in wort and fruit juice, milk bacteria in milk, and pathogenic bacteria in meat broth or blood serum. Such natural media are of variable and partly unknown composi- tion, and, therefore, do not lend themselves so well to the study of fermentations from the chemical point of view. For this purpose artificial media containing as far as possible only substances of known composition in definite proportions are the best ; for bacteria which require proteins, a preparation of peptonised fibrin known as Witte peptone is used ; unfortunately this involves the MICROORGANISMS AND FERMENTATIONS 17 introduction of a substance of which the composition is not quite constant ; only the best brands, prepared under standard condi- tions, should be used. Most milk bacteria thrive in the following solution : — Tap water, 1 litre ; spdium chloride, 2 grams ; dipo- tassium phosphate, 2 grams ; magnesium sulphate, 1 gram ; dextrose, 20 grams ; peptone, 20 grams. This solution has an alkaline reaction, and phosphoric acid should be added until it only just turns litmus paper blue. For the cultivation of water bacteria and most pathogenic organisms, the medium should be distinctly alkaUne ; for yeasts and moulds it should be acid. The media are distributed in flasks or test tubes closed with plugs of non-absorbent cotton wool and sterilised by heating in an autoclave for a quarter of an hour at 110° to 120° C. The Freudenreich flask is the most convenient vessel to use, as the contents are not so easily dried up or infected as in test tubes. If the nutrient solution is to be used for the investigation of acid Fig. 18. — Freudenreich Flask. Fig. 19. — Petnmchky Flask. formation, then 10 c.c. are introduced into each test tub^, and the inoculated solution is titrated when the maximum acidity is certain to have been reached, say, after fourteen days at 30° C. The amount of acid produced by lactic acid bacteria increases with the amount of nitrogenous nutrient material in the medium ; these organisms thrive better on casein peptone ^ than on Witte's peptone, but most of the rod-shaped lactic acid bacteria thrive best on an extract of autolysed yeast 2. In order that the cultures may be kept at definite temperatures the bacteriological laboratory must be equipped with several incvhators heated by gas or electricity, and, if necessary, cooled by water circulation, in such a way that the temperature is automatically kept constant. In order to examine microorganisms, to obtain them as pure cultures or to determine their numbers, they must first be isolated. This may- be accomphshed by Koch's method of plating, which consists in distributing a definite small quantity of the liquid containing the organisms in a solid transparent medium which 1, 2 See footnotes at end of this Chapter. DB. 2 Fig. 20.— Incubator with Petri Dishes, Hugged Test Tubes, Apparatus for Measurement of Gas Prodnotion, etc. MICROORGANISMS AND FERMENTATIONS 19 has previously been melted, and then allowing this to set in a thin layer. The isolated germs, fixed in position, wiU after some time multiply into colonies visible to the naked eye. From the number of colonies, the number of organisms per cubic centimetre of the original liquid can be calculated. Care must be taken that the tem- perature of the melted medium is not high enough to cause injury to the organisms. For milk bacteria the best media are whey or the solution recommended above, with the addition of 12 per cent, of gelatine or IJ per cent, of agar. Gelatine may be regarded as a protein, whereas agar, a product obtained from certain seaweeds, is composed of carbohydrates, known as pectins. Gelatine is to be preferred, as it can be made to yield clearer media than agar, but as it melts slightly over 20° C. or lower if it has been heated too frequently or at too high a temperature, it must be replaced by agar when deal- ing with organisms which only thrive at higher temperatures. If litmus or chalk be added, acid-producing organisms may at once be recognised. Solid media are usually allowed to set in Petri dishes, flat dishes with ' lids, which have been sterilised dry at 150° to 170° C. Organisms which do not grow in presence of oxygen are best cultivated in Burri's tubes, which are closed at one end by a rubber stopper and at the other by a plug of cotton wool ; on removing the stopper, the agar column may be shaken out and examined for colonies. Some obligate anaerobes form characteristic surface colonies ; to obtain these, the Petri dish must be kept in an atmosphere of hydrogen or in a vacuum. The oxygen which gradually leaks in may be absorbed by alkaline pyrogallol solu- tion. The simplest plan is to use a vacuuili desiccator, which is tilted during exhaustion in such a way that the chemicals (5 grams of pyrogaUol and 50 c.c. of 10 per cent, caustic potash) in the sulphuric acid container are only mixed when the desic- cator is stood straight after evacuation. As a rule, the liquids to be examined contain so many germs that they must be diluted with sterilised water before plating, in order to avoid overgrowth. The number of colonies to be aimed at is 40 to 200 per plate, and it will generally be necessary to make Fig. 21. — Desiccator for Anae- robic Cultivation. 20 DAIRY BACTERIOLOGY several dilutions to achieve the desired result. A number of 100 c.c. Freudenreich flasks sterilised in the autoclave with 50 c.c. of water should be prepared in readiness, and also some dry sterilised plugged pipettes, graduated in quarters of a cubic centimetre. When making counts in milk, the following dilutions may be made : — Dilution 1. 1 c.c. milk in 49 c.c. water, of which 0-5 c.c. in Petri dish No. 1. , „ 2. 0-5 c.c. of Dilution 1 in 495 c.c. water, of which 0-5 c.c. in Petri dish No. 2. „ 3. 0-5 c.c. of Dilution 2 in 495 c.c. water, of which 0-5 c.c. in Petri dish No. 3. The number of organisms per cubic centimetre is obtained by multiplying the number of colonies on plates 1, 2 or 3 by 100, 10,000 or 1,000,000, respectively. Butter is measured by means of a rounded platinum spoon, holding exactly 0-25 c.c. The spoon and the knife used for scraping off the surface of the butter must be sterilised by passing through a Bunsen flame before use. If the water used for dilution is warmed to about 40° C, the butter will readily melt from the spoon and become distributed on shaking. Cheese gives more trouble, as it must be weighed and comminuted ; about 1 gram of cheese may be accurately weighed into a Freudenreich flask containing 50 c.c. of water and ground up by repeatedly pressing it out in the water by means of a flamed glass rod. An alternative method, resembling that for determining the soluble nitrogenous matter, is to grind 10 grams of the cheese with sterile water at 40° to 50° C. in a sterile mortar, pouring the emulsion into a sterilised 250-c.c. measuring flask and making up to the mark with sterile water.. This method is to be preferred as a more representative sample, is obtained, a matter of some importance as the organisms are usually very unevenly distributed in cheese. Further, the cheese is more easily ground up in the mortar than in the flask, whUe the nuinber of organisms introduced by infection from the air will be negligible compared with the large number present in the cheese. In all cases uniform working methods should be adopted in order that the results may be comparable ; the colonies should be counted after incubating for a definite time, say seven days at 20° C. The counting is facilitated by placing the plate on a squared glass plate resting on a black surface. For use when travelling, Petruschky flasks (Fig. 19), sterilised with the proper ahiount of nutrient gelatine, are more convenient than Petri dishes ; they are generally used in luater examinations, which are best carried out on the spot, as an increase in the MICROORGANISMS AND FERMENTATIONS 21 number of microorganisms generally takes place durjng transit. Bacterial multiplication may, however, be avoided to a con- siderable extent if the samples are packed in ice. For counts of water samples the peptone gelatine recommended above may be used, modified as follows : the sugar is omitted, and the neutralised medium is treated with 15 c.c. of 10 per cent, soda solution per litre. In these, as in all bacterial counts, it is im- possible to get all the germs present to grow on the same medium ; as many typical water bacteria, for example, the thread-forming and sulphur organisms, will not grow on gelatine at all, the counts must not be regarded as representing the actual numbers of organisms present, though they furnish useful indications as to the relative purity of samples if the same working conditions are adhered to throughout. It must also be remembered that long chains of bacteria or large pieces of mould myceha only yield single colonies. The method is thus subject to considerable error, a fact which may easily be demonstrated by comparing the results with those obtained by direct microscopical counts. According to BartheVs investigations, 2^ to 200 times as many organisms are found by direct counts as by plating according to Skar's method *. Pasteurised milk cannot be examined by the direct method as there are no means of distinguishing by micro- scopic inspection between living and dead bacteria ^. Furthermore, one cannot be certain that any particular colony may not have arisen from several different species which may have adhered together or become intertwined in the original liquid. If, therefore, a pure culture is desired, it wiU be necessary to sow plates from a well-isolated colony, and only when the resulting colonies are found to consist exclusively of the desired organism is it possible to be sure that the individual colonies are pure. It is still safer to make a single cell the starting point, a method which is described under the next heading. Pure cultures are best preserved in Freudenreich flasks on nutrient agar. As a rule stab cultures are made by piercing the medium with a platinum wire on the point of which a trace of the culture has been picked up. Streak cultures are made of organisms which require free access to air ; the agar is allowed to solidify in a slanting position so as to ejfpose a large surface, and the infected wire is drawn lightly across the surface. In order to protect stab cultures of anaerobic bacteria from the air, the cotton wool plug is flamed and pushed down the test tube till it 1 " Milohwirtschaftliches Zentralblatt," 1912, p. 455. 2 H. W. Oorm (U.S. Public Health. Report, 1915, No. 295) gives some valuable information concerning the limits of accuracy in^the bacterio- logical analysis of milk. 22 DAIRY BACTERIOLOGY >a nearly reaches the agar surface. A plug of absorbent cotton wool soaked in alkaline pyrogallol is then inserted a short distance above the first plug, and the tube is closed by a rubber stopper or a cottoi} wool plug soaked in melted paraffin wax (see Fig. 22). As soon as the cultures have made growth they must be kept at a low temperature or they will quickly die. The acid-producing bacteria keep better, the smaller the amount of sugar contained in the agar medium. Lactic acid bacteria may be. preserved alive for years if not more than 0-25 per cent, of dextrose is employed, but it is s,afer to sow into fresh media every month. If it is desired to keep lactic acid bacteria in perfect condition with respect to their action on milk, they should always be cultivated in milk, and reinoculated into fresh sterilised milk as fre- quently as possible. Inoculation is carried out by means of a platinum wire sealed into a glass rod or fixed into a screwed aluminium holder ; the end of the wire may be shaped as desired ; when dealing with liquids it is looped. The wire is always flamed immediately before use. In order to demonstrate the presence of certain organisms which may be greatly out- numbered by other species, the method of plating fails ; it will be necessary to use the enrichment method of inoculating a small quantity of the material into a medium which the Cui- favours the growth of the organism in question to the disadvantage of others ; the cumulative effect of several reinoculations into the same medium wiU often be the production of a pure culture of the desired organism, or at any rate a culture in which the organism can easily be recognised. Such methods are employed, among others, for demonstrating the presence of pathogenic organisms. -Fig. 22. Method for tivation of Anae- robic Bacteria. (After Sidonwnaen.) METHODS OF EXAMINATION Of all the methods in use for the determination of the species of microorganisms, microscopic examination is one of the most important, for until the shape and size of the cells have been noted it will not even be possible to say definitely whether one is dealing with a yeast or a bacterium. The more highly developed the organism, the completer will be the information to be gained by microscopic examination, especially if the various stages of MICROORGANISMS AND FERMENTATIONS 23 development are studied. In this way the moulds may readily be identified. Greater difficulties are encountered in dealing with the two other groups, which are much simpler in morpho- logical structure ; in these groups cultural and especially bio- chemical characteristics are more important as means of identifica- tion. It has already been mentioned that the appearance of a stab culture will reveal the character of the organism with respect to atmospheric oxygen, and as to whether it produces proteolytic enzymes or coloured products. Sometimes even the shape and general macroscopic appearance of the colony may be so charac- teristic as to afford in itself a means of identification. By varying the composition of the nutrient medium characteristic preferences may be discovered — for example, an investigation may be made to determine which sugars are fermented with the production of acid or acid and gas. Satisfactory results, however, can only be Fig. 23, — Jiirgensen's Moist Clianihcr. got by finding out what fermentation products are formed under certain conditions. For detailed descriptions of the microscope, works on micro- scopy or optics may be consulted. It need only be mentioned here that high magnification is achieved as the product of the magnifications due to the two systems of lenses, the eye-piece (ocular) and the objective. The object to be examined is placed on a glass slide and covered with a square or round cover slip of thin glass. For high magnifications, a drop of cedar-wood oil of the same refractive index as glass is placed between the cover slip and the special immersion objective, an arrangement which entails less loss of light than when air intervenes, and which is essential when magnifications of 1,000 diameters are required. In order to determine whether an organism is motile or non- motile, it is observed in a hanging drop adhering to the under side of a cover slip placed over a hollow slide ; in this way the growth and development of the organism may also be observed. In examining moulds, which require a plentiful supply of oxygen, the cover slip is separated from the slide by a glass ring, and the whole arrangement, known as a moist chamber, is sealed together 24 DAIRY BACTERIOLOGY with vaseline ; a drop of water is placed on the slide to keep the air in the chamber moist, and to prevent evaporation of -the drop on the cover slip. The moist chamber is also employed in making single cell cultures as follows : a miniature gelatine plate is made on a special cover glass marked with numbered squares, and with the aid of the microscope the most isolated cells are sought out and their positions noted with respect to the markings on the cover glass (see Fig. 23). The colonies which grow in these positions may then be sown separately into suitable media. The first yeast culture of undoubted purity was prepared in this v&y by Emil Christian Hansen, an achievement of far-reaching im- portance in the brewing industry. The method is not adapted Fig. 24. — ^A. Comet's Forceps. B. Kiihne'e Forceps. for the preparation of pure cultures of the bacteria, as these are so much smaller than the yeasts. Bacteria may be seen more clearly if they have been stained after fixation on the cover slip. Fixation is accomplished by picking up the cover slip by the edges, between the thumb and first finger, and twice drawing it slowly through a Bunsen flame ; as long as the fingers are not burnt, the preparations wUl not be overheated ; the bacteria are killed, and on this account stain better. If the culture medium is whole milk, the_ preparation should be freed from fat by immersion in chloroform before staining. The cover glass is immersed for a few minutes in the staining solution in a small dish or watch glass, washed with pure water, laid on the slide and dried on the surface by means of filter paper ; it should be handled by means of the Kuhne's forceps (Fig. 24). The stain may also be dropped on to the cover slip held clamped by means of the Cornet forceps (Fig. 24). 'The MICROORGANISMS AND FERMENTATIONS 25 stains most used are alcoholic solutions of methylene blue or fuchsine. The former ^ which must not be diluted too much with water, is specially suited for preparations from milk, as it does not stain the casein strpngly ; if fuchsine is used, a red patch may be formed in which it is impossible to distinguish the bacteria. As fuchsine stains very deeply, it is used in dilute solution ; its staining capacity is increased by the addition of phenol {carbol fuchsine). Tubercle bacteria, which are coated with wax, and intracellular spores, which are very resistant to staining, may be stained by warming with carbol fuchsine, and when thus stained they retain the colour so persistently that mineral acids fail to remove it. Methylene blue is also suited better than fuchsine for staining broth cultures. Acid cultures should, however, first be neutralised, unless the methylene blue solution has been treated with a little alkali. Grain's method of staining is especially useful in the identification of bacteria ; it depends on the fact that microorganisms after treatment with gentian violet are stained a violet black by a solution of iodine in potassium iodide, and retain this stain more or less completely on treatment with absolute alcohol ^. Bacteria which retain the stain are known as Gram-positive, and those which lose it as Gram-negative. Most yeasts and moulds and all true lactic acid bacteria are Gram-positive, while most of the harmful milk bacteria are Gram-negative. The Gram method lends itself well to the examination of lactic acid cultures and sour milk preparations in general, as the casein is completely Gram-negative. Instead of staining, Burri's Indian ink method may be adopted ; a drop of the liquid to be examined is placed on a cover slip, mixed with a little sterile liquid Indian ink, allowed to dry, mounted in water, and examined. If the liquid is acid it should be neutra- lised, as the colloidal Indian ink is coagulated by acid. Treated in this way, the organisms show up white on a black background.' Permanent preparations may be made by mounting in Canada balsam instead of water. 1 V. Jensen ( " Hospitalstidende," 1912, No. 20) recommends a 0-5 per cent, solution of methyl violet in water, and a solution of 1 gram of iodine and 2 grams of potassium iodide in 100 c.c. of water. Notes to page 17. 1 100 gm. sugar free casein (precipitated by acid) were digested for a week at blood heat with a litre of water containing 4-6 per cent. HCl and 2 gm. pepsin. The resulting solution contained, after neutralisation, sterilisation and filtration, about 1 per cent. N and 1-2 per cent. NaCl. 2 At 50° C. the digestion is completed in 24 hours. The sugar dis- appears at the same time. The highly acid solution contains about 2 per cent. N. Chapter II Bacteria Many bacteria differ from the yeasts and moulds in being capable of independent motion, which is accomplished by means of fine threads, knotvn as flagellce, growing from the protoplasm. The monotricha have a single flagellum growing from one end, and the lofotricha a tuft of flagellss similarly situated ; the peri- tricha have flagellse aU round the cell. Motility may be restricted to certain stages of development, chiefly the period of active growth ; on the other hand, a large number of bacteria appear to be non-motile at all stages, and on this account it may be presumed that they are without flageUse ; unfortunately these extremely fine thread-like structures can only be rendered visible by means of complicated methods of staining which sometimes fail. There is, however, no doubt that the disposition of the flageUse affords Fig. 25. — Bacterimn pyocyaneum, monotrich. Fio. 26. — Bacterium nyncyaneum, lofo (After Migula.) X 1,000. trich. (After Migula.) .x 1,000. the most important of the morphological methods of distinction, and one on which the classification of the bacteria should primarily be based. Thus the monotricha and the lofotricha require com- paratively simple nutrient material, being chiefly water bacteria, while the peri trich bacteria are typical fermentation organisms ^. According to their shape the bacteria are divided into sphere, rod or screw forms. As the first-mentioned have no axis of length,- division is possible in three dimensions, and, as a matter of fact, cases are known where division takes place in either one, two or ^ Orla Jensen, " Hovedlinierne i det naturlige Bacteriesystem." Viden- skabemes Selskabs Oversigter, 1908, No. 5. Die Hauptlinien des Natiir- lichen Bakteriensystems. VerJag von Chistav Fwcher, Jena, 1909. BACTERIA 27 three dimensions, resulting in the Streptococcus, Micrococcus and Sarcina forms respectively, provided that the cocci remain united after cell division has taken place. Streptococci may be com- pared with strings of -beads. Micrococci iovia. tetrads, i.e., squares composed of four spheres, and Sarcinse form bundles of cells. FiQ. 27. — Bacterium typhoswm, peritrich. {Mtev Migula.) x 1,000. Fig. 28. — Bacterium vulgare, peritrich (After Migula.) X 1,000. Cocci which adhere together in pairs are known as Diplococci, and those forming irregular aggregates resembling clusters of grapes are named Staphylococci ; these names, however, have no systematic significance. Cocci which divide in more than one direction do not as a rule stretch before division, but form two hemispheres immediately after division. Many streptococci / t s h % / • * j> ly ' .. . '^ % I f tf.i ^ • ■•• • ••• •.1 ^ Fig. 29.— Various Cocci.' (Aiter FHigge.) x 1,000. behave similarly and resemble chains of spheroidal linlts, flattened as though compressed along the direction of growth. Other streptococci become distinctly elongated, forming oval or even rod-shaped links ; an example is seen in Streptococcus lactis, the commonest of the lactic acid bacteria. With the exception of a few sarcinse, the cocci have never been found to form spores. The rod-shaped bacteria are divided into the genera Bacterium and Bacillus. Originally the term bacillus indicated long rods, 28 DAIRY BACTERIOLOGY later, according to Migula, motile rods, while now it generally denotes spore-forming rods. The screw-shaped bacteria, which always have polar flagellse, are classified into the genera Vibrio and Spirillum. The vibrios are monotrich and form single curves, being comma-shaped ; the spirilla are lofotrich and more or less screw-shaped. Certain spirilla are said to be able to form spores. Fig. 30. — Vibrio ckokrce. (After Migula.) b. ^'VA/^^ V «\. Fig. 31. — Various Screw-shaped Bacteria. (After Flugge.) x 1,000. The above remarks may be condensed into the following tabular classification into families and genera : — Family 1. — Coccacese, or Sphere Forms. Genus Streptococcus, dividing in one direction. Genus Micrococcus, dividing in two directions. Genus Sarcina, dividing in three directions. Family 2. — Bacteriacese, or Rod Forms. Genus Bacterium, without spores. Genus Bacillus, with spores. Family 3.— Spirillaceae, or Screw Forms. Genus Vibrio, monotrich. Genus Spirillum, lofotrich. BACTERIA 29 As bacteria may lose the power to form spores, the distmction between the bacterium and the bacillus groups is not sharp. The distinctions between the micrococci and the sarcinse, and between the vibrios and other monotrich rod forms, are still more ill-defined. On the whole, the above system of classification, due to Lehmann and Neumann, is unsatisfactory, as it groups together organisms which have but little in common and separates others which should be grouped together. It is only mentioned here as being that which is most commonly used. As far as the true lactic acid bacteria are concerned, I propose to use generic terms of more essential significance. Together with the bacteria may be classified the Sulphur Bacteiia, the Thread Bacteria and the Ray Fungi. Sulphur bacteria are always present where hydrogen sulphide is pro- duced, for example in rotting seaweed, where they transform the poisonous gas into sulphates necessary for plant growth. Colourless and red species are known in forms of almost end- less variety. The thread bac- teria, like the sulphur bacteria, are typical water organisms ; they are found attached to solid ^-- ''-^^^^^^'^ ^. (^"" objects, and their outer cell walls form sheaths in which may be accumulated large amounts of iron. They may cause stoppages in water pipes, and contribute towards the formation of bog-ore. The ray fungi, or Actinomycetes, may be regarded as being intermediate between the bacteria and the moulds, especially the Mycomycetse (Mucorinas) ; on the one hand their cells are as slender as those of the bacteria, on the other hand they ramify and form oidia by constriction. They are of very common occurrence in soil, contributing to its characteristic smell. They may develop in butter, in which they produce a similar smell ^- One variety, Actinomyces bovis (Fig. 32), is the cause of actinomycosis in cattle. The organisms of diphtheria, and especially those of tuberculosis, are closely related to this group. The Spirilla are also typical water bacteria, being difficult to cultivate artificially. As representing the vibrios, only, the well- known cholera vibrio need be mentioned (Fig. 30). In dairy 1 Orh, Jensen, " Centralblatt fiir Bacteriologie," 2 Abt., 1902, Bd. VIII., p. 250. 30 DAIRY BACTERIOLOGY practice we have to deal almost exclusively with cocci and rod- shaped organisms ; special attention will therefore be paid to these groups in this work. THE LACTIC ACID FERMENTATION This is the most important fermentation in dairy practice. In the true lactic acid fermentation, lactic acid is practically the sole product arising from the fermented sugar. The chief by-products are carbonic and acetic acids. The production of carbonic acid Fio. .33. — Thfrmohaclerium bulgaricum. Grown in sterile milk. Stained with ^ methylene blue. The grains arc round and dark blue. •; 1,000. in recognisable amounts is a sign that the lactic acid bacteria are in full vigour ; acetic acid, on the other hand, only appears in appreciable quantities in stale cultures, or when the bacteria are given a plentiful supply of air, either by aerating the liquid during fermentation or by allowing it to expose a large surface to the air. Psevdo lactic acid fermentations are characterised by the formation of considerable quantities of volatile acids, gases and other by- products, such as succinic acid and alcohol. In view of the fact that in dairy practice only the true lactic acid fermentation can be turned to economic advantage, while the pseud o lactic acid fermentations generally cause trouble, the terms "true" and BACTERIA 31 "pseudo" may be taken as corresponding with the concepts of useful and harmful lactic acid bacteria. Lactic acid was first studied systematically by the Swedish chemist Scheele in 1782 i. It is a syrupy liquid, entirely soluble in water, alcohol and ether. The most convenient method of preparing it pure is to evaporate sour whey to a syrup, and to extract this with ether. If chalk is stirred into the fermenting liquid from time to time the acid will be neutralised, and thus prevented from weakening the bacteria, which will then be able to ferment the whole of the sugar. Lactic acid maj? be prepared on ■ Fig. .34. — Thermoharteriiim bulgaricum. Grown in milk pasteurised by heat- ing previously to 80° C. for half an hour. Stained with methylene blue. The grains are long-drawn and red. Capsule clearly shown X 1,000. a large scale from a 10 to 20 per cent, solution of maltose, made by saccharifying starch with extract of malt containing active diastase. The solution is treated with chalk, sterilised and fer- mented by a pure culture for a week or two at 50° C. , the culture being kept pure at this temperature. As calcium lactate is formed, the solution gradually sets to a pasty mass of crystals, which is pressed and decomposed with sulphuric acid ; after filtering off the calcium sulphate the liquid is evaporated, preferably in vacuo, till it contains 40 to 80 per cent, of lactic acid. The yield is about 75 per cent. Lactic acid and its salts are largely used as mordants in the dyeing and tanning industries. Before the War, Germany ' "' Ksl. Vetenskap.s Academieii.s nya Handlingar," Bd. III., p. 120. 32 DAIRY BACTERIOLOGY produced 1,000 tons annually. Polarimetric observation reveals three forms of lactic acid, dextro and Isevorotatory, and inactive. ~ The last-mentioned is a mixture of the two optically active acids in equal proportions, and differs from these in the solubility of its salts as well as in its optical properties ^. In order to identify a lactic acid bacterium, it is necessary to know what form of lactic acid it produces under certain conditions. In 1857, Pasteur discovered that the lactic acid fermentation was due to the action of certain bacteria ^, and soon after the discovery of the method of making gelatine plate cultures, Hueppe, in 1884, succeeded in isolating one of these bacteria^. This organism, which was named Bacillus acidi lactici, is not one of the true lactic acid bacteria, but belongs to the aerogenes group, which will be dealt ivith later. The organism which plays the principal part in the self -souring of mUk had already been correctly described by Lister, in 1878, as an oval diplococeus, and given the name Bacterium iactis *. A similar form was isolated by Grotenfeld in 1879, and named Streptococcus acidi lactici ^. The importance of this organism was, however, only recognised by Leichmann in 1894®, and by Guniher and Thierf elder'' in 1895, who demonstrated that it produced dextro lactic acid in milk. Leichmann named it very appropriately the bacterium of sour milk. Bacterium lactis acidi. The large number of lactic acid bacteria now known really only resemble one another in not forming spores, and therefore being compa,ratively easily destroyed by heat. The True Lactic Acid Bacteria. — These bacteria ferment carbohydrates and higher alcohols to lactic acid ; they only grow in presence of proteins or complexes of amino acids, and not in presence of ammonium salts or single amino acids. They are Gram-positive, non-motile, non-sporing rod or sphere forms. It will only be possible here to mention the more important character- istics by which nearly related forms are best distinguished. The following is a brief summary of the results of the author's recent researches on this subject ^ : — ^ The lactic acids are best identified by conversion into zinc salts. The active zinc lactates rotate the plane' of polarised light in senses opposite to those due to the free acids, and they crystallise with two molecules of water, corresponding to 12-9 per cent. HgO, which is not driven off below 140° C. The inactive zinc lactate is much less soluble, and crystallises with three molecules of water (18-2 per cent. HjjO), which is more readily driven ofi on heating. 2 " Comptes rendues," tome 45, p. 913. 3 ."Mitt. Kais. Ges.-Amt.," Bd. II., p. 309. « " Trans. Path. Sec. of London," vol: 29, p. 425. 6 " Portschr. Medizin," Bd. VII., p. 121. • " Milchzeitung," Bd. XXIII., p. 623. " ' " Archiv. fiir Hyg.," Bd. XXV., p. 164. * " The Lactic Acid Bacteria." Monograph published in English by the Danish Academy of Sciences, Copenhagen, 1919. BACTERIA 33 (A) No catalase formation, nitrate reduction nor surface growth. (a) Forming only traces of hy-froducts in addition to lactic acid. Rod forms . . Genera Thermobacterium and Strepto- bacterium. Sphere forms . . Genus Streptococcus. (b) Generally forming appreciable amounts of gas and other. by- 'products in addition to lactic acid. Rod forms . . B'etahacterium. Sphere forms . . Betacoccus. . (B) Usually forming catalase, reducing nitrates and showing surface growth. Rod forms . . Microbacterium. Sphere forms .. Tetracoccus '^ Group B has been included because it forms a limiting group to the lactic acid bacteria on the one side, just as the Coli and Aerogenes group do on the other, though none of these can be reckoned as true lactic acid bacteria. The last-mentioned group includes group A, and also Bacterium bifidum, which, on account of its branched form and obligate anaerobic character, occupies a unique position. Group A. — Although we may well suppose that the genera Streptobacterium and Streptococcus and the genera Betabacterium and Betacocciis are respectively more closely related to one another than the various rod forms to one another, and the various sphere forms to one another, we will, however, seeing that the rod forms as a whole are stronger acid producers than the sphere forms, keep up the old tradition and treat the rod' and sphere forms separately. ( 1 ) Rod Forms (named Lactobacilli by Beijerinck) . — Short or long , straight or curved rods which may grow out into long threads. Sometimes they contain granules which stain more readily with methylene blue than the rest of the protoplasm. They may occur in pairs or chains of varying lengths and regularity of form. To this group belong the most typical of the lactic acid bacteria, which produce, and are able to stand, greater quantities of lactic acid than the other lactic acid organisms. Some of them may form as much as 3 per cent, of lactic acid in milk. The members of the Thermobacterium genus are long rods, which thrive at 40° to 50° 0. or even higher temperatures, .but not below 22° G. ; they will generally obtain predominance in milk kept above 40° C. They form Isevo or inactive lactic acid, and with one exception, Tbm. 1 Common term for acid-forming Micrococci and Sarcinse. 34 DAIRY BACTERIOLOGY cereale (Bacillus Delbrilcki), they attack casein to a considerable extent, and thus come to play an important part in the ripening of strongly scalded cheeses such as Emmental or Gruyere. To this genus belong the strongest acid producers, e.g., Thm. helve- ticum (Bacterium casei e), which may produce over 2-7 per cent, of inactive lactic acid, and which plays an important part in the ripening of Emmental cheese. Thm. bulgaricwm. {Bacillus bulgaricus) forms up to 1-7 per cent, of Isevo acid, and Thm. jugurt forms as much inactive acid as Tbm. helveticum, and grows in peculiar feathery-shaped colonies. These two last-mentioned organisms occur in Bulgarian sour milk. Organisms belonging to the Streptobactermm, genus are short of long chains of short or long rods, which as a rule have a maximum Fig. .'15. — Thermohaclerivni helfeliciim from Emmpntal Cheese. (After Freiidfiireich.) X 1,000. Fig. 36. — Strfiptoha^teriiwi casei from Danish Dairy Cheese. ... 1,000. of 35° to 40° C. ; they gradually come to predominate in dairy products which are kept at temperatures below 35° C, and are therefore frequently found in cheese. They form inactive or dextro acid. Sinn, casei {Bacterium casei a) hydrolyses casein, while Shm. plantarum. does not do so. The Betabacteria almost always form inactive acid, and when in a freshly isolated state, perceptible amounts of by-products ; they have no action on casein, and as a rule do not grow well in milk. As examples may be named Bbm. breve and longum {Bacterium casei y and 8 respectively). The former ferments arabinose strongly, and frequently also xylose ; it has a maximum temperature of 38° C. The latter never ferments arabinose, but frequently ferments xylose and rafifinose ; its maximum tempera- ture is 45° G. Bbm. caucasicum is the chief constituent of Kefir BACTERIA 35 grains ; it forms appreciable amounts of acid together with yeast, and its optimum temperature lies below 30° G. (2) Sphere Forms. — The Streptococci are related to the Strepto- bacteria,. the points of similarity having been mentioned above. They usually grow out into long chains when cultivated in broth, but in milk and on solid media they may present varied appear-' ances. They grow well in milk, and always form dextro lactic acid, though very seldom more than | to | per cent., i.e., not much more than is required to coagulate njilk! They are better able to grow on the surface of solid media than the rod forms, being less strictly anaerobic in character, but they do not form spreading Fio. 31 .—Streptococcua cremoris. (Streptocociyas lacticus.) colonies. With the exception mentioned below, the Streptococci show very little tendency to hydrolyse casein, and completely lose the power to do so if not cultivated in mUk. Their optimum temperature is as a rule 30° C, and many strains do not grow above 37° C. Sc. thermophilus, however, grows best at 40° C. ; ^it is irregular in shape, and is easUy isolated from milk which has been kept at a fairly high temperature. The greatest interest attaches to Sc. cremoris, which is used for ripening cream in butter making ; • it forms long chains in milk and grows best at 25° to 30° C, but not at blood heat. Shme-producing strains are represented by the " bacterium of long milk," and Sc. Hollandicus. The former of these was first described by Gerda Troili-Petersson under the name 36 DAIRY BACTERIOLOGY of Bad. lactis longi^, and the latter by Weigmann^ ; this organism was to be found in the ropy whey formerly used in the manufacture of Dutch (Edam) cheese. Sc. mastitidis (8c. agalactice), which produces notable quantities of lactic acid in milk, is the cause of mastitis or inflammation of the udder in cows. It may be recog- nised by the orange colouring matter which it produces after some time in agar or broth with casein peptone and soluble starch. Unlike Sc. cremoris, it ferments saccharose, maltose and dextrin. 8c. pyogenes is a general term for a number of pathogenic streptococci which do not coagulate milk ; they cause boils and many other similar diseases in animals and human beings. Belonging to this group are also the common bacteria of sour milk, Sc. lactis {Bad. ladis acidi, Leichmann), which always obtain Fig. 38. — iSlreplococcii-s cremoris (Sturch'a No. 18). X 1,000. Fig. 39. — StreptucocrAis Ihermophilus. predominance in milk which is kept at ordinary room temperature, appearing mostly as diplococci in milk. It ferments dextrin, but not saccharose. Sc. fmcium is also a typical diplococcus form, which grows even at 50° C. , and is very common in the manure of mammals. Other related organisms appear both as diplococci, and as longer chains in the same culture. They are distinguished according to their power to ferment a number of different sub- stances such as glycerine, sorbitol, maltose, dextrin and salicin, and generally also pentoses and saccharose ; their maximum temperature is about 45° C. As an example may be mentioned Sc. liquefaciens '^, which liquefies gelatine and produces a bitter taste in cheese. 1 "Zeitschr. fiir Hyg. und Infeotionskrankheiten," 1899, Bd. XXXII., p. 361. 2 " Milchzeitung," 1899, Bd. XVIII., p. 18. 3 Freudenreifh originallv named this organism Micrococcus casei amari ("Landwirt. Jalirbuch der Schweiz," 1894, p. 136). BACTERIA 37 The Betacocci have been so named by the author because they are generally found in sugar and other beets, swedes and mangold wurzels, especially when these are in a state of decomposition. In countries where such roots are largely used as cattle fodder, the Betacocci areof very common occurrence Fin. 4((. — Strc'ijlorncni-': litfti-'!. (\ ^^ -k '. ■» Fro. 41. — Bi'lai-iirciiK from thin juice frcjm Nakskov .Sugar Factor}'. ,- ', - ." ■■•■ ■■ -% -' • Fig. 42. — Streptococcus liquefariens {Freii- dcnreich's Micrococcus eifci amari). Fig. 43. — Streptococcus tiquefaciens (Escherich's Streptococcus coli gracilis). in milk and in the cheese made therefrom. The Betacocci occur as diplococci or short chains, wliich are not to be dis- tinguished microscopically from the streptococci which have been dealt with above. They generally form Isevo lactic acid, gas and other by-products, and render saccharose broth more or less slimJ^ The slime formation may best be observed in 1 38 DAIRY BACTERIOLOGY stab cultures in saccharose gelatine. Some species liquefy this medium after some time, though they do not decompose casein. The Leuconostocs (Fig. 12, p. 7), which may give a deal of trouble in beet sugar manufacture, are Betacocci. They grow at temperatures as low as 5" 0., and some species thrive better at room temperature than at 30° C. The sour cabbage bac- terium, Sc. hrasfiicw, also belongs to this group. Fig. 44. — Various Betacoocd in Stab Cultures in Cane-sugar Gelatine. Group B. — The bacteria belonging to this group are not true lactic acid bacteria, differing from the forms hitherto described in forming catalase, reducing nitrates and generally growing well on the surface of solid media. The microbacteria are very small rods, usually only 0-3 to 0-4 /^ thick, which stand fairly high temperatures, and are therefore to be found in pasteurised milk. They form lactic acid, and some of them (Bacillus acidophilus) are common intestinal organisms. To these bacteria arc related a group of small rod bacteria which liquefy gelatine, but only produce traces of acid. The tetracocci include the acid-producing forms of the Micro- cocci and Sarcince. Division into these two groups is not feasible, and confusion may even occur with the Streptococcus group, as many of the Tetracocci appear as diplococci ; they may, however, readily be distinguished by their power to decompose hydrogen peroxide, for, as mentioned above, they differ from the true lactic acid bacteria in producing catalase. They produce less lactic acid BACTERIA 39 than the Streptococci, while they form notable amounts of acetic acid besides. They , are more aerobic in character than the Streptococci, forming as a rule large surface colonies which are often coloured yellow, orange or pink. Most of them liquefy gelatine, though generally slowly, and they will therefore also coagulate milk, though the quantity of acid formed is seldom sufficient to accomplish this. Several of the liquefying species probably play some part in the ripening of certain cheeses, for^ example Tetracoccus liquefaciens^, which forms white colonies and produces dextro lactic acid. The Tetracocci are found in great numbers in cow dung and earth, whence they find their way into dust ; they are well able to stand desiccation and common salt, and many of them will also stand heating to over 70° C. They are common skin bacteria, some giving rise to pustules and others to inflammations, which are generally not of a dangerous character. According to Beijerinck, certain lactic- acid-producing sarcinse which occur in soil, but are of no import- ance in dairy practice, produce large amounts of carbon dioxide and hydrogen. The Pseudo Lactic Acid Bacteria. — These are motile or non-motile. Gram-negative short rods with rounded ends, which seldom form chains or threads of any length. They do not require organic nitrogen, and in stab cultures show profuse surface growth. As a rule they do not liquefy gelatine, and are mainly intestinal and excremental bacteria. The gas which they produce from sugars consists of carbon dioxide and hydrogen in widely varying proportions ; according to certain American investigators, the composition of the gas formed affords a basis for the classifica- tion of these organisms ^. The aerogenes bacteria are non-motile rods which produce large amounts of gas, being able to convert most of the sugar present into gas, especially if the acid which is produced is neutralised. The gas may contain up to three times as much carbon dioxide as hydrogen, while aerogenes forms are laiown which may esven produce carbon dioxide alone. In dairy bacteriology two types may specially be distinguished. The one forms much slime which gives rise to outstanding colonies with the shjny appearance of porcelain ; appreciable amounts of alcohol are formed, but not acid enough to coagulate milk. If the acid is neutralised by chalk as fast as it is formed, the milk will gradually be converted itito a thick slime. To this type belong certain pathogenic bacteria, such 1 The author ("Landwirt. Jahrbuch der Schweiz," 1904, pp. 349 and 369) has described this organism under the name Micrococcus casei liquefaciens. " L. A. Sogers, W. Mansfield Clark, Brooke J. Davis and Alice 0. Evans (" Jouin. of Infectious Diseases," vols. 14, 15 and 17). 40 DAIRY BACTERIOLOGY as Bacterium pneumonice, and some bacteria which, according to Oilleheau, cause inflammation of the udder. The second type, to which belongs Bacterium lactia aerogenen, produces less slime, and its colonies on gelatine are often but little larger than those of the lactic acid streptococci. They generally coagulate milk by the production of succinic acid and Isevo lactic acid. The aerogenes bacteria can convert the citric acid of the milk into acetic and carbonic acids. On corn and flour aerogenes bacteria are found which produce a yellow colouring matter. Bacterium cloacae is a liquefying aerogenes bacterium. Motile forms related to this organism may be regarded as a link with the Proteus bacteria. In cheese such forms are often found, but these form spores in one end of the cell, and must there- fore be designated as aerobic Plectridia. The coll bacteria are as a rule peritrich, though often they are only slightly motile. Vf They differ from the aerogenes bacteria in producing about equal quantities of carbon dioxide and hj'drogen, and also i.- -^ K r V ** j^ "^ ' ^ V in their power to decompose casein. In the latter respect they show a certain resem- * ' blance to the true lactic acid Fig. 4.5.— The Coli Bacterium whicli spoilt bacteria, but thev carrv the the Milk and Butter at iJuclunil JJairy , .^. . /. ^, ■' ^. in 1888. (After f. 0. Je„.- — Rdttiiii; Swede rnntainiiiy nunuTous Portin-forineiitinii Fleet riilia. 1 Orln-Jenseii, " Ijandwirtsoliaftliches Jahrbucli rter Schweiz," 1905, 58 DAIRY BACTERIOLOGY able fermentations. Diffusion slices ^ are generally supposed to exert a particularly undesirable influence on milk in connection with cheese making ; but as a matter of fact, they, are harmless in a properly soured condition ; on the other hand, if they are given in spring or summer when they have begun to putrefy, they will be just as dangerous as any other putrefying roots ; the author has found that such material is the chief source of butyric acid and aerogenes bacteria in milk. It is obvious that cleanliness will be promoted by cutting the hair on the udders and hindquarters of the cows as short as possible. The condition of the ground or the covering of the stable floor has a most important bearing on cleanliness. The most favourable conditions are on the pastures in dry weather. In rainy weather the cows may be badly soiled with mud which, as has been mentioned above, is a fruitful source of sporing bacteria ; milk obtained under these conditions is on this account' difficult to steriUse. In damp meadows or during a prolonged spell of wet weather, microorganisms will grow abundantly on the surfaces of plants. The floor of the cowshed must be weU covered with fresh straw, but it is better for the cows to rest on a clean cement floor than on decaying straw or husks which give rise to dust rich in microorganisms. Peat, especially the long fibred variety, is good, but it must not be allowed to remain until it becomes sloppy. If the shed is not planned so that the cows cannot lie down in their dung, someone must be at hand to remove or cover up the dung at once. There should be a sharp fall in the floor where the dung is deposited. If once the udders have become badly soiled, it will be impossible to obtain the milk in a decent condition. Rubbing the udder with a cloth, which will very quickly be dirtied, is of little avail ; proper cleaning of the udders will be very difficult of accomplishment on large farms. Prevention will be found to be far easier than cure, and if this matter were only given the atten- tion which it justly deserves, the farmer would be rendering a most valuable service to the cause of clean milk. It should go without saying that everyone who touches food should have clean hands, but it is of little avail that the milkers wash their hands only to soil them again immediately they com- mence their work. Of course, something will be gained if the hands are thoroughly washed after the milking of each cow, provided that they are not soiled again by touching the bottom rim of the pail or the cobwebs on the beams, or by slapping the dirty flanks of the cow to make it give room for the milking. Clean hands and clean clothes are without doubt much to be 1 A by-product from beet sugar manufacture, which is used as fodder in countries where this industry has been developed, CLEANING AND THE PROCUREMENT OF MILK 59 desired, but in the first place we must insist on clean cows and clean sheds ; when these points have been gained the rest will no doubt follow as a matter of course. Dry milking is naturally more hygienic than wet milking, but as experience has shown, it is extremely difficult to carry out in practice ; it is facilitated by smearing the teats with a little vaseline or fatty material. This practice, which was first proposed by GiUebeau of Berne, has the additional advantage that the dirt is taken up by the greasy matter ; should a drop of the grease fall into the milk it will not mix readily with the latter, and it will be removed on straining through cotton wool. The hope that cleaner milk would be obtained by the use Fid. ~y.). — Jfilking Room at Faiicrliolni. of milking machines has hitherto been disappointed. These machines, with their numerous corners, cavities and rubber tubes, are so difficult to clean and sterilise that they require far more intelligent and conscientious attention than they are likely to receive at the hands of the average milker. As already mentioned, the cleaned Pails should receive a final rinsing with hot sterile water, and this is especially necessary if the farm has not a good water supply. It has often been main- tained that bad drinking \\'ater for the cows will give rise to bad milk ; this, however, onlj^ applies in cases where the cows may have contracted some disease through the water ; the direct infections caused by rinsing the pails with bad water will have far 60 .DAIRY BACTERIOLOGY more serious consequences. To avoid the introduction of dirt into the milk in filling or emptying the pails, cans or churns during weighing or measuring, care must be taken that these vessels do not become, soiled during transport, and especially that they do not become plastered with mud round the bottom rim where they will be gripped on emptying. The cans should be covered with tarpauUn during transport, and they should not be stood on the ground but on clean planks. The possibility of infection being carried by Flies is by no means to be overlooked as microorganisms may be brought from any place where the flies may have lodged. In cowsheds the danger is best avoided by removing the dung as often as possible and by hanging up, just below the ceiling, wide shallow enamelled dishes containlhg skim milk to which four tablespoonfuls of formalin have been added per litre (or quart). To ensure fresh air during milking, the cowshed must be well ventilated beforehand, and as many doors as possible be kept open during the milking ; in this way, the additional advantage of a good light wilt be secured. Cleaning operations of any kind or feeding should be avoided just before or during the milking, in order that the air may be as free from dust as possible. Naturally, it would be an advantage if the hindquarters of the cows could be brushed down just before milking ^ ; but the evil effects of the dust raised in the process are only to be avoided if a special milking room is available. Fig. 53 illustrates the room set aside for this purpose at Fauerholm, near Frederiksborg, whence the city of Copenhagen receives its highest grade milk, which is known as " Ismaelk " (ice milk), as it is received into pails specially designed by Busck, which are provided with jacketed bottoms containing a freezing mixture. . The milk is (or should be) strained on the farm, in order to remove the coarsest of the dirt particles. By means of Ulander's filter, in which the filtering medium is a layer of cotton wool, most of the finer dirt particles may also be removed. Although the renewal of the cotton wool each time it becomes choked may entail some expense, this filter is strongly to be recommended, as the benefit to be derived from the immediate removal of the dirt is incomparably greater than that derived from the subsequent cleaning which the mUk may receive on arrival at the dairy, when the soluble dirt and the bacteria contained therein will have become distributed throughout the milk owing to the shaking of the cans during transport. The best practice of all is to prevent the dirt from ever entering the milk by tying a straining cloth over 1 In the procurement of the American " certified milk " the cows are often vacuum cleaned before milking. ■CLEANING ANt> THE PROCUREMENT OF MILK 61 the pail, as is done when using Ourler's pail (Fig. 54) ; the cloth must of course be changed frequently in order to avoid the dirt particles thereon being broken up and washed through by the impact of the milk from the udder. . We need not here enter into the discussion of other refinements calculated to further the production of clean milk, regardless of cost, which have been adopted in some other countries, for the public catered for in such cases is of necessity strictly limited ; the points which do immediately concern the general public are the necessity for improved cowsheds and for more efficient and intelligent labour for the tending of the cows and the cooling of the milk. As, however, these matters also involve extra expense, the only hope of progress lies in the institution of a system of payment according to quality. The following figures illustrate the points discussed above : — Fig. 5i.~ Gurler's and Stadlmillkr's Milk Pails. (After Conn.) Cow dung contains over 1,000 million organisms per gram. - Straw and earth contain up to twenty million organisms per gram. According to Barthel, the air in a well-kept cowshed contains on an average, 300,000 organisms per cubic metre during the dinner hou^, and over a million during the feeding of the cattle. According to Harrison, 20,000 organisms fall into the milking pail per minute if the manure is removed and straw laid during the milking, but only 1,000 if this work is done one hour before the milking. However clean the cows and the shed may be kept, it is im- possible to obtain absolutely sterile milk as the udder, even when healthy, always contains some bacteria which find their way in through the opening in the teats ^. There will always be a fairly 1 A few investigators are of the opinion that bacteria also find their way into the udder through the blood. 62 DAIRY BACTERIOLOGY large number of bacteria in the milk duct, and if high grade milk is desired, the very first portion of the fore milk from each teat should always be excluded. Thus the author found the following results on milking from a washed udder and teats into sterile bottles : — In the fore milk from the four quarters, 16,000 organisms per cubic centimetre. In the middle milk from the four quarters, 480 organisms per cubic centimetre. In the strippings milk from the four quarters, 360 organisms per cubic centimetre. The number of bacteria present in the udders varies ; if the teats are regularly washed and- disinfected, and protected from dirt between the milkings, e.g., by enclosing them in a waterproof bag, the bacteria in the milk may be reduced to ten per cubic centimetre. The American " certified milk " which contains less than 10,000 organisms per cubic centimetre, keeps extremely well ; instances have been observed of this milk, keeping quite good at 0° C. for more than a month, ajid only containing 1,000 organisms per cubic centimetre after a week's keeping. According to Burri, milk fresh from the cow, obtained under ordinary circumstances, contains 3,000 to 86,000, and on an average, 21,000 organisms per cubic centimetre. Chapter II The Normal and Abnormal Microflora of Milk A. THE NORMAL FLORA Milk fresh from healthy clean cows does not contain many organisms beyond a few from the air, and those which normally occur in and on the udder and teats. These organisms are nearly all micrococci and sarcina forms, most of which are without action on milk, while a few both acidify and peptonise it. Accord- ing to Arthur Wolff ^, certain alkali producing short rod forms (Bacterium lactis innocuum) rank next to the micrococci in point of numbers, in fresh milk. In appearance, these organisms and their colonies closely resemble the aerogenes bacteria, but they do not ferment sugars, and they render milk feebly alkaline • instead of acid without causing any other change. According to Burri and Hohl, Streptococcus liquefaciens is occasionally found as a pure culture in the udders of healthy cows ^- Milk which has been less carefully handled contains, in addition to the above, coli, aerogenes, proteus and hay bacteria, ray fungi, moulds, ' yeasts, fluorescent bacteria and sometimes also butyric acid bacteria. The coli, aerogenes, proteus and butyric acid bacteria generally come from the dung, the fluorescent bacteria from the water used for rinsing the pails, and the others chiefly from the bedding and the stable dust. If the cattle are on pasture, the fluorescent and sporing bacteria may come from the ground. Curiously enough, the typical streptococci are seldom found in milk fresh from the cow. According to BartheVs investi- gations *, they occur in cow dung with which they are distributed over the fields, being therefore found on all cultivated plants. From the latter they find their way back to the cow, and mUk 1 Inaugural Dissertation, Zurich, 1908. Bacterium lactis innocuum is., probably identical with the organism known in th^ literature as Bacterium, alcaligenes. 2 " Sohweitzerische Milchzeitung," 1916, Nos. 3 to 8. ^ " Landbruks-Akademiens Handlinger och Tidsskrift," 1905, p. 403. It will, however, be necessary to revise these investigations since we have now learnt better to differentiate between the various species of lactic acid bacteria. Thus a large proportion of the lactic acid bacteria of the plants are not streptococci in the narrower sense, but betacocci. 64 DAIRY BACTERIOLOGY may thus be infected with streptococci not only direct from the manure, but also from the bedding and the dust from the fodder. The milk pails are equally important .as sources of infection, for they will as a rule be impregnated with lactic acid bacteria. These bacteria may also be introduced by flies. The bacteriological state of milk -will be (Jetermined at the outset according to the degree of care with which it has been handled, and the nature of the bedding and the feed. If, how- ever, milk is kept for any length of time, the temperature at which it is kept will play an all-important part as it is the factor which determines which groups of microorganisms shall develop in preference to other groups. Not only the various groups of bacteria, but also the milk itseK rnust take part in the struggle for existence, for milk contains bactericidal substances which, however, are gradually weakened in their action, presumably by the bacteria themselves ; only at low temperatures, at which bacterial develop- ment is inhibited to a considerable extent, can these substances retain their activity for any length of time, and at these tem- peratures it is found that the number of organisms decreases at first instead of increasing. In the following table are given the numbers of organisms after twenty -four and forty -eight hours in the same milk kept in sterile flasks at different temperatures. The milk originally contained 84,000 organisms per cubic centimetre, of which 2,000 were liquefying. 0°0. 12° C. 20° C. 30° C. 38° C. 45° C. After twenty-four hours keeping. Grelatine— Total Liquefying Agar total . 52,000 18,000 8,200,000 1,600,000 163,000,000 6,000,000 * 380,000,000 200,000 460,000,000 * 17,400,000 20,000,000 * 12,000,000 100,000,000 After forty-eight hours keeping. Gelatine — total Liquefying Agar total . 252,000 60,000 27,000,000 1,800,000 * 350,000,000 2,000,000 * 380,000,000 500,000,000 * 3,000,000 - 60,000,000 1,200,000 222,000,000 * The milk was clotted. It will be seen that at 0° C. the number of microorganisms was lowered in twenty-four hours from 84,000 to 52,000 owing to the action of the bactericidal constituents of the milk. At the same time the number of liquefying bacteria had increased, and Ihe NORMAI^ AND ABNORMAL MICROFLORA OF MILK 65 examination after forty -eight hours showed the total number to have increased. ChiUing to 0° C. will therefore not prevent bacterial development indefinitely. At temperatures above 10° C, bacterial multiplication is rapid ; thus in the present example, after twenty- four hours at 12° C, the number of organisms had increased one hundred fold, and at 30° C, five thousand fold. Thirty degrees to thirty -five degrees is the optimum for most inilk bacteria ; already at 38° C, many of them cease to grow and, moreover, the inhibitory effect of the acid which is formed increases with the temperature, as is shown in the table, particularly in respect to the liquefying organisms. In order to make bacterial counts of samples kept at the higher temperatures, it was necessary to employ agar, prefer- ably in BuYri's tubes (see p. 19) at 40° C, as organisms would be present which would not grow at ordinary temperatures. A comparison between gelatine and agar plates from milk kept for forty-eight hours at 38° C. showed that the original flora was being suppressed, while a new flora consisting entirely of rod-shaped lactic acid bacteria was making its appearance. At 45° C, a temperature unfavourable for the development of the common milk bacteria, this- change proceeds much more rapidly. The lactic acid xods which develop at this temperature are mostly thermobacteria producing Isevo lactic acid. The 'results of investigations of the flora of milk at different temperatures, by Conn and Esten ^, Arthur Wolff, Luxwolda ^ and the author, are as follows : — 1. Under 5° C. the fluorescent bacteria predominate. 2. Between 5° C. and 10° C, in addition to fluorescent bacteria, proteus bacteria, micrococci, alkali producing rods, and, as Beijerinck has shown, also some rods which produce an aroma of fruit. 3. Between 10° C. and 15° C. betacocci, streptococci, and some species of aerogenes bacteria in addition to the above. 4. Between 15° C. and 30° C. the streptococci, especially Sc. lactis, predominate. 5. Between 30° C. and 40° C. coli and aerogenes bacteria and lactic acid forming rods in addition to streptococci. 6. Above 40° C. lactic acid forming rod bacteria and Saccharo- mycetes, which ferment lactose, predominate. The streptococci which are found are now chiefly Sc. thermophilus and 8c. fcBcium. It is well known that the lower the temperature to which milk is cooled the better it keeps ; in this connection, some results obtained by Kjaergaard Jensen may be quoted ; they repre- sent the bacterial counts of the same milk kept for eighteen 1 Ann. Rep. Storr's Exp. Station, 1904. 2 " Centralblatt f. Bakt.," 2 Abt., 1911, Bd. XXXI., p. 129. 66 DAIRY BACTERIOLOGY hours at the temperatures which are most general in actual practice. Number of bacteria per CO. Immediately after milking . . 1,480 After standing eighteen hours at 9° C. . 2,100 jj a 12° C. . 5,600 , 33 55 15° C. . 156,000 55 35 18° C. . 550,000 55 JJ 21° C. . . 6,750,000 Naturally, the cleaner the milk the greater will be the benefit derived from cooling. As already pointed out, it is not advisable to keep milk longer than twenty-four hours, even if cooled to 0° C. ; if it is to be kept longer it must be frozen in order to avoid the risk of the development of fluorescent and other water bacteria which produce an unpleasant taste ; at somewhat higher tem- peratures certain toxic proteus bacteria will also develop. For these reasons cooled milk or cream which has stood for any length of time are to be regarded with suspicion, even if apparently unchanged. As a rule milk does Tiot become coagulated when kept at temperatures below 10° C, but above this temperature coagulation takes place in the course of a few days owing to the action of rennet and acid forming bacteria. At 20° C. milk quickly becomes coagulated, obviously owing to the formation of acid ; at this temperature the streptococci, especially 8c. lactis, develop so freely that after a time they will come to con- stitute about 90 per cent, of the bacterial flora. The presence of large amounts of lactic acid inhibits the growth of other milk bacteria, for which reason the sour milk thus produced is harmless as an article of food. As already mentioned, at higher tempera- tures the streptococci give place to rod bacteria which produce higher concentrations of lactic acid. As regards the gas forming lactic acid bacteria, a few of these grow even below 10° C, but as tjrpical intestinal bacteria they have as a rule a high optimum temperature and will most readily obtain predominance at 38° to 40° C. ; this temperature is somewhat high for the strepto- cocci, and the slowly growing lactic acid rod bacteria will only come to exercise an inhibitory effect on the other forms at a later stage. For similar reasons the temperature mentioned is also the most favourable for the development of the anaerobic sporing bacteria which form butyric acid. On the other hand, the aerobic sporing hay and potato bacilli, which often constitute the main flora of pasteurised milk, are practically inert in raw milk ; their spores do not germinate at the ordinary temperature, and at higher temperatures the growth of their vegetative cells is. inhibited NORMAL AND ABNORMAL MICROFLORA OF MILK 67 by the lactic acid formed by other organisms ; as a group, they are extremely sensitive to acid. There are, howeyer, varieties of Bacillus mycoides which can become accustomed to fairly large amounts of acid and can even be induced to produce acid them- selves. As the aerobic organisms grow best near the surface of the milk, and the anaerobic near the bottom, the pseudo lactic acid bacteria will be more plentifully represented in the cream, and the true lactic acid bacteria will predominate near the bottom of the vessel 1. Hence the spontaneous curdling of milk by souring always starts from the bottom. While several of the pseudo lactic acid bacteria produce Isevo lactic acid, the common lactic acid streptococci produce the dextro acid only ; the result of the combined action of the two groups is a mixture of inactive and dextro acids. After some time, these organisms are supplanted by lactic acid forming rods, not only at higher temperatures (thermobacteria), but also after keeping at lower temperatures {strepto- and betabacteria) ; as many of these rods form inactive acid, the so-caUed fermentation acid will consist chiefly of this variety. In the spontaneous souring of milk at the ordinary temperature, three stages may be distinguished. At the outset the original flora of the milk develops rapidly ; as this is poor in true lactic acid bacteria, the milk will only acquire a slightly unpleasant smell and taste which may be described as stale. Only by degrees do the streptococci gain the upper hand, whereupon the milk becomes coagulated, and the acid which is formed masks the first taints. Finally comes the third stage in which the lactic acid rods predominate and in which the concentration of the acid may rise from 0-6 per cent, to well over 1 per cent. Before this happens however, a plentiful development of yeasts and moulds (especially Torulse and Oidium lactis) will have taken place, and these consume the acid or neutralise it by- producing ammonia or other basic products of protein hydrolysis ; in the latter case their action is similar to that of chalk excepting that they destroy most of the acid instead of conserving it. Through the joint action of all these organisms the milk sugar will be fermented and the lactic acid destroyed, so that the way is .prepared for the putrefactive organisms. As a rule, however, many weeks must elapse before this state of affairs is brought about. The shallower the layer of mUk, the quicker wiU the acid disappear, provided, of course, that the milk does not dry up in the meantime. ^ When whipped cream, i.e., cream containing numerous air bubbles, is kept at the ordinary temperature, coli and aerogenes bacteria will obtain predominance in it. 6— a 68 DAIRY BACTERIOLOGY B. THE ABNORMAL FLORA The changes brought about by the normal flora in milk cannot be looked on as dpfects unless they appear at too early a stage. Thus the fact that milk turns sour on standing is in itself not a defect, but if the milk arrives at the dairy in a sour condition it is certainly defective. Defects of milk we understand to be not the normal but the abnormal changes. Defects of milk may be spoken of as primary if present from the outset, or secondary if they appear at a later stage in the history of the sample. In the former case they may simply be due to changes to which the milk is naturally subject at different stages of the period of lactation, or they may originate from the fodder or diseases of the cows ; in the latter case they will be due to the action of microorganisms which find their way into the milk either during the milking or at a later stage. Primary Milk Defects. — It is well known that just after calving or towards the end of the period of lactation the milk is abnormal in composition and coagulates badly with rennet. While colostrum has a strongly acid reaction, milk obtained towards the end of the lactation period is rather neutral than acid. Owing to the rapid growth of the foetus, much potash and especially phosphoric acid are used up instead of passed into the milk. On the other hand, the percentage of sodium chloride often increases to such an extent that the milk tastes salt or bitter salt. It may be pointed out that so long as the cow is not in caH, the milk rhay be suitable for cheese making during two to three years ^. The feed may have an undesirable effect on the milk by impart- ing to it an abnormal taste and smell, the best known examples being a taste of onions through feeding with onions, a turnip-like taste through giving liberal amounts of turnips and swedes, mustard and rape seed cake containing mustard, and a bitter taste from lupins (unboiled) or large amounts of vetches. Further- more, poisonous substances may be derived from certain plants which, however, are generally avoided by the cows when on pasture. Greater danger attaches to such poisons as iodine, arsenic and mercury compounds w^ich may be given as medicines and thus passed into the milk. For this reason no milk should be sent from any farm where the cows are given medicine of any kind, without the sanction of a veterinary surgeon.- Similarly, disinfectants such as carbolic acid may pass through the blood into the milk, or they may be absorbed direct from the air, in which case the milk will also be unsuitable as food. Finally the 1 Orla Jensen, " Ltodwirtschai'tliches Jahrbuoh der Schweiz," 1905, p. 542. NORMAL AND ABNORMAL MICROFLORA OF MILK 69 milk may be poisonous on account of toxins which have passed into it as the result of fevers or serious digestive troubles. An indirect influence may of course be exercised by diseases of the udder, ^mong which inflammation of the udder or mastitis and tuberculous udder are the most important. The alterations in the composition of the milk are bacteriological as weU as chemical. In the first place the reaction of the milk is altered ; frequently it becomes alkaline rather than neutral, due to the inflammation produced by Streptococcus mastitidis, though as a rule it eventually becomes acid as this organism produces appreciable amounts of lactic acid. The milk acquires a bitter, salt or other unpleasant taste, and the percentage of lactose, the most constant factor in the milk as long as the udder remains healthy, falls off appreciably. Flakes and lumps of pus and casein will be seen in the milk, and the colour changes. With streptococcic mastitis the milk becomes yellow, and with tuberculous udder bluish. Occasionally the milk may be coloured red by blood, and generally speaking, increasing quantities of the constituents of the blood pass into the milk while the normal constituents of the milk fall off. At last a watery secretion containing pus is obtained which can no longer be described as milk. Inflammation of the Udder. — The most dangerous form of this disease is that caused by 'Streptococcus mastitidis. It is very infectious, being easily transmitted from one cow to another, so that for this reason alone it is desirable that the milker's hands should be washed after milking each cow. Inflammation of the udder is also occasioned by Bacterium pyogenes and certain coli and aerogenes bacteria and micrococci. B. pyogenes is a very small rod form which may produce a very unpleasant smell in the inilk. A similar form, Bacterium minimum mammce, which decomposes casein, but does not liquefy gelatine, and which produces small amounts of lactic acid, has been found repeatedly by Oorini ^ in the udders of cows which have not been milked properly. Under these conditions, micrococci, possibly the same as those which are normally found in and on the udder, may gain predominance. This may also happen if the udder is in an unhealthy state owing to chills. The micrococci generally only produce light catarrhs. Bacterium pyocyaneum may also be found in inflamed udders. As most of the bacteria mentioned here may give rise to stomach and intestinal diseases, milk from inflamed udders must be regarded as dangerous. Udder and other Tuberculosis. — Owing to the prevalence of 1 " Eevue g6n6rale du Lait," 1907, Vol. VI., No. 24. 70 DAIRY BACTERIOLOGY bovine tuberculosis in general (in Denmark 30 to 50 per cent, of - the cows are affected in one form or another), it is no wonder that tuberculosis of the udder is often met with. Generally speaking, large herds seem to be affected the most. As udder tuberculosis makes rapid progress, the cows from which nursery milk is obtained should be examined by a veterinary surgeon at least once a fortnight. Owing to the dangerous nature of this disease, the Danish law orders the slaughtering of cows with tuberculous udders. Tuberculosis of the udder is, however, not the only form of the disease which may involve the infection of the milk with tubercle bacteria. This may also very well happen in cases of tuberculosis of the uterus and kidney or the intestine, and even tuberculosis of the lungs may be dangerous in this respect as the animals swallow most of the slime which they bring up, with the result that the bacteria pass into the manure. Every form of open tuberculosis must therefore be regarded as a source of danger as far as the milk is concerned. As tubercle bacteria do not grow at temperatures much below blood heat, they will not multiply in milk or milk products under normal conditions, but as they are not kUled by small amounts of lactic acid they can live in butter- milk. In the separating of milk the majority of the tubercle, bacteria are removed with the separator slime, though appreciable numbers 'pass into the cream while only very few remain in the separated milk. Raw milk is on this account less dangerous than raw cream or butter. Tubercle bacteria can live in butter for a much longer period than it is usually kept nowadays. In cheese making the great majority of the bacteria as well as fat globules are precipitated with the curd, for which reason milk and especially fresh cheese may be far more dangerous than whey. According to Harrison^, the hard cheeses may contain virulent tubercle bacteria even after keeping for two months. The latest researches have established that the organisms of human and bovine tuber- culosis are different varieties, the latter being less dangerous to adults than was formerly supposed though dangerous to children. Tuberculosis is usually contracted by adults through inhaling the dried saliva of consumptive persons. On the other hand, bovine tuberculosis is very dangerous to calves and pigs, for which reason a law was passed in Denmark at the suggestion of Professor B. Bang, ordering all dairies to heat separated milk and buttermilk to 80° C. before returning it to the farmers, who use it chiefly for feeding pigs. It is regarded as a matter of the greatest importance that pathogenic germs are excluded from Danish butter in a similar manner. 1 " Landwirtschaftliches Jahrbuob der Sohweiz," 1900, p. 317. NORMAL AND ABNORMAL MICROFLORA OF MILK 71 Other Diseases of the Cow. — Just as milk from cows suffering from tuberculosis of the uterus and intestine may easily become infected with tubercle bacteria, so milk from cows affected by other diseases of the sexual and digestive organs may become infected with the corresponding organisms. Experience has shown that the milk should not be used for cheese making until ten days after calving, the reason being that not only is the chemical composition abnormal, but also that during this period the milk is especially subject to infection from the uterus, and to taints from the disinfectants often used during calving. The small rod form. Bacillus abortus, which causes abortion, may occasionally be found in milk^ The consequences of ordinary diarrhoea have already been mentioned. Chronic diarrhoea, when the motions contain blood or are otherwise abnormal, is far more dangerous, for in such cases we are dealing with an infectious disease and the organisms in question (according to C. 0. Jensen, often coli bacteria of the swine fever group) may cause similar intestinal trouble in human beings, especially in children. Mor- tality among calves will always be a danger signal for human beings. Finally there will always be a danger of infection through the milk in cases of anthrax and all skin diseases such as foot and mouth disease, cowpox, etc., more especially if the udder is affected, and possibly also in cases of udder actinomycosis, acute lung diseases and rabies. The chemical composition of the milk is affected through several of these diseases. Secondary Milk Defects. — Milk may also be infected with pathogenic organisms from human beings, and any one suffering from an infectious disease or living in the same house with sufferers should be forbidden to handle or seU milk. It is generally accepted that tuberculosis, diphtheria, scarlatina, cholera and typhoid fever may be transmitted by milk. Epidemics of typhoid, cholera, scarlet fever and diphtheria have undoubtedly been caused re- peatedly through milk infections. Although Bacterium typhosum does not ferment lactose, it grows freely in milk, and it is also stated to be able to live for some time in butter and cheese ; like the organisms of the other diseases mentioned above with the exception of that of anthrax, it is kiUed by pasteurisation at a low temperature. 1 Sehroeder and Cotton, U.S. Dept. of Agric, Bur. of Animal Industry, 28th Annual Report, 1911 ; AUee 0. Evans, " Journal of the Washington Academy of Sciences," Vol. "V., No. 4, 1915, and " Journal of Infectious Diseases," Vol. XVIII., No. 5, 1916. According to the latter paper, Bacillus abortus and other similar forms seem to be fairly common udder bacteria, to be found in over 20 per cent, of the milk fresh from the cows. It appears to be chiefly a fat-spHtting variety (var. Upoh/tieus), which is killed after warming only to 52° C. for thirty minutes. 72 DAIRY BACTERIOLOGY While many of the above mentioned defects, however dangerous they may be, will not be readily detected and indeed are only to be demonstrated with great difficulty, the following defects will hardly esqape observation, and it is accordingly these which are commonly alluded to in speaking of milk defects. We will begin with those which are visible and then pass on to defects of taste and smell. Fermenting and Gassy Milk. — Milk may be described as fer- menting if it shows copious evolution of gas even before coagula- tion sets in, and gassy if this only occurs after keeping for some time at a high temperature (see the fermenting test). Both defects are du^ to coli and aierogenes bacteria (more seldom to yeast), and the difference between them is only determined, by the degree of infection. Fermentation may arise through direct infection from fermenting fodder or when the cows are suffering from violent diarrhoea or aerogenes mastitis. Both fermenting and gassy milk should be avoided in cheese making. Fermenting cream may give trouble in churning. Prematurely Coagulating and Cheesy Milk. — ^Badly-cooled milk may coagulate when only a few hours old ; as the milk will not be appreciably sour in such cases the phenomenon must be ascribed -to an action resembling that of rermet ; it is always due to excep- tionally active development of peptonising lactic acid bacteria (Streptococcus liquefaciens or Tetracocci), which will probably have started to secrete coagulating enzymes in the udder. The weU-known phenomenon of milk being specially liable to curdle in thundery weather does not seem to be ascribable to any other reason than the high temperature which usually precedes a thunderstorm. Milk> which has been coagulated by rennet is generally described as cheesy ; it can easily be distinguished from sour milk by the separation of clear whey and the contraction of the coagulum. If gas-producing enzymes are present as weU, the curd will become flaky or lumpy owing to'the disturbance of the milk by rising bubbles. The same effect may be produced in practice in presence of coagulating bacteria alone, if the milk is stirred or shaken. Slimy and Ropy Milk. — Milk may become slimy on standing, and sometimes the effect is so pronounced that the milk may be pulled out into threads a metre long. The change is most marked at 18° to 20° C. At higher temperatures the slimy organisms may be suppressed by the lactic acid, but even in pure cultures they form more slime at the temperatures mentioned. According to Gillebeau, one of the commonest slime-producing organisms is Micrococcus Freudenreichii, a large coccus ^ 2 /x thick, which 1 " Landwirtschaftliohes Jahrbiioh der Schweiz," 1891, p. 135, and 1902, p. 342. NORMAL AND ABNORMAL MICROFLORA OF MILK 73 liquefies gelatine and produces an appreciable effect in milk within five hours. Like several allied forms, it may occur in bad water. If a stable once becomes infected with these organisms, nothing short of a very thorough disinfection will eliminate them. The author has isolated a non-liquefying micrococcus, which, being an obligate aerobe, only makes the surface of the milk slimy ; in pure cultures,- the sliminess is preserved for many weeks ; the milk becomes faintly alkaline, but is not peptonised. It turns the surface of agar brownish black. Storm has isolated a motile rod which similarly only turns the surface of the milk slimy ; it coagulates and peptonises the milk. As already mentioned, the slime is derived from the outer portion of the cell membrane. While the organisms mentioned can form slime from the protein of the milk, the lactic acid bacteria which produce slime require sugar in order to do so, but, on the other hand, they make the milk slimy throughout. As has been mentioned, the most important slime-producing lactic acid bacteria are certain varieties of Sc. cremoris. As far as the Swedish " long milk " or the Dutch " long whey " .are concerned, slim.iness is a desirable characteristic, but otherwise it is highly undesirable, for slimy cream gives a bad yield of butter, and slimy whey is difficult to press out of cheese, collecting under the rind, and, as Bwrri has shown, causes the cheese to crack at a later stage ^. For this reason slimy bacteria are no longer used in making Dutch cheese, as other lactic acid bacteria have been found to possess the same advantages without the disadvantage in question. Further, slimy organisms easily lose their character- istic property, especially if grown at higher temperatures ; con- versely, the common Strepto60QCUs cremoris is inclined to make milk slimy if cultivated at lower temperatures for any length of time. Other lactic acid bacteria, pure (certain thermobacteria), as well as pseudo (certain aerogenes bacteria), may cause sliminess. Colofired Milk. — In the old-fashioned process of allowing milk to stand in order to let the cream rise, coloured spots often appeared on the cream, or the milk would gradually become blue or red throughout. As these defects have not been met with in practice since the introduction of the centrifuge, they need not detain us here. They are due to the colour-producing organisms already mentioned (see pp. 44, 45 and 46). Milk with an Unclean Sour Taste. — There is no defect more commonly met with in dairies than this one, as it is in no way due to foreign or rare milk organisms. It is simply the stale stage prematurely reached owing to insufficient cooling. 1 " Centralblatt f. Bakteriologie," 2 Abt., 1904, Bd. XII., p. 192. 74 DAIRY BACTERIOLOGY Milk with Stable or Grass Taste. — Formerly it was supposed that a stable smell was exclusively due to air absorbed in the stable. The taste, however, usually becomes worse after the milk has left the stable, and it has been shown to be due to the bacteria which produce the smeU in the stable, i.e., the intestinal bacteria. A heavy infection with manure will not only give the milk a taste of manure by direct means, but will introduce a number of bacteria which will continue the decomposition of the specific constituents of the manure and, naturally enough, do not neglect the con- stituents of the milk itself. In the same way, milk may acquire an unduly strong aroma of grass in spring. In the usual course some principles of colour' and taste will always pass into the milk from the young grass, but the strong odour of herbs, which is appreciated by some people, will only arise on infection with the liquid secretions of grass. Milk with a Tasfe of Turnips. — As already mentioned, the milk acquires this taste when the feed includes too great a proportion of the pungent principles characteristic of the Cruciferse, turnips and swedes being especially dangerous if given in a rotting con- dition or too cold, so as to cause digestive troubles. Under these conditions other roots may, of course, also' have an undesirable effect ^- The taste-producing constituents of the roots need not necessarily pass into the udder, but may also_ be introduced into the mUk with the manure, and, still worse, they will then be accompanied by the microorganisms which vegetate on the roots, and which, therefore, are equipped with those enzymes which are capable of hydrolysing such substances as the glucosides of mustard oil. According to Weigmann, the active organisms in such cases are chiefly coli bacteria, Penicillium brevicaule and certain species of oidium. C. O. Jensen found in the course of his weU-known researches at Duelund dairy, which led to the pasteurisation of cream for butter making^, that a coli ^jpacterium living in water could produce in the milk a very un- pleasant turnip-like taste even when the cows had not been fed on roots at all. Bacterium fluorescens liquefaciens also produces a taste of turnips, and this defect is accordingly often met with in milk which has been kept for any length of time at a low tem- perature. Weigmann has isolated a non-liquefying fluorescing bacterium. Bacterium carotce, which produces a strong smell of carrots in all nutrient media. The author has cultivated this 1 According to Johannes Bolle, " Zeitschrift f. Untersuchung d. Nahrung u. Genussmittel," 1915, Bd. XXX., p. 361, feeding with liberal amounts of beets may cause betain to pass into tbe milk. As this substance is a base, it will delay the coagulation of milk by acid. ^ C. 0. Jensen and Lunde, " Forsogslaboratoriets," 22 Beretning, 1891. NORMAL AND ABNORMAL MICROFLORA OF MILK 75 organism for years, the smeU of the cultures remaining as strong as ever. Milk with a Soapy Taste. — ^As is well known, this taste arises when milk is neutralised with alkalies, and is therefore produced by bacteria which chiefly produce ammonia. As the neutralisa- tion of the milk is counteracted by acid production, and the ammonia-forming bacteria grow better at low temperatures than the acid-forming bacteria, it will readily be understood that the defect in question is most noticeable in cooled milk. Bacterium sapglacticum, isolated by Eichholz, can grow even at 5° C. ; it is a non-liquefying fluorescent organism. Bitter Taste in Milk. — This taste nearly always occurs in milk which has not been completely pasteurised, being produced by the peptonising sporing bacteria. Bitter substances are nearly always formed in the first stages of protein hydrolysis. The defect may also be due to the cow, to certain feeding stuffs, yeasts {Torula amara) and peptonising cocci, especially Strepto- coccus Uquefaciens. According to Weigmann, Bacterium Zopfii, Bacterium lactis innocuum and aerogenes bacteria may turn mUk bitter. Metallic and Tallowy Taste in Milk. — Badly-tinned vessels will always impart a metallic taste to milk which is acid or fairly warm. In Denmark the pasteurised separated milk is sent back warm from the co-operative dairies to the farms, and in such cases it is difficult to prevent the cans from being attacked to a somewhat" greater extent than usual, and thus giving the milk a metallic taste. The metallic taste must not be confused with the tallowy taste which may arise when the cream layer is exposed to direct sunlight, or through the action of certain microorganisms. As the metallic taste which is caused by copper is not to be distinguished from the tallowy taste, it may be presumed that copper salts promote by purely catalytic means the oxidation processes owing to which the tallowy taste arises. According to Rosengren ^, it is only milk or cream which is or has been heated which gets a foreign taste from the copper. The same holds good for butter made from the cream. According to Storch, certain lactic acid bacteria may cause a tallowy taste. Most liquefjdng rod bacteria (the hay baciUus, etc.), may produce a sickly, tallowy^ taste at first, but the more ra^pidly they peptonise the mUk the sooner will the taste become bitter. The author has found that some aerobic non-sporing rod bacteria occasionally occurring in water are generally the cause of the tallowy taste which may arise in milk on long standing *. ' " Landbrugsforsogsmeddelelse," No. 197, 1920. ^ As an instructive example it may be mentioned that a dairy which 76 DAIRY BACTERIOLOGY Bacteria are known which form hydrogen sulphide from sulphur, and therefore also from vulcanised rubber. Milk which has passed through rubber hose pipes, such as in milking machines, may therefore, on warming to a relatively high temperature, acquire a Smell of rotten eggs. s supplied Copenhagen with pasteurised cream which tasted good on arrival, but which became tallowy before it reached the consumers, eliminated this defect by rinsing the transport cans with boiled water. The rod bacteria mentioned above were found both in the cream and the water from this dairy. Chapter III The Preservation of Milk and its Treat- ment for Direct Consumption PRESERVATION The general methods used for the preservation of foods are constant cooKng, short heating, concentration, and the addition of bactericidal substances. All four methods or combinations thereof are applied to milk. Cooling is most used because it is the cheapest method and because the milk suffers no chemical change in the process. The principle of the method has been discussed above, and it is well- known that the sooner and the more thoroughly the milk is cooled the better will be the result. Even in cold weather the milk cannot be cooled quickly enough by simply allowing it to give up its heat to the air. It is absolutely necessary to stand the pails in cold water which is frequently changed ; the level of the water must be higher than that of the milk in the paU. The water container should be fitted with a grid or the bottom should be fluted so that the water may circulate freely under the pails. The same object may also be achieved by standing the pails on a flat surface if their lower rims are provided with holes. The water inlet pipe should reach the bottom while the overflow should be at the top. On large farms it is best to have a tank above the cowshed, which is filled just before milking, and from which the water slowly fiows through wooden troughs situated on the coolest side of the shed; the coohng may thus be started during the straining process. The pails are first placed in the end of the trough nearest to the outlet and are moved by degrees towards the inlet end, whence they are removed from the trough. The troughs must be covered over by a lean-to roof sufficiently high to allow a man to stand under it. The used cooling water may with advantage be used for drinking water for the cows. When the milk has been cooled to the temperature of the water, it may be further cooled by standing the pails in ice water or by passing it over a cooler through which a mixture of four parts of ice in small lumps and one part of salt is circulated. In summer time the cooler may be moved out into the fields, though if this is done the 78 DAIRY BACTERIOLOGY consumption of ice will be unreasonably large. The accompany- ing illustration shows the arrangement of the cooling apparatus at Fauerholm dairy which supplies Copenhagen with milk. Here the milk is cooled to 3° C. By allowing the milk warm from the cow to flow over a cooler or a special aerating apparatus, some of the unpleasant animal odour is eliminated, but at the same time the milk loses carbon dioxide and takes up oxygen, with the result that the development of the aerobic putrefactive bacteria will be favoured at the expense of the true lactic acid bacteria. As l'"i» u >, >> >', >, ■r* 2 >, tA ti >t 1 ^ Is 1 i i 1 -3 P" It & ek S5 1 2 p. c it u p. 'S Q. o ft Ph ■5 P* & ■< ■< •«! < MS .5 ' < ■n -< ->! 17 34 39 f 12 f 16 g2 44 85° , 17 ^^ 35 39 ,, 12 J, 26 gr 80 90° , 14 33 36 jl 13 JJ 16 BlSl 36 95° . 1* ji 33 37 „ 13 „ 16 . g2 21 Sterile. 30° , 19 " 35 38 40° , " 22 " 35 60 Raw. Vigorous n 41 gl 36 gl 40 Thermo- g2 71 gici 64 g3 80 60° starter. „ -40 34 39 bacte- J 15 gl 51 gl. 64 6.5° ., « ^ 35 41 rium 16 glCl 41 48 70° ,. 41 jj 35 42 bvlga- ,, 19 f 18 82 27 75° A 30 „ 42 jj 34 41 Hcum.' ,, 19 J, 20 gl9l 28 80° „ 44 36 42 18 flgl 19 01 27 85° B30 „ 40 '35 40 fgl 19 Bl 20 11 90° „ 42 32 41 gl 21 21 g'l 95° „ 43 jj 33 41 24 „ 36 89 Sterile. „ 43 " 36 42 40° " 63 " 60 It 70 f = Fluid. ' g = Gelatinous curdled, s = Spongy curdled, clear whey), fg = Seml-solld. See further under the " Fermentation Test." The acidity is given in Soxhiet-Henkel degrees (see p. 161). The original acidity of the milk was 7 ; tbe acidity of the sterilised milk was, however, 8. Cheesy (casein contracted and much APPLICATIONS OF LACTIC ACID FERMENTATION 115 The table first shows the behaviour of the uninoculated mLLk on standing ; at 20° C. it was practically unaltered after twenty-eight hours, which is explained by the slow growth of the heat-resisting bacteria at the ordinary temperature, whereas at 40° C. it was affected fairly quickly. The sudden interruption of the increase in acidity on heating the milk above 7'6° C. is explained by the fact that the heat-resisting lactic acid bacteria will survive heating to 75° C, but not to 80° C. ; microscopic examination confirmed this view, as streptococci were found in great numbers in the mUk heated to 75°C., but none were found in that heated to 80° C. or more^. From the results already considered, we may draw the conclusion that it is impossible to be sure of the exclusion of foreign lactic acid bacteria from the starter unless the milk has been heated to at least 80° C. Microscopic examination also shows the milk pasteurised at 75° C. to contain most hay bacilli after standing, 75° C. being the lowest temperature at which the bactericidal substances of the milk are completely destroyed. These points are particularly well illustrated in the case of the milk which was kept at 64° C. ; in this milli only those thermophile bacteria grew which were not in the slightest degree affected by the pasteurisation, but which, on the other hand, seem to be fairly sensitive towards the bactericidal substances in the milk. This explains the somewhat paradoxical result that under these conditions the milk which has been heated to 75° C. or over is that which is most rapidly affected. The conditions are still more complicated in the inoculated mUk; In the raw as well 'as in the low temperature pasteurised milk the lactic acid fermentation is inhibited by the bactericidal substances but encouraged by the lactic acid bacteria of the mUk itseK, and in the milk heated up to 70°, 75° and 80° C, a sharp struggle for existence takes place between the lactic acid and the hay bacteria, which often results favourably for the latter at the outset. Finally, it must be borne in mind that the higher the temperature to which the milk has been heated, the less oxygen will it contain. As the different species of lactic acid bacteria are differently affected by the conditions under consideration, no definite rule for pasteurisa- tion can be laid down on this head. Thus it was found that Streptococcus cremoris and the nearly related Sc. thermophilus develop most slowly in the milk heated to 75° C. (and the latter also in the milk heated to 70° C.), while Sc. lactis shows a minimum capacity for souring (though not very pronounced) in the milk which has been pasteurised at low temperatures and retained its bactericidal constituents. The point- of special interest is that the vigorous lactic acid culture (starter) is only slightly affected by the 1 Living micrococci are, however, still found in milk pasteurised at 80° C. ; they grow best at 20° C. 8— a 116 DAIRY BACTERIOLOGY previous treatment of the milk (perhaps a maximum capacity for souring is found in that heated to 80° C.)- On the other hand, the two thermobacteria which are distinctly anaerobic in character are greatly influenced by the previous treatment of the milk, as is seen from the acidity and also, in the case of Thermobacterium bulgaricwm (of Yoghurt), by direct microscopic examination (c/. the illustrations, p. 30). Both these organisms grow best in raw milk, where the oxygen is quickly consumed by other organisms, and in sterile milk which is quite free from oxygen. Thermo- bacterium helveticum also shows a maximum of souring in the milk heated to 75° C. in which the bactericidal substances have been destroyed, but wHich still contains a little dissolved albumin. Of the lactic acid bacteria employed in these experiments, this was the only one which was influenced by the albumin, all the others growing better in milk which had been heated to 80° C. or above. From what has been said we may draw the conclusion that no harm will be done by pasteurising the starter milk thoroughly. All that is necessary is to avoid discolouration or burning, which may cause difficulty in judging the aroma of the starter or impart a cooked flavour to the butter in cases where large amounts of starter are used. Experience shows that pasteurisation for one hour at 85° gives good results. The milk is heated in a water bath, stirring frequently, and it is cooled as quickly- as possible to the souring temperature, stirring carefully, the pail or can being placed in running water. Aeration by pouring the milk from vessel to vessel is unnecessary ; the risk of infection is only increased by exposure to the air, and especially by contact with more vessels than is strictly necessary. It is a great mistake to pass the pasteurised milk over a cream cooler. ,As in the case of cream, undesirable fermentations are best avoided by keeping the souring temperature low, and moreover, it is unsound as a matter of principle to accustom the bacteria to a sensibly higher tem- perature in the starter than that at which they are required to act in the cream. A temperature of 22° to 23°'C. is quite satisfactory if only this is kept constant by placing the starter cans in a sufficiently large water-bath at the right temperature. Boekhout's and Starch's aroma bacteria, it may be noted, do not thrive well at higher tempera.tures. On the other hand, it is not advisable to lower the temperature further, as Sc. cremoris nearly always degenerates on propagation several times at temperatures below 22° C. As cream is always soured under this temperature, we find here a new and hitherto unknown reason why the use of butter- milk for this process is unsatisfactory in the long run. The water- bath may be made of wood or galvanised or tinned iron, and covered in in the same way as the culture apparatus, so that only APPLICATIONS OP LACTIC ACID FERMENTATION 117 the tops of the cans project. The cans are covered with steam sterilised double covers which allow of air circulation. . As\iothing is gained by keeping the starter cold overnight, there is no reason to add more culture than is necessary to ripen the starter before the following morning ; about J to 1 per cent, is sufficient. Im- mediately before use the starter is skimmed, as the top- layer will be richest in foreign germs if infection has taken place i. The starter is given a more liquid consistency by stirring it vigorously, after which it is poured into the cream ripening tank . As the starter is more easily titrated than cream, its acidity should be checked from time to time. Experience has shown that the best results are obtained with an acidity of 90° to 100° (number of cubic centimetres of normal soda per litre, corresponding to 18 to20c.c. of decinormal soda per 20 c.c. of milk). This corresponds to 36 to 40 Soxhlet-Henkel degrees. If the starter becomes weak and shows a lower acidity although it has been kept at a tempera- ture over 22° C. all the time, the culture used is weak, while if the acidity is too high the milk has probably been oversoured, and the most beneficial bacteria will not be in full vigour. The culture will thus often contain rod-shaped lactic acid bacteria, in which case it will be best to use a new culture. What has been said regarding the starter also applies to the culture from which the starter is made, only in this case still greater care is necessary, for an infection in the starter need only degrade the quality of a single churning, while an infected culture may cause repeated trouble. Fortunately it is easier to keep the culture pure, and there is not the same difficulty in obtaining the small amounts of finest grade milk required for this purpose as may be experienced in obtaining sufficient quantities of milk in the condition in which it should be used for the starter. Herein lies the advantage of propagating the culture and the starter separately instead of simply inoculating the new starter milk with some of the old starter. It is a good plan to keep two or three cultures going independently of one another, inoculating daily into fresh milk, for by comparing the tastes of the different cultures the detection of defects in taste is much facilitated and as it would be a very exceptional misfortune if all the cultures became bad at once, at least one good culture will always be available if the precaution is taken of renewing any bad cultures as soon as possible. Several forms of culture making apparatus are on the market ; it is important that means should be provided 1 If the milk is stirred during the souring there is no benefit to be derived from this precaution. According to Bostrum's experiments at Alnarp dairy, the lactic acid bacteria thrive better if the milk is stirred occasion- ally. By this means local over- or under-souring is avoided. 118 DAIRY BACTERIOLOGY for keeping the temperature of the milk as constant as possible during the souring period.. Before leaving the subject of cream ripening, it may be pointed out that the so-called souring defects need by no means be ascribed to infections of totally foreign groups of microorganisms such as pseudo lactic-acid bacteria, yeasts' and moulds ; as often as not they may originate from the true lactic acid bacteria. Thus Shrch once isolated a lactic acid bacterium which was able to produce a tallowy taste in cream and butter, and C. 0. Jensen found, almost simultaneously, lacjtic acid bacteria, some of which gave the butter an oily taste, and others which imparted a hurnt or malty taste ^ The last-mentioned taste is often produced by strains of 8c. lactis. From the foregoing it will be seen that the" species of lactic acid bacterium chosen for the culture is by no means a matter of indifference. Buttermilk is obtained as a by-product from butter making ; its good qualities have already been mentioned. It becomes particularly good when the cream is pasteurised and soured with a pure starter. As the most favourable stage of the lactic acid fermentation has always been reached before the churning, the buttermilk quickly deteriorates in taste and throws ou.t large lumps of casein unless it is kept quite cold. It foUows as a matter of course that water should not be added to buttermilk which is to be retailed in towns ; under these circumstances all washing and rinsing of the butter should be done with ice-cooled butter- milk from a previous churning. Separated milk is not so suitable for this purpose, as it causes the buttermilk to curdle more quickly. The undiluted buttermilk will always have a lower acidity than the starter which has been used in souring the cream from which it is made, even if the cream and the starter milk have been soured in exactly the same way. Tholstrup Pedersen ^ has shown that the difference is largely due to the fact that the buttermilk loses its carbonic acid during churning ; the sour starter will also show a lower acidity if it is shaken before titration. The Souring of Separated Milk. — In the manufacture of mar- garine, separated milk is soured and churned into an emulsion with melted fat. The aroma of the soured milk is taken up by the fat in much the same way as occurs in butter making ; the difference between the two cases lies in the fact that while the aroma is taken up by the fat globules in the cream during the ripening process, this occurs in margarine only during the churn- ing, or emulsifying process, and the ensuing operations. As regards the actual souring of- the separated milk, this subject has '■ " Porsogslaboratonets,"' 22 de Beretning, 1891. » " Maelkeritidende," 1916, p. 65. APPLICATIONS OF LACTIC ACID FERMENTATION 119 been discussed at some length in dealing with the preparation of starters for the ripening of cream, and the process does not differ materially from that described under the souring of cream. Blichfeldt ^ has devised an appliance for the continuous souring of separated milk, consisting of a closed cylindrical vessel into which fresh separated milk is introduced, and from which soured skim milk is withdrawn simultaneously. The contents of the vessel are kept stirred; and by regulating the temperature and the rate of output, the acidity of the product may be kept constant. The apparatus is worked under sterile conditions as far as the avoidance of infection from outside is concerned, while the fresh separated milk must be efficiently pasteurised before use, which of course is also the case in the tank souring process. The continuous process has the advantage that by its means a large amount of milk may be treated in a relatively small space. The Preservation of Stable Manure by addition of Whey. — This process has been proposed by Barthel^, and vriirhave economic value where whey is plentiful and peat is unobtainable. The lactic acid fixes the ammonia- in a form easily available for the nutrition of. plants (or the nitrifying bacteria). By using 50 to 100 litres of whey per 1,000 kilos of manure, at a cost of about Id., the increased yield to be obtained from good soil will amount to the value of 6s. M. In this connection it may be mentioned that all dairy refuse, even the washing water, has great fertilising value in virtue of the nitrogenous matter which it contains. The washing water should therefore, wherever possible, be used for surface irrigation on the fields instead of, as is sometimes done, run into ditches, where it will putrefy. In this case the milk sugar, or rather the lactic acid formed therefrom, is a drawback, but as a rule the latter will be neutralised by the large amounts of lime used in cleaning. 1 English patent 4504, 1912. The patent covers continuous fermenta- tions. 2 (;_ BartJiel and Sigurd Bhodin, Meddelande Nr. 57 fr^n Centralanstalten for forsoksvasanet pa jordbruksoniradanet, 1912. Chapter V The Normal and Abnormal Microflora of Butter THE NORMAL FLORA Fresh butter from unpasteurised cream will naturally contain the same microorganisms as milk, and the bacterial changes which take place on keeping will be the same as those which occur in milk kept at the same temperature. Thus at low temperatures water bacteria will tend to predominate, while at the ordinary temperature the fresh butter will soon become sour owing to the rapid development of lactic acid bacteria, and, in particular, streptococci. Later on, lactic acid rod bacteria, yeasts and moulds appear. As the moulds, which hydrolyse the fat, only appear on the surface, the keeping qualities of the butter will be greatly enhaijced by packing it in large casks instead of in small fiat slabs. Such small pieces will, unless frozen, be subject in the course of a few days to the same changes, originating on the surface and gradrually woriiing towards the centre, as octiur in a mouldy soft cheese. Butter from pasteurised ripened cream will have a much simpler flora to begin with, as Strepto- coccus cremoris, used in the ripening process, will generally be the principal organism present. This organism, however, does not seem to be capable of living long in butter, and is gradually replaced by yeast, and generally also by lactic acid rod bacteria. Moulds, which are an unavoidable infection from the air, gradually appear on the surface. As the yeasts and bacteria only develop in the small drops of water which constitute only about one-sixth of the weight of the butter, it is obvious that butter will never show such high bacterial counts as milk and cheese. The number of microorganisms in butter will depend on the amount of nutrient matter and antiseptic substances present in the water droplets^ i.e., on the washing, working and addition of salt, boric acid or other preservatives ^. Properly treated butter seldom contains 1 The preserving action of salt is more pronounced the lower the per- centage of water in the butter. Thus in butter containing 2 per cent, of salt, the aqueous portion will contain 12-6 per cent, of salt if the water percentage is 16, but 20 per cent, if the water percentage is 10. The development of microorganisms is only completely inhibited when the MICROFLORA OF BUTTER 121 more than a few million bacteria per cubic centimetre. In butter from unripened cream, the bacterial count increases during the first few days ^, after which it decreases. In butter from ripened cream the count is highest when the butter is freshly made (ten to twenty million lactic acid bacteria per cubic centimetre), and decreases steadily in the course of a few weeks to a few hundred thousand, and sometimes even to as low a figure as a few thousand, ±he reason, being that the original lactic acid bacteria die off at a quicker rate than their successors develop, for the available nutrient matter, especially lactose, is continually falling off. In cases where fat hydrolysis is taking place, this source of carbon will be replaced by glycerine, and the lactic acid bacteria will then again fare better. On keeping for any length of time, butter acquires a stronger flavour, and it becomes rancid quicker than other fatty materials on account of the large amounts of water and nutrient substances which it contains. Before the introduction of margarine, com- pound and other substitutes, it was a common custom in Central Europe to preserve the butter fat by melting and separating off the other constituents ; the pure butter fat kept good if preserved in well closed stone jars in a cool place. Duclaux 2 was the first to study the changes which take place in butter on keeping. He found that direct sunlight promoted the action of atmospheric oxygen, i.e., its function was similar to that of the oxidases, causing oxygen to combine with the con- stituents of the butter-fat, especially the olein. Duclaux also found that butter was spoiled by various moulds to which he ascribed an action similar to that of sunlight in oxidising the fat. The author's investigations^ show that there is an important difference between the action of sunlight and that of micro- organisms. While /ate are principally oxidised under the action of sunlight, the iodine value being reduced, thsy are hydrolysed by the microorganisms into fatty acids and glycerine, the acid value being increased. Oxidation causes changes in taste ^hich are much more undesirable than those caused by hydrolysis ; if butter is concentration of salt reaches 25 per cent., e.g., with 13 per cent, of water -and 3-3 per cent, of salt (tinned butter). If permissible by law, it is specially recommended to add 0'75 per cent, of benzoic acid, or 2 per cent, of sodium benzoate, or a mixture of 0-5 per cent, of benzoic acid and 05 per cent, of sodium benzoate. As benzoic acid is a physiological product (it is trans- formed to hippuric acid), it may be regarded as one of the less objectionable preservatives. 1 The highest count ever obtained by the author from butter made from unripened cream was 59 millions per cubic centimetre. 2 " Ls lait," Paris, 1894. ' " Studien iiber das Eanzigwerden der Butter" ( " Centralblatt f. Bakt.," 2 Abt., 1902, Bd. VIII., p. 1 1). References to earlier literature given here. 122 DAIRY BACTERIOLOGY exposed to strong sunlight for only one hour, its surface becomes bleached and perfectly uneatable. The taste thus produced bears the closest resemblance to that of bad tallow, and is generally described as " tallowy." The rancid taste proper is only produced by the microorganisms, and is due to the lower members of the fatty acid series, i.e., the volatile fatty acids, as well as certain esters which have a fruity odour, such as ethyl and amyl acetate. The glycerine, which . is usually completely transformed by the- microorganisms, is the starting substance in the formation of the esters as well as of the alcohols which may be formed. As most fat hydrolysing organisms require air for their development, the exclusion of air is the most important precaution necessary to protect the butter from the changes under discussion. . For this reason, butter intended for use in warm climates is packed in hermetically sealed tins. Heat, like sunlight, promotes the oxidation of the fat. According to Ritsert ^, carbonic acid, even in darkness, will impart a tallowy taste to butter ; this point is not without bearing on the ripening of cheese, as considerable quantities of carbonic acid are often produced in cheese. The most important fat hydrolysing microorganisms are Bac- terium fluorescens liquefaciens. Bacterium prodigiosum, Oidium lactis, Penicillium glaucum and Cladosporium hutyri. As they do not form spores, they are all easily destroyed on heating ; of , the two bacteria, B. fluorescens liquefaciens is, as mentioned above, very widely distributed in water, for which reason there is ' a danger in letting butter come into contact with water or ice. In many cases the advantages gained by pasteurisation are nullified when we introduce bacteria with the washing water, which have a worse effect on the keeping qualities of the butter than those which were destroyed in pasteurising. It should be made a principle to pasteurise (in a regenerative appa^ratus) all the water used for rinsing the cream cans, ripening tanks and chums, and for washing the butter. Moulds may come from the starter (or from the milk it the cream has not been pasteurised), from the air or from the packing material. By treating the casks and parchment paper for twenty-four hours or longer with con- centrated brine, nutrient substances are extracted and the mould spores are considerably weakened. It is better to treat the inside of the casks with melted paraffin wax immediately before use ; they wiU then be sterilised and rendered airtight. If this is done,' parchment paper may be dispensed with, and the wood will not soak up brine and increase the tare at the expense of the nett weight ^. Although Oidium lactis and Penicillium glaucum 1 Inaugural dissertation, Berne, 1890. 2 Rogers, U.S. Dept. of Agric, Bureau of Anim. Ind., 1906, Bull. No. 89. MICROFLORA OF BUTTER 123 are the most active in hydrolysing fat, they do not spoil the butter to the same extent as the above-mentioned bacteria. Not only do the moulds hydrolyse the fat, but they also consume part of the free fatty acids, especially the lower memibers of the series ; the acids produced by the bacteria, however, are allowed to accumulate to such an extent that the fat hydrolysing bacteria, which, curiously enough, are fairly sensitive to acid, are destroyed. This explains the fact that very rancid butter may occasionally be sterile some distance from the surface. - The formation of the esters which are so characteristic of rancid butter is due to Penicillium glaucum and especially to Cladosporium hutyri. The former, however, only forms esters in symbiosis with Oidium lactis, which mould edso promotes ester formation by Cladosporium hutyri. Certain mycodermse which do not themselves hydrolyse fat may also promote the formation of esters. According to the investigations of H. C. Jacobsen^, exactly the same microorganisms are responsible for rancidity in margarine. All the above-mentioned organisms grow better and quicker in unsalted butter than in salted butter. The water bacteria are particularly sensitive to salt, and by packing the butter in large casks the moulds, which require air, will obtain the smallest possible surface for attack ; Danish butter and similarly prepared butters therefore contain, as a rufe, only lactic acid bacteria and yeast. Among the yeasts, however, there are certain torulse which are by no means innocuous and which may, especially in symbiosis with lactic acid bacteria, hydrolyse butter-fat to varying extents. The reason why the lactic acid ^bacteria promote hydrolysis in such cases is that the yeasts in question thrive best in presence of a little lactic acid *. While the lactic acid bacteria, generally speaking, promote the growth of y6asts and moulds, they inhibit the action of the fat hydrolysing bacteria to a considerable extent, so that it is difficult on the whole to say whether thg souring of cream will as a general rule contribute towards the preservation of butter. According to the observations of Sogers and Gray^, butter from pasteurised sweet cream keeps better than that from pasteurised sour cream, and the addition of a little lactic acid to the pasteurised cream has the same undesirable effect as souring with a starter. In all circumstances it is essential that the starter used should contain only pure cultures of good lactic acid bacteria, and no yeasts or moulds. The safest method of preventing yeasts and moulds as well as other harmful organisms from developing to any extent in 1 "Folia Microbiologica," 1918, V. 2. 2 OrU Jensen, " Maeikeritidende," 1910, p. 965. '^ Experimental Station Record, 1909, No. 5. 124 DAIRY BACTERIOLOGY the butter is to wash it so thoroughly that the organisms will hardly find any nutrient matter therein^. THE ABNORMAL FLORA As in the case of milk (and, as we shall see later on, of cheese), the normal changes have been dealt with before the abnormal changes, though in the present instance one may note the im- portant difference that while the souring of milk may occasionally be a desired change, the turning rancid of butter is under all circumstances a defect, and if the defects of butter are classified in the same way as those of milk, i.e., into original and secondary defects," most of the latter will be found to be closely connected with the normal process of turning rancid, although in their first and indeterminate stages they may go under many different names. The original butter defects may often be divided into defects in appearance and defects in taste ; they originate in the milk, in the ripening process or in the processes connected with churning, and they may be of a chemical or of a biological nature. As the nature of the feed largely influences the melting point of the butter -fat, it has an important bearing on the consistency of the butter. The consistency is also affected by the water percentage and the way in which the water is distributed. It is the fine state of division of the water in the butter which renders the product pliable and easily spread on bread. Starch's re- searches ^ have shown that the water globules in butter should neither be too large nor too small. If they are too small, the butter will be dull in appearance and " thick," and if too large, they will tend to coalesce, giving a wet butter. Normal good butter contains about 3| million water globules per cubic millimetre, while " thick " butter contains about 12| millions in the same volume. Starch has found that during the souring of cream bacteria may develop, which make the butter thick, but this defect may also be due to causes, chiefly chemical and mechanical, which give rise to a high water percentage. This is especially brought about by churning and working at too high temperatures *- If the cream is oversoured the casein may coUecJ in large lumps, which may persist in the butter in cheesy lumps and spoil its appearance and keeping properties. '^ In Weigmarm'g work, " Versuolie zur Bereitung von Dauerbutter " ( " Milchwirtschaf tliches Zentralblatt," 1915, p. 353), many valuable hints will be found. 2 " Porsogslaboratoriets 36 Beretning," 1897. » OrU Jense/n, " Maelkeritidende," 1907, -p. 943. MICROFLORA OF BUTTER 125 Thje original defects in taste are due to original milk defects (and therefore also possibly to the feed), to milk defects arising at a later stage, i.e., secondary defects, faulty ripening and impure salt. What has been said under the heading of milk regarding stable, grass, turnipy and bitter tastes, applies also to butter. When butter has a strong taste of grass it often contains numerous small gas bubbles, which show the defect to be' due to gas-producing organisms, i.e., intestinal bacteria. Yeasts which ferment lactose may produce gas bubbles and give to the butter a peculiar yeast- like ,taste. Defects of this nature are generally to be avoided by pasteurising the cream. On the other hand, pasteurisation will not prevent a metallic taste (which may arise in butter through washing with ferruginous water), and secondary defects due to faulty souring. A cooked taste is not so often due to heating the cream to too high a temperature as to heating it for too long, as may occur if the cooling after pasteurisation is too slow ; the taste often arises through the milk having too high an acidity, which causes the proteins to separate and burn on the pasteuriser. A burnt taste may also arise in this way ; as already mentioned, a burnt, oily or tallowy taste may also be due to faulty souring, while the effect of sunlight or copper salts in producing tallowiness has also been alluded to. If the salt used contains appreciable amounts of ma^gnesium salts, it may give a bitter taste. Although for the sake of uniformity distinction has been made between original and secondary defects, it must be admitted that the line of demarcation between the two is by no means sharp. Thus a defect such as unclean taste, which is produced by various putrefactive bacteria (proteus, coli bacteria, etc.), may develop sooner or later in the history of the butter. The term original defects will be appHed to .such defects as come out immediately or during the first few days after the butter has been made, and then disappear either partially or completely ; by secondary defects will be understood those which develop gradually and become worse as time goes on. Secondary defects are of course counteracted by any condi- tions tending to increase the keeping powers of the butter, i.e., good raw material, efficient pasteurising, ptu-e starter, proper churning, thorough washing with good water, proper working and salting, clean air, sterile and airtight packing, and the most. thorough cooling possible. Defects in appearance include mouldy spots which are generally accompanied by a mouldy smell ; it must be mentioned that many kinds of moulds may grow on butter besides those which generally cause rancidity, and although most of these organisms may be able to hydrolyse fat, moulds are known which do hot do so. Oidium lactis cannot as a rule be seen in butter 126 DAIRY BACTERIOLOGY with the naked eye, but most of the other moulds form green, brown or black spots. As has already been mentioned, butter from sweet cream may be coloxired red by certain torulse, which in symbiosis with lactic acid bacteria hydrolyse fat -and also give an oily taste in butter. Actinomyces chromogena turns butter brown and gives it an unpleasant earthy smell. Among the secondary defects of taste, the sour cheesy taste deserves special considera- tion. The acid is chiefly due to lactic acid rod bacteria, so that the defect is particularly likely to arise when the butter has not been properly freed from buttermilk or when it contains lumps of casein in which these cheese bacteria may initiate a cheese- ripening process. Th& defect, however, only attains its worst form when a symbiosis with yeast ^ves rise to fat hydrolysis ^. Certain yeasts may producea fishy or train-oil taste. In marshy districts or where the land is occasionally inundated by sea or brackish' water, this defect may appear in fresh' butter, and is then due to the grass or small crabs which are found in great numbers in the grass. Certain bacteria are also said to be able to produce a fishy taste by forming trimethylamine from lecithin ^. Some of the defects which may appear in butter after keeping for any length of time are of a purely chemical nature, like the oxida- tion process discussed above, and their appearance may be accelerated by iron and possibly other salts. It is therefore of importance that the salt used in butter should be chemically pure ^. It has been said that dairy salt may contain fat hydrolysing bacteria *. Fresh pUre salt is oi course sterile, but when kept in the dairy, numerous organisms (thousands per gram) may collect on its surface, and Weigmann has therefore proposed to dry the salt in an air oven at 100° C. before use. As butter defects which are apparently of the same nature may arise in different ways, and conversely, as is often seen to be the case in , butter grading, the same defect may pass under different names, it is hardly possible in the present state of our knowledge to go into, further detail as regards the secondary defects. As, " moreover, most of the defects sooner or later pass into the stage 1 The author was the first to show that the ability of certain yeasts to hydrolyse fat is promoted in the presence of lactic acid bacteria : " Bak- teriologisohe Studien iiber die danische Butter" (" Centralblatt f. Bakt.," 2 Abt., 1911, Bd. XXIX., p. 610). This was later confirmed by Sandelin : "Die Hefen der Butter," Helsingfors, 1919. 2 Thus, Ousick (" Journal of Dairy Science,'' 1920, Vol. IJI., p. 194) is of the opinion that this defect can be produced by Bad. ichthypsmius, a motile Gram -negative rod showing dirty white surface growth, peptonising milk Avhile producing slight amounts of acid, and producing gas from cane sugar, but not from laptose. ' Sogers, Berg and Potteiger, U.S. Dept. of Agric, Bureau of Anim. Ind., 1913, Bull. 162. « Wolff, " Milchzeitung," 1914, p. 545. MICROFLORA OF BUTTER 127 known as rancidity, it is probable that they are only forerunners of this principal defect. The dairies can easily control the keeping qualities of the butter which they produce, and thus of any possible defects, by making a practice of keeping samples of the freshly worked product in small jars at about 8° to 14° C, and tasting these after a week and a fortnight. Chapter VI The Ripening Processes of the Different Cheeses The methods of preservatdon, involving the use of preservatives or bactericidal substances, include the use of harmless acids such as acetic or, better, lactic acid. The latter is not added but produced by allowing the material to be preserved to set up a lactic acid fermentation. This method is applied to the preserva- tion of beet slices, white 9abbage (sauerkraut) and other sugar containing fodders and foodstuffs which contain too much water to be dried without, the aid of artificial heat. In such cases it is only necessary to exclude air as completely as possible so that the acid-consuming moulds are kept down. The same process may also be applied to milk. If the yeasts and moulds are destroyed by pasteurising in bottles, and the milk is inoculated with a vigorous culture of lactic acid bacteria, it will remain unchanged after the souring process has been completed ; a simUar principle is applied in the making of cheese, which is always based on a vigorous development of lactic acid bacteria. Under normal conditions the acid which is produced will always inhibit the growth of other bacteria, and in the closely pressed mass of cheese no moulds will develop, owing to the absence of air. If, as in the case of the hard cheeses, an additional protection is afforded in the shape of a firm cheese rind, all risk of infection is excluded, and a permanent product is obtained. The origin of cheese making was without doubt a desire to preserve the valuable constituents of milk in a permanent and easily market- able form. The primitive process, therefore, only involved the drying and salting of the curd, a process which is still employed in several places in the East. Subsequently it was discovered that the curd would also keep without drying, and that with suitable treatment it would acquire other valuable properties into the bargain. The art of cheese making thus became not only one of mere conservation, but also the production of a palatable food, and in the case of the soft cheeses it may well be said that attention has been concentrated on the latter~point. The curd is separated by the action of either rennet or lactic acid. ; we may commence with an examination of the mechanism of the two processes. RIPENING PROCESSES OF CHEESES 129 The active principle in rennet is a -proteolytic enzyme Chymosin, the action of which does not cease with the coagulation of the milk and the contraction of the casein, but continues in the cheese with the formation of soluble proteins. The author's researches have established that cheese rennet exerts a powerful solvent action on the proteins of milk, and that this action is promoted by the addition of small amounts of acid i. American researches ^ have shown that the ripening of cheese is accelerated by increasing the amount of rennet used. As pepsin is also present in rennet, it was formerly supposed that the solvent action on the proteins was exclusively due to this enzyme. Careful investigation, however, has conclusively shown that chymosin itself is a proteo- lytic enzyme which can act in the presence of smaller amounts of acid than pepsin *. The addition of pepsin to the milk used for cheese making appears to have no influence on the ripening, process. Trypsin, on the other hand, has a decided influence on cheese, but may easily cause a bitter taste *. In this connection, mention may be made of galactase, a proteolytic enzyme which, according to researches by Babcock and Russel ^, is a normal constituent of milk, and which just after its discovery in 1897 was assumed to play a most important part in the ripen- ing of cheese. Subsequently it transpired that galactase does not play any notable part in the ripening of soft cheeses, in which, however, it is particularly plentiful ® ; and the fact that this enzyme is not indispensable to the ripening of hard cheeses is amply demonstrated by the fact that good cheese has been produced on a large scale from milk which has been heated to over 80° C, at which temperature the enzyme is destroyed. As regards the action of lactic acid, this is of interest not only in the making of sour milk cheeses, but also in the making of rennet cheeses. As is well-known, casein occurs in milk as a calcium salt, a dicalcium caseinate (the calcium compounds of the proteins usually form milky solutions in water), and when casein is precipi- tated by acid it is not due to transformation into paracasein as occurs with rennet, but simply to the abstraction of lime by the acid. At the same time a little lactoglobulin is precipitated. The greater the percentage of casein in the milk, the more acid 1 " Landwirtschaftb'ohes Jahrbuch der Schweiz," 1904, p. 404, and 1907, p. 97. ^ Seventeenth Keport of Wisconsin Agricultural Experiment Station, Madison, 1900. " Petry, "Wiener Klinische Wochenschrift," 1906, p. 143. 4 OrU Jensen, " Nyt Tidskrift for Fysik og Kemi," 1897, p; 92, and " Landwirtschaftliches Jahrbuch der Schweiz," 1901, p. 197. ' Wisconsin Agric. Expt. Station, Bull. 14, 15 and 19. « Oria Jensen,^' Centralblatt f. Bakt.," 2 Abt., 1900, Bd. VI., p. 793. D,B. 9 130 DAIRY BACTERIOLOGY will be required for complete coagulation. The higher the tem- perature, the easier the coagulation. Thus, at 18"°, 30°, 40° and 100° C, 0-6, 0-5, 0-25 and 0-1 per cent, respectively of lactic acid will generally be required, corresponding to 80, 72-5, 53-5 and 27-5 c.c. of decinormal caustic soda per 100 c.c. respectively. For this reason, milk is often warmed when the casein is to be precipitated. If a temperature of 70° C. is exceeded, appreciable amounts of albumin are also precipitated ; larger yields of cheese are therefore oTatained by using milk pasteurised at high tem- peratures. On heating to over 90° C, all the albumin and globulin are precipitated ; the more completely this takes place the more easily will the milk -coagulate, for aU solid particles, including fat globules, serve to stiffen the coagulum. The same milk will therefore coagulate at a lower acidity when pasteurised at a high temperature than when pasteurised at a low temperature. Acid coagulum differs from rennet coagdlum in not contracting so much, so that it does not separate such large quantities of whey when allowed to stand undisturbed. This affords a means of distin- guishing between the two types of coagulation. In the case of a bacterium which coagulates milk, a titration will at once decide whether a quantity of acid sufficient to be entirely responsible for the coagulation has been produced ; if not, then the bacterium must also have produced a coagulating enzyme like rennet. The coagulating power of rennet is increased by the addition of acid ; the reason for this is not only that the acid promotes the action of the enzyme, but also that it forms soluble calcium salts which facilitate the precipitation of the paracasein. The coagulation of pasteurised milk by rennet may thus be promoted by the addition of a fair amount of acid, e.g., in the fofm of buttermilk ^, and also by the addition of calcium chloride (100 c.c. of a 40 to 50 per cent, solution to 100 litres of milk). The contraction of the rennet coagulum is also promoted to a certain degree by the addition of acid, the whey separating most readily at a concentration of about J per cent, of lactic acid, when all the casein is converted into the mono-calcium salt ; for this reason, the cheese dries better if prepared from slightly acid milk than if prepared from fresh milk. If the milk is so sour that there is a suspicion that the resulting cheese will be too dry, all that is necessary is to dilute it with water before adding the rennet ;\ in this way, the concentration of both the free acid and the soluble calcium salts will be decreased. Conversely if a higher acidity is desired, the 1 In the making of Cheddar cheese from pasteurised milk, Sammis and Bruhn (Bureau of Anim, Ind., 1913, Bull. 165) recommend besides the addition of starter milk containing j per cent, of acid the use of I part of normal hydrochloric acid per 100 parts of milk. RIPENING PROCESSES OF CHEESES 131 milk may be allowed to ripen at a temperature favourable to the development of good lactic acid bacteria (15° to 20° C), or an appro- priate amount of buttermilk, starter or other lactic acid cultures may be added. The Ipnger the time occupied in making the cheese, the sourer will be the curd. The temperature of the curd when placed in the cheese press determines the species of lactic acid bacteria which shall obtain predominance. If scalding is omitted, and the curd is cooled by kneading after the whey has been run off, the bacterial flora will be quite different from that which results when heat is applied and the curd is taken direct from the warm whey. By scalding and carefully cutting the curd, the separation of the whey will be facilitated ; a means is thus afforded of shortening the curd forming process, and of regulating the degree of acidity of the cheese. As nearly half the natural acidity of milk is due to the casein, it follows that the whey must have a much lower acidity than the milk from which it was made. In order to control the process of souring during cheese making, the whey may he titrated at different stages. Although the indications thus obtained will be of some value, they are far from accurate, and should be supplemented by a careful examination of the consistency of the curd. On coagulation, with careful treat- ment of the milk, most of the bacteria become enclosed in the curd and consequently lactic acid fermentation will take place far more rapidly within the curd than in the whey^. In the curd, however, most of the acid produced will be neutralised by the lime of the casein and the phosphates ; accordingly it will sometimes be found that in spite of a vigorous lactic acid fermentation the acidity of the whey may remain unaltered during the process, or even decrease as happens in the making of Emmental cheese, for owing to the fact that the whey is scalded at a fairly high temperature, the loss of carbonic acid more than counterbalances the gain in lactic acid taken up by the whey from the curd. If the bulk of the whey is removed at an early stage, the remaining whey wiU be more acid than usual at the end of the process as the lactic acid which has diffused out of the curd will have been diluted to a less extent. When the casein loses its lime it becomes, as van Slyke and Hart were the first to show^, fairly readily soluble in a 5 per cent, solution of sodium chloride, especially at 50° to 55° C, but this property is lost in the presence of an excess of acid. The author has shown ^ that this 1 Orla Jensen, " Uber die im Emmentlialerkase stattflndende Milch- sauregarung." " Landwirt. Jahrbuch der Schweiz," 1906, p. 287. 2 New York Agric. Exp. Station, Bull. No. 261, 1905. 3 " Zeitachrift f. physiologische Chemie," 1914, Bd. XCIII., p. 283. 9—2 132 DAIRY BACTERIOLOGY is due to the easy solubility of monocalcium easeinate and para- caseinate, while free casein and paracasein are practically insoluble in salt solution. As cheese is always salted, these observations have an important bearing on cheese making ; it will now be understood why the acidity of the curd and the way in which it is salted come to have such an important effect on the, consistency of the cheese. When a slightly acid curd is salted or placed in brine, it will swell as if tending to dissolve, becoming elastic and semi- transparent. On the other hand, on excessive acidification or slow dry-salting, the curd will retain its original crispness for a long time. Monocalcium easeinate, though soluble in dilute salt solution, is insoluble in strong brine, for which reason the brine used in the cheese making dairies should contain at least 25 per cent, of salt ; as lactic acid is constantly diffusing into it from the cheeses, it should be neutralised and filtered from time to time, as is done in Hollaixd. According to Rosengren, 1 to 1-2 per cent, of salt in cheese can be considered normal ; 2 per cent, or more makes the cheese dry, and inhibits the fermentation processes. The action of acid has been dealt with at some length not only because the degree of acidity influences the consistency of the cheese, but also because it has a determining in- fluence on the course taken by the ripening process which, owing to the empirical methods of treatment in vogue, deter- mines more than anything else the nature of the resulting cheese. As the maximum acidity attained by the cheese de- pends flrst and foremost on the amount of whey which it con- tains, the classification into more or less sour cheeses accords fairly weU with the division into hard and soft cheeses. In the former the production of acid wiU practically be limited to the amounts required to neutralise the lime, and the cheese will therefore ripen uniformly throughout. On the other hand, the relatively large amounts of acid present in the soft cheeses will only be neutralised after a considerable lapse of time, and as a rule the process is only completed by the aid of the ammonia which is formed on the surface. It follows that in such cheeses the ripening process will start at the surface and work inwards by degrees ; for this reason the ripening may be accelerated by giving the cheeses a large surface relative to their bulk. The foregoing points are illustrated by the accompanying table which gives the percentages of soluble proteins and their decomposition products in different cheeses. Sol. N. stands for soluble nitrogen, i.e,. the nitrogen of the soluble proteins, plus that of the protein decomposition products present, Dec. N. for the nitrogen of the protein decomposition products, or amino acids which are not RIPENING PROCESSES OF CHEESES 133 precipitated by phosphotungstic acid^. Am. N. stands for ammonia nitrogen. Per cent, of total Nitrogen. Per cent, of soluble Nitrogen. Sol. N. Dec. N. Am.N. Dec. N. Am.N. 1 Emmental cheese, five months old. Interior. 35-82 17-36 — 48-47 — Exterior. 29-22 12-57 — 43-02 — 2 Emmental cheese twelve months old, ripe. Interior; 33-15 17-35 2-37 52-34 7-15 1 Edam cheese four months old, ripe. Interior. 26-90 3 00 0-60 11-15 2-23 1 Prize Danish dairy cheese from pasteur- ised milk, three months old. Interior. 35-5 9-7 0-22 27-6 0-6 2 1 Prize Danish dairy- cheese from pasteur- ised milk, six months old. Interior.. 34-9 90 0-17 25-8 0-5 Swiss skim milk cheese, eight months old, ripe. Interior. 41-51 7-90 6-40 19-03 15-40 Exterior. 35-90 7-40 5-^0 20-61 14-50 Swiss skim-milk cheese, sixteen months old, over-ripe. Interior. 43-54 6-66 6-85 15-29 15-73 Exterior. 53-59 911 5-37 17-00 10-02 1 Roquefort cheese, ripe. The whole bulk. 52-50 23-64 4-99 45-03 9-51 i 03 1 Brie cheese, not quite ripe. Interior. 47-10 7-58 5-14 16-10 10-91 Exterior. 53-50 21-33 12-37 39-87 23-12 1 Camembert cheese, ripe. Interior. 95-52 8-71 8-71' 9-12 912 1 Limburger cheese, six weeks old. Interior. 24-82 5-27 4-37 21-23 17-60 Exterior. 55-10 12-58 4-51 22-83 7-85 Limburger cheese, ripe. Interior. 99-82 4-33 11-97 4-52 11-99 ^^ S CO H 02 1 Schabzeiger. The whole bulk. 37-35 16-58 5-80 44-39 15-53 1 Norwegian Gammelost (old cheese), ripe. Interior. 51-35 31-99 4-23 62-33 8-23 Exterior. 69-47 38-57 7-42 55-52 10-68 The chief feature of the ripening process is the conversion of insoluble proteins into soluble substances. The table shows that 1 For cheese analysis, see " Zeitschr. f. Nahrungs und Genussmittel " 1906, Bd. XII., p. 193. 134 DAIRY BACTERIOLOGY in the hard cheeses usually only one-third of the casein becomes converted into water soluble proteins, whereas in the soft cheese's nearly all the casein undergoes conversion. This explains the apparent richness in fat of the soft cheeses, for when anything gives the sensation of melting in the mouth distinction is as a rule not to be made between melting proper, as in the case of butter, and a solvent action. Further examination of the soluble sub- stances in hard cheeses shows that a larger proportion of these have been converted into amino acids thian in the soft cheeses ; these conditions may be summed up by saying that in hard cheeses the ripening is less extensive hiit more thorough, while in the soft cheeses it is extensive but not so thorough. This definition, however, cannot be regarded as a rigid one, for when the hard cheeses are not much older than the soft ones (see, for example, Edam cheese), the proportion of Dec. N. to Sol. N. is not large, and if the soft cheeses do only contain small amounts of amino acids, this is because these are quickly broken down into ammonia on the surface ^. Now as ammonia readily combines with the lime free casein to form soluble salts, the high proportion of Sol. N. in the soft cheeses is partly attributable to the thorough- ness of the ripening process. The most important factor in the ripening of the rennet cheeses, besides the various microorganisms, is the rennet. It is the rennet which produces the perceptibly soluble proteins and the microorganisms carry the degradation further. Further, as the action of the most important of the enzymes of the cheese ripening bacteria is inhibited, while that of rennet is promoted by the presence of acid, it will easily be under- stood why the practically neutral hard cheeses contain larger proportions of amino acids than the soft cheeses. In the ripening of cheese, not only' is the casein converted into easily digestible and palatable products, but the fat and the lactose also undergo changes. As might be expected, the fat is hydrolysed most rapidly in cheeses like Roquefort which are permeated with moulds. Rapid fat hydrolysis also takes place in cheeses made from separated milk, for here the fat globules are very minute and thus expose a large surface for attack. On the other hand, fat hydrolysis proceeds with extreme slowness in the common hard rich cheeses. As, however, butyric, caproic and capric acids have a very persistent taste, they contribute very largely to the aroma of the cheese even though they may only be present in small amounts. The reason why the choicer cheeses must pass through a long period of ripening in order to attain their characteristic piquant taste to the full, is that the processes 1 Orla Jensen, " Centralblatt f. Bakt.," 2 Abt., 1900, Bd. VI., p. 773. RIPENING PROCESSES OF CHEESES 135 of fat hydrolysis and ammonia formation to which the production of the sharp taste (not to be confused with the salt taste) is due proceed with extreme slowness in these cheeses. Both of these processes seem also in the case of hard cheeses to start at the surface and gradually penetrate inwards. While the fat is usually slowly decomposed in cheese, the lactose is the first constituent to undergo change. In hard cheeses it usually disappears in a few days and in soft cheeses in a week or two. Normally it is completely converted into lactic acid, which is neutralised by the lime and other bases present. Calcium lactate is not necessarily the end product, for' it is quite frequently more or less completely converted into propionic acid by the fermentation process described on p. 41, whereby the normal " eyes " or cavities are produced in cheese. The butyric acid fermentation of calcium lactate however, must be looked on as a disease of cheese, for the evolution of gas will be too vigorous, while unpalatable or even poisonous substances may be produced. The author's researches on the volatile acids of cheese, summarised in the accompanying table, show that normally no more butyric acid is found in the ordinary rennet cheeses than will originate from fat hydrolysis. After these general remarks, we may pass on to consider the microorganisms which play the chief part in the ripening of the different kinds of cheese. The flora of the hard rennet cheeses will be dealt with first. Duclaux, who was the first to investigate this field, found in cheese various sporing rod bacteria which he named Tyrothrix, i.e., " cheese threads." He found both aerobic and anaerobic forms, i.e., what now would be called hay and potato bacilli and anaerobic putrefactive bacteria. As the aerobic forms were able to decompose and dissolve casein, and the anaerobic forms produced the odour characteristic of cheese, Duclaux and all other contemporary investigators were agreed that the ripening of cheese was due to the combined action of these bacteria ^. The constant success of Duclaux in isolating Tyrothrix bacteria from cheese was due to the fact that he employed an enrichment method which particularly -favoured the growth of these bacteria. He introduced a small piece of cheese into broth in which the lactic acid bacteria did not thrive, and it was therefore only necessary to have a few Tyrothrix spores present from the outset in order that a Tyrothrix film should form on the surface of the- liquid after some time, after which the anaerobic sporing bacteria, protected from the air, could grow rapidly. The careful researches carried out by Freudenreich over a period 1 Duclaux, " Le Lait," Paris, 1894, pp. 213—258. 136 DAIRY BACTERIOLOGY of many years ^ have established that the Tyrothrix bacteria are comparatively rare in cheese, that normally they cannot develop in cheese even if introduced in large numbers, owing to their sensitiveness to acid, and finally that if conditions favourable to their growth are secured by using pasteurised milk and omitting the addition of lactic acid bacteria, they produce a disgusting taste of putrefaction in the cheese. The bacteria living in the Found in 1,000 grams Cheese. Kind of Cheese. As cc. normal. In grams. 1 1 Produced by fat hydrolysis. Produced by the splitting up of the Casein (paracasein) and lac- tose (lactic acid). 1 V 1 1 1 1 1 1 o *3 1 1 1 •J 1 o Emmental cheese. Interior. 880 75-0 0.116 0-176 — — 4-218 1-680 — 6-190 1-276 Exterior. 75-0 55-0 0-928 1-232 — — 2-812 0-900 — 6-872 0-936 Edam cheese. Interior. 15-6 16-0 — -, — — 0-224 0-678 0-057 0-959 0-256 Swiss skim- milk cheese. Interior. 81-6 267-5 0-986 1-496 — 2-405 1-200 0-138 6-225 4-548 Exterior. 100-0 207-5 1-682 2-552 — — 2-775 1-080 0-046 8-135 3-528 Koquefort cheese. Whoie buik. 380 115-0 0-928 1-672 — — — 0-640 0-092 3-232 1-955 Camembert cheese. Interior. 6-6 176-0 0-081 0-246 - — — 0-069 0082 0-478 2-975 Brie cheese. Interior. 11-3 96-0 0-139 0-572 — — — 0-204 0-008 0-923 1-615 Exterior. 8-7 217-5 0-128 0-466 — — — 0-120 0-013 0-727 3-698 Limburg cheese. Interior. 1110 200-5 0-058 0-440 1-681 — 5-180 1-140 0-046 8-445 3-409 Exterior. 104-5 220-0 0-232 1-003 1-550 — 4-629 0-822 0-046 8-182 3-740 Giarner Schabzelger. Whole bulk. 258-2 215-0 1-195 1-848 — 4-452 9-102 3-198 — 19-795 3-655 interior portions of the cheese are nearly exclusively of the lactic acid group, and it must consequently be these which are principally responsible for the protein hydrolysis which takes place. Freudenreich, who devoted special attention to Emmental cheese, found chiefly the rod-shaped species, and succeeded in showing that these were able to attack casein if only the lactic acid which they produced was neutralised by chalk as completely as it is neutralised in the hard cheese. This forms the basis of a 1 The first of Freudenreich' s papers on this subject appeared in " Annales de Micrographie," 1889, p. 257, while the subsequent ones are nearly all published in " Landwirtschaf tliches Jahrbuch der Schweiz," 1891 to 1906. RIPENING PROCESSES OF CHEESES 137 correct understanding of the ripening processes which take place in these cheeses. According to the author's researches, the proteolytic enzyme of the lactic acid rod bacteria must be regarded as an endoenzyme, as it is not liberated by the living cell, and its action recalls that of erepsin, for it produces amino acids from the casein without forming albumoses and peptones as intermediate products ^- The large amounts of amino .acids which are found in many hard cheeses are principally due to the action of this endoerepsin^. Th^ bacteria themselves take no part in the process, for most of them will be dead before the bulk of the amino acids have been produced ^ ; they do not thrive without sugar, and as all the lactose in hard cheeses will have been fermented in the course of a day or two, the ripening bacteria will already have reached their maximum (over 100,000,000 per gram) by then, after which they gradiially fall off. Dead cells which are not exposed to desiccation generally digest themselves more or less completely owing to the action of the intracellular enzymes which thus set themselves free, so that they become able to exert their digestive action on the surrounding medium ; this is what happens in cheese where the endoenzymes act under favourable conditions, not involving too great dilution. The author has demonstrated the presence of such enzymes in both hard and soft cheeses *. The fact that the ripening of hard cheeses depends on enzyme action pure and simple, and is not directly dependent on the action of living bacteria, is also demonstrated by the ripening of these cheeses at temperatures far below the minimum for cheese bacteria ^ Of the various rod-shaped lactic acid bacteria isolated by Freudenreich from Emmental cheese, one particular species, Thermobacterium helveticum, seems to be indispensable for the production of the tjrpical sweetish taste, and it is extremely interesting to see how the methods of manufacture, arrived at by practical experience, favour the development of this bacterium throughout the process. As the organism occurs in the fourth stomach of the calf, it will develop freely when the stomach is extracted in a warm place with " Schotte " (see p. 50). This is the usual practice in the Swiss dairies, the ripening bacteria thus 1 " Centralblatt f. Bakt.," 2 Abt., 1900. Bd. VI-., p. 840, and 1904, Bd. XIII., p. 521. ^ Freudenreich and Orla Jensen, "Landwirt. Jahrbuch der Schweiz," 1899, p. 167. * Orla Jensen, " Landwirt. Jahrbuch der Schweiz," 1906, p. 303. * " Studien iiber die Enzymen im Kase," " Centralblatt f. Bakt.," 2 Abt., 1900, Bd. VI., p. 734. ^ Babcock and Bussel, Eighteenth Annual Eeport of the Wisconsin Agricultural Experiment Station, 1901, p. 136. 138 . DAIRY BACTERIOLOGY being introduced with the "natural rennet"^. As the stomachs, especially those of bad quality, also contain many harmful bacteria, it is advantageous to inoculate the rennet at once with a mixed culture of Thermobacterium helveticum and the mycoderma men- tioned on p. 50 2, or to secure favourable conditions for the lactic acid bacteria simply by adding lactic acid (e.g., some boiled acid Schotte) to the rennet. It is safer to use the pure factory made rennet, together with pure cultures of Thermobacterium helveticum, in sterile milk ^. In order to secure the development of this organism to the exclusion of other bacteria, the cheese must be made warm and kept warm in the presses. This is achieved oy scalding at a comparatively high temperature, taking the curd out of the warm whey in a lump, and above all by making the cheeses so large that they will retain their heat for a long time. Cheeses made in this way maintain a temperature of 50° to 35° C. during the first twenty-four hours, and under these conditions Thermobacterium helveticum is certain to obtain predominance. At the same time a lactic acid streptococcus (Sc. thermophilus) having a high optimum temperature grows luxuriantly *. As this organism does not attack casein, it hardly plays any important part in the ripening of the cheese beyond the souring process, and possibly assisting in the production of favourable conditions for the growth of the lactic acid rod bacteria, in much the same way as occurs in Yoghurt and similar products. WhUe the drastic scalding weakens the gas -producing bacteria, the high temperature of the cheese while in the press favours the growth of the pseudo lactic acid bacteria, and if the milk was dirty there will already be a very vigorous development of blow holes. (Such a cheese {Presslis) develops a' large number of pin- holes.) Coli and aerogenes bacteria, as well as butyric acid bacteria, may also develop at a later stage, and it is therefore advisable to keep the cheese as cold as possible after it has left the press, and until all the lactose has been fermented. Any evolution of gas will do far more harm in the compact mass of . Emmental cheese than in most other cheeses from which the gas can partially escape through the narrow fissures which mark off the ofiginal particles of curd. For this very reason the normal cavities or " eyes " are able to attain to a much larger size in 1 Freudenreich and Orla Jensen, " Centralblatt f. Bakt.," Abt. 2, 1897, Bd. III., p. 545. I 2 J. Thoni, " Bacteriologische Studien iiber Labmagen und Lab." ("Landwirt. Jahrbuch der Schweiz," 1906, p. 181.) 3 Eosengreen and Haghind, " Meddelande No. 101 Iran Centralanstalten for forsoksvasandet pa jordbruksomradandet," Stockholm, 1914. * Orla Jensen, ■" Uber die in Emmenthalerkase stattfindende Milch- sauregarung." " Landwirt. Jahrbuch der Schweiz," 1906, p. 437. RIPENING PROCESSES OP CHEESES 139 Emmental cheese than in other makes. The eyes should only begin to form when the cheese has ripened to such a degree that it is sufficiently plastic to allow of their rounding off ; if they form too soon, they become irregular in shape, and if the mass is made too dry, it will never become sufficiently plastic ; in this state the cheese is known as " Gldsler," having elongated cavities or clefts instead of proper eyes. In order to promote the formation of normal eyes, the cheese is brought into a room at 18° to 22° C. when it is two weeks old. Here the ripening process is accelerated and the propionic acid bacteria will gradually develop so that in the course of four to six weeks the eyes will have been fully formed. The cheeses are then brought into a cold place again. As the propionic acid bacteria are very sensitive to sodium chloride, it is possible to regulate the formation of eyes by the addition of more or less salt ^ ; the chief reason for the adoption of the somewhat troublesome method of dry salting in the case of Emmental cheese, instead of methods by which the cheese receives its full amount of salt at an earlier stage, is doubtless that the 6heeses would, in the latter case, only develop small eyes or none at aU, as often occurs with snijall cheeses, which naturally tend to become salted too quickly. If the cheese has not been made sufficiently dry, or contains too many propionic acid bacteria at the outset, the development of the normal eyes will be excessive, and this is a defect which may be just as objectionable as the blowing at an earlier stage, which has been described above. On the other hand, if neither the ripening process nor the development of the propionic acid bacteria have progressed sufficiently in the " warm cellar," it may happen that the eyes wiU suddenly begin to form at a still later stage ; an after fermentation of this nature will always tend to give a variable product. The presence of unusually large numbers of butyric acid bacteria will also give rise to an abnormal eye formation which, it may be noted, will be of the worst possible type^ In such cases a very energetic butyric fermentation sets in when the cheeses are from ten to fourteen days old, and under these circumstances they will not stand exposure to the temperature of the warm ceUar. - Closely connected with the ripening process is the formation of drops of liquid or " Tears " in the eyes, a process which often first starts when the Emmental cheese is eight months old. The conditions determining the collection of this liquid are, first, that it shall not be too viscous, apd second, that the pores of the cheese shall riot be too fine. In the ripening of the cheese an appreciable proportion of the dissolved proteins are converted into amino 1 Orla Jensen, " Landwirt. Jahrbuch der Schweiz,'' 1906, p. 437. 2 J. Thoni, " Landwirt. Jahrbuch der Schweiz," 1906, p. 157. 140 DAIRY BACTERIOLOGY acids, whereby the viscosity of the liquid is decreased, and as increasing amounts of the curd become soluble the pores in the cheese will become larger. The salt which at an earlier stage tended to causejthe cheese to swell now has the opposite effect, but as the rind prevents contraction of the mass as a whole, the pores must become still larger. The salting and drying of the cheese finally bring about the precipitation of the sparingly soluble amino acids as white crusts, and these so-called " salt stones " may be distributed throughout the whole mass. The reason why the ripening of Emmental cheese has been discussed so thoroughly is that this cheese has been studied more thoroughly than any other. In this respect, the large-holed Swedish manor farm cheese, which resembles the Danish-made Swiss cheese, comes next. -According to Gerda Troili-Peterson's researches 1, the ripening of this cheese depends on peptonising tetracocci in addition to the lactic acid rod bacteria, and the formation of eyes on certain glycerine-fermenting aerogenes bacteria as well as the propionic acid bacteria. According to Oorini ^, peptonising tetracocci also play an important part in the ripening of Parmesan cheese, in the making of which the curd is scalded at a high temperature ; this cheese differs from Emmen- tal in being prepared without the addition of strongly acidifying lactic acid rod bacteria, a dough made from chopped calves' stomachs' being • used instead of the acid "natural rennet" de- scribed above. The peptonising cocci, just mentioned, generally develop freely in the fresh curd, for, unlike the tyrothrix bacteria, they can grow in the presence of acid. They are, however, quickly suppressed if too much acid is present, and will therefore be most prominent in cheeses which sour slowly or in cheeses made from milk which has been ripened at a temperature below 15° C, as they grow better than the true lactic acid bacteria under this temperature. The proteolytic enzyme secreted by the peptonising tetracocci is less sensitive to acid than the erepsin of the lactic acid rod-bacteria, and its mode of action is intermediate between that of the latter and that of rennet. It would therefore appear to be best defined as an " exotrypsin." The best investi- gated of these cocci is Tetracoccus liquefaciens, which produces the characteristic taste of Tilsit cheese or Russian Steppe cheese, and there is no doubt that it plays an important part in the 1 " Centralblatt f. Bakt.," 2 Abt., 1909, Bd. XXIV., p. 343. 2 Among the numerous papers by this investigator, special mention may be made of " Eecherches sur les coccus producteurs d'acide et de pressures du fromage " (" Eevue g6n6rale du lait," 1910, vol. 8, p. 337). The true significance of the action of the peptonising micrococci in the ripening of cheese was first pointed out by Weigmann, 1896 (" Centralblatt f. Bakt.," 2 Abt., Bd. XL, p. 151). RIPENING PROCESSES OF CHEESES 141 ripening of these cheeses. . Similarly, it is probable that it assists in the ripening of Gouda cheese, which is made on the smaller Dutch farms from perfectly fresh milk without the addition of lactic acid bacteria ; under such conditions these organisms will have ample opportunity for development. Boekhout and de Vries have shown that the pe^onising bacteria do not assist to any appreciable extent in the ripening of Edam Dutch cheese, which is nowadays generally made with a lactic acid starter ^, and they have not succeeded in demonstrating other specific ripening bacteria ^. As Edam cheese is on the whole poor in typical pro- ducts of bacterial action (amino acids and volatile acids), it would appear that the bacteria here play only a subordinate part, and that the principal changes are due to the rennet, as is indicated by • van Dam's researches^. *' Salt stone" is occasionally found in Edam cheese, but in this case it consists of calcium lactate and phosphate. Again, in Cheddar cheese, the peptonising bacteria do not bring afeout any changes of importance ; here also a great deal of the action is due to the rennet, and the main feature in the making of this cheese is the production of a curd having a fairly high acidity at the outset, so that when the curd is pressed, against a hot iron it can be drawn out into long silky threads (the hot- iron test). American investigators have found the flora in Cheddar cheese to consist almost exclusively of the true lactic acid bacteria, chiefly streptococci. The bacterial count reaches its maximum immediately after the cheese has been made and then decreases steadily, the rate of decrease being quicker the higher the temperature at which the cheese is kept*. By allowing the curd to become strongly acid through the agency of different streptococci, the author has succeeded in producing cheeses resem- bling Cheddar in texture and taste, so that there can be no doubt that in this case such bacteria actually accomplish more than the mere production of acid ^. The streptococci cannot, however, be responsible for the large amounts of amino acids found in ripe Cheddar, so that here also we have evidence of the action of lactic acid rod bacteria. As a matter of fact, the author has never met with any kind of cheese in which lactic acid rod bacteria (strepto- 1 " Kevue g6n6rale du lait," 1910, vol. 6, p. 1. 2 " Centralblatt f. Bakt.," 2 Abt., 1905, Bd. XV., p. 323, and 1906, Bd. XVII., p. 149. These authors have described a diplococcus-like rod bacterium which converts lactic acid into acetic acid, carbon dioxide and hydrogen, and which is said to have some significance in the forfeation of eyes in Edam cheese. It will stand 4| per cent, of salt, and its optimum tempera- ture is 21° C. {ibid., 2 Abt., 1918, p. 130). a "Centralblatt f. Bakt.," 2 Abt., 1910, Bd. XXVI.. p. 189. 1 Harrison, " Centralblatt f. Bakt.," 2 Abt., 1904, Bd. XI., p. 637. Sub- sequently Harding and Prucha made a thorough study of the flora of Cheddar cheese (New York Agric. Exp. Station, Bull. No. 8, 1908). 5 /" Centralanstalten's 97 Meddelse." 142 DAIRY BACTERIOLOGY bacteria) could not be demonstrated in large numbers, a natural result of the ability of these organisms to overgrow all other bacteria under circumstances favourable to themselves. Thus, they also obtain predominance by slow degrees in the Danish dairy cheese, although this cheese, like Cheddar, is freely inocu-' lated with lactic aci(J-producing streptococci by the addition of buttermilk." Barthel ascribes to the streptococci a more important part in the ripening of cheese than has hitherto been done. He has shown that certain strains form, at ordinary temperatures, quite appreciable amounts of Sol. N. (see table, p. 143). The author has also found, with fair regularity, streptococci, especially strains • of Sc. cremoris, which are conspicuously able to split casein into soluble products ; as these bacteria gradually lose this power when cultivated on artificial media, it may be surmised that streptococci which do not hydrolyse casein to any appreciable extent may acquire this property when cultivated in milk and cheese. It must also be pointed out that many lactic acid bacteria grow better in milk to which rennet has been added than in ordinary milk ; that is, their action is promoted by the rennet just as, conversely, the action of the rennet is promoted by the lactic acid. In addition to the action of certain lactic acid streptococci which hitherto have not been further investigated, the chief factors in the' ripening of the hard rennet curd cheeses are seen to be the rennet, the exotrypsin of the peptonising tetracocci and the endoerepsin of the lactic acid rod bacteria. The relative importance of each of these factors varies considerably in different cheeses, and determines the characteristic propertites of each variety. Further differences arise owing to the fact that the above-men- tioned groups of lactic acid' bacteria include many different species, an illustration of the production of special characteristics owing to the action of a particular species of organism being seen in the case of Emmental cheese. The table on page 143 has been drawn up to illustrate the action of the several factors in ripening ; the amounts of soluble nitrogen (Sol. N.), nitrogen of decomposition products (Dec. N.), and ammonia nitrogen (Am. N.) formed in milk after two months are given (cf. table, p. 133). All the cultures were treated with chalk and shaken regularly so that the lactic acid produced was neutralised. Taking a wider view of the ripening process, as including not only the decomposition of the casein, we must also consider the propionic acid bacteria, as a ripening factor. Weigmann is of the opinion that the aromas of the various kinds of cheese are princi- pally due to the action of bacteria other than the lactic acid RIPENING PROCESSES OF CHEESES 143 organisms^, but as yet he has not been able to adduce any evidence as to the correctness of his theory. Bodella's^ contention that the specific-taste-prodTicing bacteria are obligate anaerobic sporing organisms can hardly be supported, for it has never been proved that these bacteria reproduce themselves in rennet curd cheese ^, and when on rare occasion they have been found in large numbers, a harmful action has also been observed. The interior of a hard cheese should have a clean taste and smell, but putrefactive pro- cesses in the rind cannot be avoided unless special precautions are taken, and if the cheese becomes very old the interior may also Milk with Chalk. Percentage of Total Nitrogen. Sol. N. Dec. N. Am. N. Streptococcus lactis .... Rennet ...... Streptococcus lactis and rennet Tetracoccus liquefaciens Thermobacterium helveticum 2-51 11-75 60-56 75-70 36-12 2-02 0-00 5-32 12-12 34-60 0-23 0-00 0-36 1-89 3-91 become affected. An ordinary dairy cheese, not too poor in fat, may even be made to acquire quite a piquant flavour in the course of three to four months, if only certain changes are promoted in the outer layer, as is done in the case of the " smeared " soft cheeses. This method was at one time successfully applied by Fru Hanne Nielsen. Certain hard cheeses, e.g., Tilsit cheese, have a tendency to undergo similar changes spontaneously, and may therefore be regarded as intermediate between the hard and the smeared soft cheeses. On the other hand, those hard cheeses which become permeated with moulds form a link between the hard cheeses and the soft mouldy cheeses ; they will therefore be dealt with before passing on to the soft cheeses. Cheeses like Stilton, Gorgonzola and Roquefort, which are per- meated with moulds, resemble the other hard cheeses in their mode of ripening, i.e., the process does not originate on the surface and work inwards. The surface is kept as clean as possible, and the cheeses are not made flat ; on the contrary, they are shaped so as to expose a relatively small surface. In Stilton and Gorgon- zola the moulds only develop slowly, as they owe their presence to 1 " Mykologie der Milch," Leipzig, 1911, p. 223. 2 " Centralblatt f. Bakt.," 2 Abt., 1903, Bd. X., pp. 499, 753: also 1906, Bd. XVI., p. 52. ' Burri and Kursteiner, " Landwirt. Jahrbuch der Schweiz," 1909, p. 442. 144 DAIRY BACTERIOLOGY casual infection and not to artificial inoculation (at any rate, up tUl recently they were not purposely introduced) ; the initial stages of the ripening processes in these cheeses, therefore, foUow much the same course as in the typical hard cheeses. On the other hand, Roquefort is inoculated with pure cultures of the required mould, Penicillium roqueforti, which means that a new factor comes into play at the outset, with the result that the ripening of this cheese, although accomplished at a low tem- perature (6° to 8° C), only requires as many weeks for its com- pletion as the ripening of the above-mentioned cheeses require months. The piquant taste and smell of this type of cheese are principally due to fat hydrolysis, and it is therefore possible to produce a similar aroma by inoculating the corresponding moulds into butter. These organisms also affect the casein to a consider- able extent, as is shown in the table on p. 133. Roquefort, in common wjth the Norwegian Gammelost (the latter an acid curd cheese which is permeated with mould), contains more amino acids than any other cheese. The development of the moulds is pro- moted by making the fresh cheese strongly acid, for which reason the curd is not submitted to a prolonged working, the treatment resembling that applied to the soft cheeses, while during the early stages of the ripening the cheese is kept at 18° to 20° C. in order to promote lactic acid fermentation. As air is necessary for the growth of the moulds, the cheeses must be stabbed immediately after salting ; before doing this the surface of the cheese must be cleaned carefully to avoid the transference of the organisms growing thereon to the interior by means of the needle ; some of these organisms may colour the cheese red Or turn it bitter. This is of particular importance if the cheeses are stabbed at later stages. They are stored in a comparatively dry room and placed on edge so that the holes shall not be closed up again. If it is desired to produce a typical Gorgonzola cheese in which certain bacterial fermentations will have taken place before the mould has com- menced its action, the stabbing should not be commenced before the cheese is two months old. The bluish-green veins of Gorgon- zola owe their origin to the practice of interlaying the fresh curd with acid curd which is twelve hours old and which has become strongly infected with Oidium lactis and various species of Peni- cillium on the surface. According to the results of the author's investigations with butter ', the above-mentioned mixture of moulds seems to be particularly well adapted to produce the desired taste and appearance of Gorgonzola. In the case of Stilton, the moulds simply penetrate through fissures in the 1 " Centralblatt i. Bakt.," 2 Abt., 1902, Bd. VIII., p. 369. RIPENING PROCESSES OP CHEESES 145 surface. The species of Penicillium found in Stilton and Gorgon- zola appear to be mainly Penicillium roqueforti. This circum- stance is explained by Thorn and Currie ^ by the fact that P. roqueforti can thrive with less oxygen than the other Penicillia, and therefore obtains predominance in the interior of the cheese, even though the cheese has not been inoculated with it. In the making of Roquefort cheese the veins are produced where desired by dusting with mouldy bread, of which about 0-1 per cent, of the weight of the cheese is used ; the mould grows not only in these places, but also in the holes made by the needle ; it is able to overgrow the cheese ba,cteria owing to the low temperature (8° C.) at which the cheese is ripened. The bread used for the cultivation of the mould is best made from a dough containing equal parts of rye, barley and wheat flour, and a little lactic or acetic acid, in which acid fermentation is induced -by the addition of some sour dough. The bread is very thoroughly baked, which, in conjunction with the action of the acid which has been added, destroys bacterial spores which otherwise would develop when the bread is set aside for the development of the moulds. It is then cut into slices and dipped into sterile water containing J per cent, of acetic acid, into which a culture of the mould has been stirred. The bread slices are placed close together on sterile shelves in a damp room and covered with sterile filter paper. As the change in temperature caused by the growth of the mould, when this sets in, is consider- able, the initial temperature should be kept down to 9° to 10° C. When in the course of three to four weeks the bread has become thoroughly mouldy, the crusts are removed and the slices are dried for about ten days at 30° to 32° C, after which they are ready to be ground and sifted. The yield is 45 per cent, of the original weight of the bread. Even if the work is carried out in rooms which are as sterile as- possible, it is difficult to avoid infection with foreign moulds. The author has accordingly worked out a method ^ in which the strain of P; roqueforti which is used is little by little accustomed to stand comparatively large amounts of formaldehyde. Formalin can be added to the water in which the slices are dipped, whereby the development of the foreign moulds which cannot stand formalin is inhibited. A mould powder made in this way is sold by Messrs. Blduenfeldt and Tvede, of Copenhagen. As already mentioned, the ripening process of the soft rennet curd cheeses starts at the surface and works inwards. At the same time, a ripening action due to the rennet takes place throughout the whole mass,^even though it may not be very obvious. While the amounts 1 " Journal of Biol. Chem.,?' 1913, vol. 15, pR. 249 and 259. ? " Maelkeritidende," 1919, p. 277. S.B. 10 146 DAIRY BACTERIOLOGY of acid present in the interior of soft cheeses inhibit the action of the ripening factors hitherto considered, they promote the proteo- lytic action of the rennet and conserve its enzyme in an active state for a much longer time than is the case in the hard cheeses ^. As will have been gathered from the preceding remarks, the soft cheeses may be divided into two groups : (a) The " smeared " cheeses ; in these the moulds are suppressed and prevented from forming aerial hyphae by daily smearing the cheeses either with a wet cloth or by hand, keeping them moist and protecting them as much as possible from the air by packing them closely together. (6) The mouldy cheeses ; in these the growth of moulds is favoured' by touching the surfaces of the cheeses as little as possible, keeping them dry and providing for free access of air. The copious production of ammonia on the surface of the best- known of the smeared cheeses, Limburger and Bomadour, has been found by the author ^ to be due to a symbiosis of peptonising tetracocci and Bacterium casei limburgensis. The latter is a non-motile^ very irregular short rod which does not ferment lactose ; it forms a film on media containing calcium lactate, and oxidises the lactic acid to acetic and carbonic acids ; it does not attack casein, but forms traces of ammonia in milk, from the amino acids, which has a slight solvent action on the casein. This organism is therefore to be classed with the bacteria which produce a soapy taste in milk. Its most characteristic property is its ability to carry further the decomposition of the products of protein hydrolysis which have been produced by other micro- organisms ; this is most strikingly illustrated by inoculating it into milk together with Tetracoccus liquefaciens. Milk without Chalk. Pei-ceatage of Total N. Sol. N. Dec. N. Am. N. Bacterium casei limburgensis Tetracoccus liquefaciens Bad. casei limburgensis and Tetracoccus liquefaciens ..... 0-60 61-61 67-59 2-36 8-73 32-18 1-30 1-43 14-99 The smeared crust of Limburger cheese consists, according to the unpublished researches of Frevdenreich, chiefly of Bacterium casei limburgensis together with smaller numbers of peptonising organisms (chiefly Tetracoccus liquefaciens, a small spore-forming > OrU Jensen, " Centralblatt f. Bakt.," 2 Abt., 1900, Bd. VI., p. 795. * " Studier over de flygtige Syrer 1 Ost, etc.". Doctoral theeis, 1904, p. 74. RIPENING PROCESSES OF CHEESES 147 rod bacteriunij and yeast) ; in view also of the results set out in the table on p. 146, there can hardly be any doubt as to the cause of the thorough-going decomposition processes which take place in this cheese. A similar flora will also develop on the rind of hard cheeses if they are kept damp, and it will be understood how the ripening of these cheeses may be modified so as to conform with the type generally associated with Limburger cheese. The ammonia formed on the surface gradually diffuses into the interior, and not only does it neutralise the lactic acid, thereby rendering possible the activity of the bacterial, enzymes, but it also converts the casein into the readily soluble ammonium caseinate. The ripening of the soft smeared cheeses involves further complexities. In the ripe state these cheeses contain appreciable' amounts of valerianic acid, an acid which is only produced by the above- mentioned bacteria in very small amounts, while on the strongly - smelling surface there are formed various • typical products of putrefaction, such as hydrogen sulphide and indole. It is obvious that under normal conditions organisms other than those mentioned above participate in the process — Weigmann mentions Plectridium foetidum— but whether their activity is to be regarded as at all desirable may be an open question ; it is quite possible that Lim- burger cheese might have a wider market if it contained no products of putrefaction. According to Laxa^, Oidium lactis participates in the ripening of certain Bohemian cheeses, which resemble Limburger cheese (Harrach and Knoppist) ; these cheeses therefore form ajink with the mouldy cheeses. The moulds play a part in the mouldy cheeses similar to that of the peptonising bacteria in the smeared cheeses. As a type we may take Camembert, which, thanks to the researches of Roger, Maze ^, and Thorn ^ is one of the most thoroughly investigated cheeses. The moulds which develop in this cheese are Oidium lactis (according to Maze, Oidium cam,emherti, other oidium species, and a mycoderma), Penicillium camsmherti, and P candidum. The moulds of the Oidium grdUp grow the quickest, while the Penicillium group first begins to develop after five to six days, when the sm-face has become a little drier. The chief difficulty lies in maintaining the proper humidity. If the air is too damp, the surface of the cheese becomes too slimy, as4n the case of the smeared cheeses, and bacteria, yeasts, oidium, and even certain mucors, gain the upper hand ; if too dry, on the other hand, the PeniciUia grow too freely, causing the rind to shrivel ; all the green PenicUlia which develop (not excepting P- roqueforti) produce > " Centralblatt f. Bakt.," 2 Abt., 1899, Bd. V., p. 755. 2 " Annales de I'lnstitut Pasteur," 1910. ' U.S. Dept. of Agric, Bureau of Animal Industry, Bull. 115, 1909. 148 DAIRY BACTERIOLOGY an undesirable taste. The moulds should not completely over- grow the surface, neither should they form too . many conidia. The drying is regulated by providing for a suitable draught and by laying the cheeses on straw mats ; after three weeks, when the cheeses have softened at the corners, they are transferred to another room and placed direct on the shelves, or sometimes on mats infected with bacteria. The moulds are now displaced by rod bacteria resembhng Bacterium casei limburgensis, which appear as red and brown spots beWeen the mouldy ridges, and the ammoniacal fermentation which starts under these spots gradually penetrates to the centre, resulting in the production of ammonium caseinate and other soluble proteins. The quicker the cheese ripens, the quicker will it become perfectly liquid and putrefying. A good saleable cheese should, therefore, be made firm and ripened at a low temperature ; during the first few days, when the cheeses are salted and develop acid fermentation, they should be kept at 18° to 20° C. ; the drying room should be kept at 13° to 15° C, and the ripening room, to which the cheeses are finally transferred for the chief fermentation to take place, at 10° to 12° C, or even lower. Frequently the process is completed in the boxes in which the cheeses are packed for transport. Cheeses never get the proper taste if they are put in the boxes too early. As soon as the interior of the cheese has become neutral in reaction the lactic acid bacteria will be able to act as in hard cheeses, but they act slowly and have no time to produce any noteworthy change before consumption. While Oidium lactis and the necessary bacteria will establish themselves of their own accord, there is some difficulty in establishing the desired species of Penicillium in places where the manufacture of Camem- bert is to be started. Thorn recommends their cultivation on dry, sterilised rusks, wetted with a suspension of the conidia in water. After keeping for ten days at 20° C, the rusks will be overgrown by the mould ; these mouldy rusks are then shaken vigorously with water, in which the cheeses are dipped immediately before salting. The French mode of procedure is more scientific ; the Institut Pasteur (Service des vaccins, 35, Rue Dutot, Paris) supplies three different cultures for the purpose, consisting of an ordinary lacti® acid starter, a culture of moulds, and a culture of the ammonia-producing bacteria. The two first-mentioned of these, or sometimes all three, are added to the milk before the rennet. The mould culture need only be used for the first ten days, after which it will have established itself in the mats used in the drying room, from which subsequent batches of cheese will become inoculated. The third culture is applied directly to the mats on which the cheeses rest during the last stage of RIPENING PROCESSES OF CHEESES 149 ripening. The cultures may also be obtained in powder form . for sprinkling on the cheeses ^. Only the acid curd cheeses remain to be discussed. These include both hard and soft varieties, and the latter include both smeared and mouldy cheeses — even cheeses which resemble Roquefort in being permeated with moulds, e.g., the Norwegian Gammelost. The making of the hard acid curd cheeses ^, the Danish cheeses, Appetitost, Knapost, the Norwegian Pultost, and the Swiss Green Alpine Cheese (Schabzeiger) presents several points of interest; they are scalded at a high temperature or for a long time and ripened before they are shaped (the Norwegian Pultost is not shaped at all). The scalding produces the same results as in the pasteurisa- tion of milk : the development of the sporing bacteria and the suppression of the lactic acid bacteria ; as the author has shown in the case of Schabzeiger^, and v. Klecki in the case of another acid curd cheese*, the curd develops a vigorous butyric acid fermentation ; in both cases the butyric acid bacteria were motile ; the butyric acid gives these cheeses a sharp taste. The ripening proper is due to lactic acid rod bacteria, which possibly act in conjunction with other microorganisms. As regards the decoinpo- sition undergone by the casein, the hard acid curd cheeses do not differ greatly from the hard rennet curd cheeses (see table, p> 133). According to Olav Johan-Olsen^, the ripening of Pultost, in which the author has found a fair amount of valerianic acid, is accomplished by yeast, Oidium lactis, and especially by a species of Mucor. Johan-Olsen has also carried out a detailed investi- gation of the ripening of the Norwegian Gammelost. Here the active moulds are especially species of Penicillium and Mucor, which turn the cheese green and brown respectively. As the curd, or the cheese itself is strongly heated, it is improbable that the moulds are derived from the sour mUk ; they must find their way into the cheese at a later stage, and gradually penetrate from the surface throughout the whole mass. This already takes place during the first six weeks, as the curd is fairly acid and porous. If it is desired to utilise separated milk for cheese, this is best done by turning it into one of the above-mentioned' acid 1 According to our experience, it is sufflc'.ent to infect the mats with P. Gandidum. The bacteria come of their own accord while the Oidia only do harm, ^ Several of these cheeses contain, in addition to casein, smaller or larger amounts of albumin (Zeiger), which also has an influence on the ripening process. 3 " Gentralblatt f. Bakt.," 2 Abt., 1904, Bd. XIII., p. 755, and 1907, Bd. XVII., p. 225. « "Gentralblatt f. Bakt.," 2 Abt., 1896, Bd. II., p. 169. 6 " Undersogelser over Ost og OstegaeriSg," Kristiania, 1905. 150 DAIRY BACTERIOLOGY curd cheeses, for, in spite of the lack of fat, a piquant product will result, owing to the formation of the sharp-tasting substances. In the ripening of the soft acid curd cheeses, e.g., Harz Cheese, the following organisms appear to play the principal part : — Oidium lactis, and, possibly, also certain yellow cocci ^, a mycoderma ^, yeast, and lactic acid bacteria. In the ripe state these ' cheeses generally contain more amino-acids and less ammonia than the soft rennet curd cheeses. Oidium lactis has a more pronounced action on casein in presence of large amounts of lactic acid ; it forms only small amounts of ammonia. 1 Eckles, "Landwirt. Jahrbuoh der Schw'eiz," 1905, p. 503. s Bahn, " Centralblatt f. Bakt.," 2 Abt., 1906, Bd. XV., p. 786. Chapter VII Defects of Cheese From the scientific point of view, the defects of cheese must be classified according to their origin. They may be due to milk of abnormal composition, faulty treatment in the manufacture (including production and treatment of the curd, pressing, salting, and treat- ment during ripening) or to bacterial action. Only the last- mentioned cause comes within the scope of this work. In practice, however, the various causes are so interdependent that it is difficult to make any hard and fast distinction. By judicious treatment of the milk and the cheese, it will generally be possible to avoid the development of harmful organisms, whereas with careless treatment, even when starting with good milk, their development may easily be encouraged. To take an instance, if the milk has curdled badly (a fault which might have been corrected by raising its temperature or adding a little calcium chloride), owing to the presence of raw milk or milk from cows which are getting towards the end of their lactation period, the curd will not dry readily and the cheese will tend to become spongy, even though the milk used could be described as good from the bacteriological point of view. Sponginess may thus be due, not merely to the presence of large numbers of gas- producing organisms, but to an excess of lactose in the curd. As has been mentioned, gas-producing organisms may come from the udder (Aerogenes mastitis), though in the great majority of cases they owe their presence to diarrhoea among the cows and unclean milking ; the acuter the digestive trouble, the richer will the manure be in gas-producing bacteria and the greater will be the difficulty in keeping the milk free from infection through the manure. The gases which are formed consist chiefly of hydrogen and carbon dioxide, the former being the more objection- able, for the water in the cheese will absorb large amounts of carbon dioxide before the slightest tendency towards sponginess becomes apparent. At 15° C, and at atmospheric pressure water will absorb its own volume of carbon dioxide, at two atmospheres pressure twice, and at three atmospheres three times its volume, 152 DAIRY BACTERIOLOGY etc. ; not only are the hard cheeses submitted to great pressures in the press, but any tendency towards expansion from within will meet with considerable resistance, owing to the close texture of the curd, and particularly the rind. By storing at a low temperature, a tendency towards sponginess may be kept in check in two ways, for not only is the absorption of carbon dioxide by water increased by lowering the temperature, but the development of the gas -forming bacteria is checked. It will thus be understood why sometimes bacterial growth may appear to commence afresh when the cheese is taken out of a cold cellar on a warm day, even though no new development of gas-forming bacteria actually takes place. It will also be understood why eyes are more readily formed in Emmental cheese when the curd has been saturated with carbon dioxide during the fermentation of the lactose ; under these conditions the distinction between normal and abnormal eyes may not become so sharp as might be expected, even though the two processes are due to totally different bacteria. Unlike carbon dioxide, hydrogen is only very sparingly absorbed by water ; it follows that those organisms which produce the most hydrogen are capable of doing the most harm. Thus the butjn-ic acid organisms may transform the cheese into a large-holed, spongy mass in the course of two days ; the' non-motile butj^ric acid bacteria are especially dangerous, owing to their ability to ferment calcium lactate, and thus to cause damage after all the lactose has been fermented. The aerogenes bacteria, especially the colon bacteria, also produce hydrogen. On the other hand, these organisms are able not only to effect respiration by means of atmospheric oxygen, but they will transfer loosely -bound oxygen from oxidising agents, like saltpetre, to sugar, and thus consume the sugar completely, so that no hydrogen is liberated or lactic acid formed. Saltpetre is thus an eifcellent preventative of the harmful effects of these bacteria ; as a rule it will be sufficient to add 30 to 50 grams of potassium nitrate per 100 litres of milk ^- As the propionic .acid bacteria and the lactose fer- menting yeasts do not form other gases than carbon dioxide, they are less dangerous to the fresh curd. The yeasts can, however, blow the soft cheeses in which there are particularly large amounts of sugar to be fermented ; their growth is pro- moted by small amounts of free lactic acid. The most natural means of preventing sponginess, as well as most other cheese defects, is the use of a vigorous lactic acid starter consisting, as "■ According to Bosengren, it is dangerous to use saltpetre in Emmental cheese, even in. small amounts ; 10 grams per 100 litres may produce an unclean taste and turn the cheese red. Feebly acid cheeses, like Gouda, are best able to stand the addition of larger amounts of saltpetre. DEFECTS OF CHEESE 153 far as possible, of a culture of the specific organisms of the cheese in question ; in this way the ripening of the cheese will also be accelerated. Saltpetre in small amounts is without effect on the ripening process, while a low temperature and plentiful salting delay it. The last-mentioned expedient is very effective in regulating the formation of normal eyes, but it is too slow in operation to prevent the defect of sponginess unless the curd is salted direct, .before moulding, though this method is not appli- cable to the choicer varieties of cheese. (See the " Ripening of Emmental Cheese.") As was mentioned above, the degree of plasticity of a cheese is determined by its content of acid and salt. If the curd is too acid or if it has' been made too dry, e.g., by over-scalding, it will become crisp and, therefore, easily craxk or crumble to pieces on rough treatment, and particularly if much gas is produced. A hard coat or rind produced by injudicious salting or over-drying will, of course, easily crack. The coat wiU also tend to crack when large amounts of whey collect beneath it ; this may occur when the cheese is pressed too hard to begin with, so that the outer layer becomes too compact before a sufficient amount of whey has been expelled, or, again, as mentioned above, the trouble may be due to slimy whey. If the cheeses are too damp without, however, being particularly acid, they will flow or 6e liquefied, especially if the temperature is high. Sc. liquefaciens, if abundant, will always cause this defect. It has a strong peptonising action on the curd and produces, at the same time, a bitter taste. While defects in colour no longer play any important part as far as milk is concerned, they are of great importance in the case of cheese making, for in cheese they have ample time to develop. Pistinction may be made between cases in which the colour appears evenly or in spots throughout the whole cheese, and those cases in which it only appears on the surface. Light spots in the interior are due to the reduction of the colour which has been added to the cheese ^. The commonest colour defect is the turning grey or blue of the curd, due to admixture of salts of iron or copper. Iron may come from the water, rusty pails or, if the milk is heated by direct steam, from the steam- pipes. Copper may come from the cheese vat or, in the case of Parmesan cheese, from the untinned copper vessels in which the 1 According to Campbell ("Trans, of the Highland Agric. Soc. of Scot- land," 1898) this defect may be avoided by the use of a good lactic acid starter. The reducing organisms may be colon bacteria {Harrison, " Eevue G6n6rale du Lait," 1902, vol. 1, p. 457) and torulae {Harding, Sogers and Smith, New York Agric. Exper. Station, Geneva, 1900, Bull. No. 183). 154 DAIRY BACTERIOLOGY evening milk is often kept for eighteen hours. In the case of iron the colour is due to ferrous salts, for which reason the outer, portion of ^ the cheese wUl not be coloured, while the colour disappears from a slice of the cheese which is exposed to the air. In the case of copper the conditions are reversed ; as the colour is due to the green cupric salts, the outer portions will be the most affected, and a slice cut out of the cheese will only develop the full colour after exposure to air for some time. Metallic sulphides, stable in air, will be produced where hydrogen sulphide is formed. The minute coloured spots which are sometimes found distributed throughout the whole mass are of greater interest from the bacteriological point of view, as they are colonies of chromogenic organisms, which develop in the same way as on other solid media, such a.§ nutrient gelatine or agar^. The conditions of bacterial growth consequent on the cheese being a solid medium are not so strikingly illustrated in the case of the lactic acid bacteria, which grow rapidly throughout the fresh curd, and thus appear to be evenly distributed ; but the slow-growing organisms which appear at a later stage will appear in this characteristic manner. Thus Bacillus cyaneofuscus (which, however, dies out before the cheese has fuUy ripened) forms blue spots in Edam cheese, while the chromogenic propionic acid bacteria form red and brown spots in Emmental cheese. In all probability the eyes in Emmental cheese are formed in those places where the colonies of propionic acid bacteria are particularly abundant. According to Connel ^. the so-caUed rusty spots in Cheddar are caused by Bacillus rudensis, an acid-producing organism, which may possibly belong to the •propionic acid group, as it is generally found in or near the eyes of Emmental cheese ; being particularly prevalent in spring, it is supposed to originate from the fresh grass ; if once established in the dairy, it will appear in successive batches of cheese unless all the appliances are sterilised. The form of discoloration resulting in the production of a red colour jy.st inside the rind, but not in the rind itself, may be regarded as intermediate between interior and exterior discolora- tion. -It is said to be due to the diffusion of colouring matter from the shelves into the cheese ; shelves of white pine, but not of red pine or fir, are said to be objectionable in this respect. This explanation hardly holds good in all cases, for the red zone may spread after the cheeses have been removed to another place, and 1 The staining of cheese sections to show the natural position of the bacteria was first accomplished by Miss. Oerda Troili-Petersson, 1904 (" Centralblatt f. Bakt.," 2 Abt., Bd. XI., p. 212). 2 "Discoloration of Cheese," Canadian Dept. of Agrlc. Bull., 1897, and Harding and Smith, New York Agric. Exper. Station, Geneva, 1902, Bull. No. 225. DEFECTS OF CHEESE 155 the. coloration is generally accompanied by an unpleasant taste. In the latter event the trouble is most likely due to chromogenic organisms which have penetrated the rind. Among the bacteria which produce a red colour in cheese, both cocci and rod forms are known ^. Several of them liquefy gelatine, and many of them are chromogenic on. cheese, but not on the common media, and some of the organisms which produce a red colour on the surface of the soft mouldy cheeses are only chromogenic in presence of t;Jie decomposition products formed from the casein by the moulds which are characteristic of the cheese in question. Bacteria are known which colour the rind yellow ^ and brown ; in fact, the rind always becomes brown when kept damp. Atmospheric oxygen plays an important part in all these colour" changes. Many moulds also produce surface colorations, e.g., Oidium aurantiacum, Penicillium casei, Cladosporium herbarum, and Monilia nigra. Red and yeUow Torulae are also believed to play some part in the process. Many moulds penetrate the rind and make the surface ' uneven. In this connection mention may be made of cheese mites and maggots, though their detailed descrip- tion does not come within the scope of a work on bacteriology. In order to avoid- the transference of harmful organisms from cheese to cheese, the uninfected cheeses should always be washed before those which are infected, and the cloth should be boiled for a quarter of an hour daily. The only efficient means of preventing the' defects under consideration is the thorough disinfection of the. ripening room and the shelves (see p. 56). Defects in taste and smell, originating from the fodder, will generally disappear in time. Similarly, the bitter taste due to Streptococcus liquefaciens and Torula amara, and sometimes also that due to certain lactic acid rod bacteria^, may disappear during a later stage of the ripening. Many of the chromogenic organisms produce unpleasant tastes. The tallowy taste sometimes found in rich cheeses (e.g., Gouda cheese) is probably due to those organisms which turn milk and butter tallowy. Bitter and tallowy tastes are defects which chiefly occur when fresh cheeses are kept too ^ Thus Adametz has described two micrococci which do not liquefy gelatine, or, at any rate, only do so very slowly ; they form red colonies on gelatine and agar. Gratas has isolated a liquefying bacterium. Micrococcus rubri casei, which forms pink colonies, and Wmgmann has isolated two liquefying organisms. Micrococcus .ohromoflavus and Bacteriurn, casei fusci, which form chrome yellow and cream-coloured colonies respectively on the common media, but which turn the surface of the cheese red. The red Bacillus firmitatis, isolated by Roger from Camembert cheese, grows only in the decomposition products produced by moulds. 2 Thus Barthel has found that yellow spots may be produced by Micro- coccus flavus, a liquefying organism commonly found in air. 3 Harding. Bogers and Smith, New York Agrio. Exper. Station, 1900, Bull. No. 183, 156 DAIRY BACTERIOLOGY damp and cold ^. In the French soft cheeses Penicillium brevicaule produces a taste of cabbage. The organisms which produce spongy cheese may also give rise to unpleasant, mostly bitter-sweet tastes. Spongy cheeses, more- over, dry too readUy, so that they ripen too slowly. As cheese poisoning, like meat poisoHmg, is chiefly due to certain colon bacteria and sometimes to non-motile butyric acid bacteria, spongy cheese must always be- regarded with suspicion ^. 1 At a low temperature the peptonising bacteria develop more rapidly than the good lactic acid bacteria, or the bacterial metabolism may be too sluggish to cause the disappearance of all the atmospheric oxygen in the cheese. Sunlight promotes the oxidation of the fat in cheese, as in butter. As already mentioned, copper salts and carbonic acid which latter is copiously produced in the ripening of cheese, may turn the fat tallowy. " H. KvM (" Zeitschrift f. Untersuchung der Nahrungs und Genuss- mittel," 1913, Bd. 25, p. 193) reports on a case of poisoning by cheese which was due to an aerogenes bacterium. A number of references to the literature of this subject are given in this paper. Chapter VIII The Grading of Milk This chapter deals with the more important methods of judging of the cleanness and freshness of milk and its suitahility for the making of good dairy 'products. EXAMINATION FOR TASTE AND SMELL As the senses of taste and smell are our best aids in avoiding putrid or harmful food, every careful examination of milk should include tasting and smelling. Unfortunately the test is of a decidedly subjective nature, as the senses in question are soon duUed. Further, considering that the temperature of the milk varies considerably on arrival, and that the smell and taste are most pronounced when the milk is warm from the cow, it is evident that milk can only be graded very roughly by this method, and that the detailed classification in fifteen grades according to the taste and smell alone, as was previously carried out by the Danish milk grading associations, was futile. ESTIMATION OF DIRT Milk which contains visible amounts of pus, blood, manure, etc., must of course be regarded with suspicion from the outset. It may justly be demanded "that milk retailed in towns shall show no sediment when 1 litre is allowed to stand at rest for two hours in a vessel of colourless glass. It is advantageous to use vessels tapering to the bottom, so that the sediment may readily be collected for examination after the milk has carefully been decanted off. As only the heaviest particles will separate, it is more usual nowadays to filter a definite quantity through a disc of cotton wool, which will become more or less dirty according to the state of the milk. The discs may be dried and kept, so that a "dirt scale " may be prepared for future reference and comparison i, and the dirtiest discs may perhaps produce some moral effect if sent to the suppliers responsible for them. As it is impossible to take an average sample of dirt from a large quantity of milk, it 1 See, for example, Hoyberg's Scale in " Maanedsskriftet for Sund- hedspleje," 1910, p. 49. 158 DAIRY BACTERIOLOGY will be necessary in dairies to filter the contents of each can or churn through a separate cotton wool disc, and also to examine the empty cans carefully, for most of the dirt, usually remains in them. As has been previously pointed out, the chief impurity of milk is cow manure, which contains 80 per cent, of water, and soluble matter, both of which are completely incorporated with the milk owing to the shaking up which occurs in transit ; at the same time, the bacteria which constitute an appreciable proportion of the solid matter, are distributed throughout the milk. The above estimate applies to normal dung ; if the cows are suffering from diarrhoea, the dung wiU contain a still larger proportion of soluble matter, and consequently the filtration test will show less dirt. Further, the more liquid the dung the greater will be the propor- tion of dangerous bacteria introduced into the milk. The estima- tion of dirt therefore furnishes no measure of the bacterial contents of the milk, and it must also be remembered that very dirty milk which has been well cooled may contain fewer bacteria than less dirty milk which has been inadequately cooled. In reality, therefore, the only sure indication afforded by the dirt test is whether the mUk has been properly cleaned or not; either by straining, filtering or centrifuging. TROMMSDORFF'S LEUCOCYTE TEST The particles of dirt and foreign bodies which are found in suspension in milk are removed much more completely by centri- fuging than by sedimentation or filtration. Thus, of the white blood corpuscles or leucocytes which normally do not separate out when the milk is allowed to stand for a relatively short time, 3 to 50 per cent, are separated by centrifuging, or even more if the milk is warm^- The heating should, however, not exceed 70° C, for otherwise precipitation of the proteins may occur. As was first shown by Barthel, th« centrifuge slime therefore consists very largely of leucocytes^. By direct microscopical counts, normal milk has been shown to contain | to IJ million leucocytes per cubic centimetre ^. The number of leucocytes increases as the jdeld of milk decreases, being particularly high at the beginning and towards the end of the lactation period. It is still higher in cases of udder disease, and on this fact Trommsdorff has based his test, the object of which is to ascertain whether or not the milk has been -derived from healthy cows. The test is carried out as 1 Campbell, U.S. Dept. Anim. Industry, Bull. 117, p. 19. 2 "Revue g6n6rale du lait," 1901, vol. 1, p. 193. > Prescott and Breed, " Journal of Infectious Diseases," 1910, vol. 7, p. 632 ; Breed and Stiger, ibid., 1911, vol. 8, p. 361. THE GRADING OF MILK 159 follows : 10 c.c. of the milk are introduced into a glass tube, the end of which is drawn out into a capillary graduated in thousandths of a cubic centimetre. The tube is closed by a rubber stopper and whirled at a speed of 1200 revolutions per minute. Normal milk will generally yield a .sediment measuring 0-002 to 0-004 c.c, while milk drawn from diseased udders will yield a sediment measuring 0-01 c.c. or even more. The amount of the sediment is, however, not a decisive criterion by itself ; the test must be supplemented by a microscopic examination, and only in cases where large numbers of bacteria characteristic of udder disease, .^7S2^M1m^ Fig. (34. — Leucocyte Sediment from the Milk of a Cow suffering from streplocorcic masliiis. (After Ernst.) ■ 1000. e.g., streptococci, are found can definite conclusions be drawn. As these bacteria cannot be distinguished from the harmless milk bacteria by direct inspection \ the test is only of value when applied to milk fresh from the cow, and can therefore only be applied as an aid to veterinarj' control at the farm. In mixed milk the characteristic features are completely lost on account of the dilution alone. THE CATALASE TEST This test is supplementary to the leucocyte test. The various constituents of blood, especially the corpuscles, are rich in catalase ; 1 Capsule formation and disc-like cells, which by some authors are regarded as characteristic of Sc. mastitidis, can be observed in all strepto- cocci. On the other hand, as already mentioned under Sc. ■mastUidu. the red colour in casein starch stab cultures is very characteristic. 160 DAIRY BACTERIOLOGY n n for this reason, milk drawn from cows with diseased udders or from cows which are approaching the end of the lactation period, or colostrum, will liberate large amounts of oxygen from hydrogen peroxide. As the fat globules carry with them large numbers of leucocytes, unpasteurised cream, obtained either by spontaneous separation or by the use of the separator, will be richer in leucocytes than the corresponding skim milk. Many ingenious and sometimes complicated forms of apparatus have been devised for the carrying out of the test. In the author's laboratory Lind's apparatus is used ; this consists simply of a graduated tube of 20 c:c. capacity, into which 15 c.c. of milk are introduced; sufficient hydrogen peroxide (1 to 3 per cent.) is added to fiU the tube, the rubber stopper carrying the bent tube is inserted, and the apparatus is inverted as shown in the illustration. The maximum amount of oxygen is obtained at 20° to 25° C, so that no water bath or thermostat is necessary. The number of cubic centimetres of oxygen evolved in six hours is taken as the catalase number. Fresh milk from healthy cows will not yield more than 2-5 c.c. In mixed milk the blood corpuscles will be too sparsely distributed to produce any recognisable effect. The milk must be perfectly fresh when tested, as many bacteria decompose hydrogen peroxide. The common sarcinse and micrococci (see p. 38), e.g., Micrococcus candicans, and most of the putrefactive bacteria are particularly active in this respect, while the true lactic acid bacteria and the butyric acid bacteria ^ do not produce catalase. Milk which has stood for any length of time at a low temperature, or old pas- teurised milk, wiU accordingly show high catalase values. Hesse ^ has proposed to apply the catalase test to butter as follows : 100 grams of butter are warmed to 45° C. and shaken with 40 c.c. of water at this temperature ; the aqueous liquid is separated and tested in the same way as milk. It is obvious that butter which has been made from pasteurised cream ripened with a pure starter of lactic acid bacteria will show a low catalase figure ; if the butter has been washed with bad water it may show a high catalase figure, for Bacterium fluorescens liquefaciens is particularly active in decomposing hydrogen peroxide. Fig. 65. — Catalase Test Apparatus. ^ Orla Jensen, " Det. kgl. danske Videnskabers Selskabs Oversigter " (Danish Academy of Sciences), 1906, No. 5, p. 306. 2 " Molkereizeitung," Hildesheim, 1912, No. 6. ■ THE GRADING OF MILK 161 THE RENNET TEST As milk drawn from diseased udders generally coagulates badly with rennet, this test affords the same indications as the two tests just described ; it has also a practical significance, for milk which coagulates badly can hardly be made into good cheese, even if it is satisfactory from the bacteriological point of view. The test may be applied with advantage when difficulty is experienced in making the curd sufficiently dry, so that the particular consign- ments of milk which are at fault may be detected. In the labora- tory the test is generally carried out by means of Schajfer's apparatus, which consists simply of a shallow water bath with a false bottom on which a number of beakers can be placed. One cubic centimetre of one of Hansen's rennet tablets. No. 2, in 500 c.c. of water is added to 100 c.c. of mUk which is kept at 35° C. ; normal milk wiU coagulate in nine to nineteen minutes . Marschall's apparatus is better suited for use in the dairy ; this consists of a graduated enamelled iron cup having a fine opening in the bottom, The hole is closed by a finger, the cup is filled, and milk is allowed to run out until the level comes to the top graduation mark in the cup. One cubic centimetre of the rennet solution is rapidly and thoroughly mixed with the milk, and the finger is removed from the opening. The milk will cease to run out the moment coagula- tion sets in, so that the capacity for coagulation will be inversely proportional to the amount of milk which has run out. Con- versely, this apparatus may be used for the estimation of the coagulating capacity of rennet, which is a test of some importance in the Swiss dairies which make their own rennet. The means available for correcting a deficient capacity for coagulation are discussed on p. 130. DETERMINATION OF ACIDITY The degree of acidity is generally understood to be the number of cubic centimetres of standard sodium hydroxide solution required to neutralise a given volume of milk, with phenol phthalein as indicator. The Soxhlet-Henkel degrees, which are largely used on the Continent, express the number of cubic centimetres of quarter normal alkali per 1 00 c .c . of milk . In British and American works, degrees of acidity are understood to represent the number of cubic centimetres of normal sodium hydroxide per Mtre of milk ; the titration is often carried out with decinormal alkah, using smaller quantities of milk, the results being calculated to the above standard ; as little as 10 c.c. of milk is sometimes used, but more exact results can be got by using larger quantities. No water should be added to the milk or cream before titration, as D.B. 11 162 DAIRY BACTERIOLOGY this will lower the acidity. Ellbrecht's titration paper " Exact " has been designed as a colour standard in order to ensure that the same end-point is reached each time. Richmond gives the follow- ing method in his " Dairy Chemistry " : " 10 c.c. of milk are titrated with decinormal baryta, or 11 c.c. with eleventh normal strontia, using 1 c.c. of | per cent, phenol phthalein ; as a standard, an equal volume of milk is coloured with one drop of 0-01 per cent, rosaniline acetate in 96 per cent, alcohol." The degree of acidity of normal milk generally lies between 16 and 19 (6-4 to 7-6 Soxhlet- Henkel). If under 15 (6 S.-H.), the milk is probably derived from cows which are sick or approaching the end of the lactation period, or it may have lost a portion of its natural carbonic acid by having been kept in shallow vessels, shaken or warmed. If the degree of acidity is over 21 (8-4 S.-H.) the milk may be derived from cows suffering from streptococcic mastitis or it may contain colostrum the acidity of which may be as high as 55. As a rule, however, high acidity will be due to incipient lactic acid fermenta- tion. Mixed milk having an acidity of over 21 wiU usually coagulate on mixing with an equal volume of 68 per cent, alcohol ; this is the basis of the so-called alcohol test. The boiling test is based on the fact that milk having an acidity of over 27-5 coagu- lates on boiling. It is, however, impossible to be certain that the milk will stand pasteurisation if the acidity exceeds 22-5 ^. Fresh milk shows an amphoteric reaction towards litmus, i.e., it turns red litmus blue and blue litmus red ; if the degree of acidity is under 12-5, the reaction towards litmus will be alkaline. Accord- ing to Hoyberg ^, the rosolic acid solution proposed by Hilger for the detection of added soda may be used with advantage in testing milk from the individual quarters with a view to detecting udder disease. If 5 c.c. of 96 per cent, alcohol and 0-5 c.c. of a 1 per cent, rosolic acid solution are added to 5 c.c. of milk, an orange colour wiU be obtained with normal milk, and a red colour with alkaline milk. Eugling * has shown that a saturated alcoholic solution of alizarin may be used for the same purpose ; if 5 to 10 drops of the solution are added to 50 c.c. of milk, a red-violet colour will be produced with normal milk, a violet-blue colour with alkaline mUk, and a yellowish colour with sour milk. Morres * combines this test with the alcohol test in the Alizarol test, 0-05 per cent, of alizarin being dissolved in the 68 per cent, alcohol, so that an indication may be obtained as to whether the coagulation is due to acid- or rennet-producing bacteria. If narrow test tubes 1 Henhel, " Milchwirtsohaftliches Zentralblatt," 1907, Bd. III., p. 378. " " Skandinavsk Veterinaertidsskrift," 1911, p. 23. " " Handbuch f. die praktische Kaseriei," Leipzig, 1901, p. 20. * " Oesterreichsche Molkerei-Zeitung," 1912. A colpur scale for this tftst is supplied by Dr. N. Oerhers Co., Zurich. THE GRADING OF MILK 163 are used, 2 c.c. of milk and 2 c.c. of alcohol wiU sufSce for this test. It is recommended that the dairies should apply it to each can of milk and reject all milk which shows a precipitate or an abnormal colour. THE FERMENTATION TEST This test shows if the milk has become infected with an undue proportion of gas-producing organisms, which first and foremost include the pseudo lactic acid bacteria. We have already seen that these are among the most objectionable organisms that can be met with in dairy practice, as they cause trouble in various ways, including the spoiling of milk for the purpose of cheese making. As the pseudo lactic acid bacteria are usually brought into the milk with the cow dung, and are particularly plentiful when the cows are suffering from diarrhoea, the fermentation test will give evidence of undue contamination and thus give warning that the milk may possibly be dangerous for human consumption. This test is of value not only in judging of the suitability of milk for cheese making, but also in the laboratory control of retail milk, particularly that which is to be used for infant feeding. While most of the milk bacteria (excepting the thermobacteria, which, however, are rare in fresh milk) grow best at about 30° C, many of them ceasing to develop at temperatures above 38° C., the pseudo lactic acid bacteria, being tjrpical intestinal organisms, have their optimum at blood heat, and will gain predominance most readily at a slightly higher temperature, for which reason the fermentation test is carried out at 38° to 40° C. It is important that the tem- perature should not be allowed to vary beyond these limits, as at a higher temperature good milk may appear to be bad, while at a lower temperature bad milk may appear to be good. If fine distinction is made between the different types and degrees of fermentation, these temperature limits are too wide, and the temperature should be kept constant at 38° C. In order that the results of the test may be strictly comparable, it is advisable always to use tubes of a certain diameter (about 2 cm.), into which 40 c.c. of milk are introduced. The shape of the tubes is shown in the accompanying illustration ; they should be strongly made and provided with a graduation mark at 40 c.c, and with a frosted square so that they can be marked in pencil. An average sample of each supplier's milk should be taken, preferably from the weighing or measuring vessel, by means of a small measure furnished with a pointed spout. The measure should be rinsed several times with the milk which is being sampled before taking the actual sample. The tubes are marked, covered with a small cap of aluminium or zinc, placed in stands and brought into the 164 DAIRY BACTERIOLOGY water bath. Failing a proper thermostat, the temperature may be kept fairly constant by means of a spirit lamp or gas burner, provided that a large well-insulated water bath is used, and that the temperature of the room does not vary too much. The samples are examined after twelve and twenty to twenty-four hours. If quite fresh, the milk will remain liquid for twelve hours. The sooner visible alteration occurs, the greater the importance to be attached to the results of the test. On the other hand, milk Fig. 60.- Vt (Is -Gelatinous Types. *i 62 63 Fig. ()7. — Blown Types. containing relatively few bacteria gives unreliable indications, duplicate tests often showing different tj'pes of fermentation. According to Peter's classification, we may distinguish between the following types in the fermentation test: — Fluid (/), gela- tinous (g), hloivn (/;), spongy {s) and cheesy (c) (see his illustrations). In the gelatinous type the true lactic acid bacteria predominate ; a perfectly homogeneous coagulum is denoted hy g^, a coagulum with very few streaks and bubbles by g'2, and one with few streaks and bubbles by g^. In the blown (gassy) milk the pseudo lactic THE GRADING OF MILK 165 acid bacteria predominate ; b^, b^ and 63 indicate progressive degrees of intensity of gas evolution. Wiiile there is only a difference of a degree between g.^ and b^, all the casein will have been driven to the surface in 63. The spongy types, Sj, Sj and s^, differ from the blown types in being finer in curd texture. In s^ the coagulum forms so fine a network that it may easily be mistaken for g^^. Milk which is poor in true lactic acid bacteria often becomes spongy in the fermentation test, owing to the gas production ^^^K' ^^^^^■i' 1 1 1 ' i \ \ i HP' ^ ^H ^ V ^^n. 1 1 ,;>>. ■ 1 ' 1 Fin. 68.— Spongy Types Fig. (39. — Cheesy Types. being in full swing before coagulation occurs. In this case the gas-producing organisms may sometimes Ije lactose fermenting saccharomycetes. The cheese' type is distinguished bj' a -tt'ell- marl<;ed separation of clear whey, due to organisms which secrete rennet-like enzymes, especially peptonising lactic acid strepto- cocci ; Cj, c, and C3 indicate progressive degrees in the contraction of the curd. If at the same time much gas has been formed, this type cannot well be distinguished from the spongy or the blown type. In actual practice distinction is only made between the 166 DAIRY BACTERIOLOGY highly objectionable blown types b^, 63 and Sg on the one hand, and all the remaining types on the other. In examining the fermentation types the following should also be watched for : sediment (and possibly pus), sliminess, and evil-smelling milk. Alkaline milk keeps fluid for a long time, and often putrefies before going sour. The combined rennet and fermentation test suggested by Fr. Jos. Herz is a special form of the fermentation test, 2 c.c. of the rennet solution mentioned above being added to each test tube. Milk which coagulates badly will have separated compara- tively little whey and formed a soft and non-coherent coaguluih after twelve hours. On examination after twenty to twenty -four hours the coagulum should have assumed the form of a smooth cyhnder, which only shows small holes in longitudinal section. If the coagulum contains large holes, and especially if it forms a screw-shaped sponge floating on the surface of the whey, the milk must be considered unsuitable for cheese making. In the Emmental dairies, where a home-made rennet is used which, at the same time, is also a cultmre of the more important ripening bacteria, but which not infrequently contains colon and aerogenes bacteria, it is of the greatest importance to test the milk, both with and without the addition of rennet. If a bad result is obtained without rennet and a good result with rennet, the milk is certainly bad, though the lactic acid bacteria in the home-made rennet will be able to counteract the defect. The mUk is only unsuitable beyond doubt if the test with rennet turns out badly. If the last-mentioned result is obtained in spite of the fact of the milk bein'g good, then the rennet wUl be known to be unfit for use. The tubes and caps used in the fermentation test must be cleaned immediately after use by rinsing carefully with hot soda solution, after which they should be placed in the stands and covered completely with water into which steam is then passed for about fifteen minutes. Finally, they are to be dried in a warm place. THE REDUCTASE TEST AH living cells, including microorganisms, have reducing properties, which are well illustrated by their behaviour towards methylene blue. The rate at which milk decolorises methylene blue will, therefore, depend on the number of microorganisms in it. The reductase test devised by Barthel ^ and the author ^ is based on this fact. 1 " Kungl. Landtbruks-Akademiens Ilandlingar ocli Tidskrift," No. 6, 1907. 2 " Maelkeritidende," 1909, p. 359. THE GRADING OF MILK 167 It must not be assumed that the time taken to decolorise methylene blue (reduction time) under standard conditions is an accurate measure of the number of microorganisms in the milk ; for, in the first place, all microorganisms do not reduce with equal rapidity, and, second, the milk itseK, as obtained from the cow, contains reducing substances. Of the milk bacteria examined by the author Streptococcus liquefaciens appears to have particu- larly marked reducing powers, while the true lactic acid bacteria are among the organisms which reduce slowly. The obligate anaerobic bacteria reduce rapidly, a fact easily explained on considering that they derive their energy from reduction processes. The reduction time is shortened by the addition of a little alkali, and lengthened by the addition of a little acid. As regards the reducing substances natural to milk, the most important is aldehyde reductase, an enzyme which appears to be associated with the fat globules^, but which has no significance in the present connection, as it only decolorises methylene blue in presence of formaldehyde. Greater interest attaches to the leucocytes, which, like other living cells, are able to reduce methylene blue \ They will, however, only exert an appreciable effect on the reduction time if present in large numbers, while it can hardly be considered a drawback to the test that milk rich in leucocytes should appear to be worse than its bacteriological condition would warrant, inasmuch as such milk should always be regarded with suspicion. Of still greater importance is the fact that milk contains substances other than enzymes which exert a reducing action in the absence of oxygen. Thus Burri and Kursteiner have shown ^ that newly- sterilised milk which has been prevented from absorbing oxygen decolorises methylene blue rapidly, and Barthel * has shown that raw milk behaves similarly when the dissolved oxygen is expelled by a current of hydrogen or carbon dioxide, from which he concludes that the decolorisation of methylene blue in milk is due to the action of the mUk itself, and that the microorganisms only act indirectly by consuming the dissolved oxygen. This 1 Orla Jensen, " Uber den Ursprung der Oxydasen und Eeduktasen der Kuhmiloh," Det Kgl. Danske Videnskabernes Selskabs Oversigter (Danish Academy of Sciences), No. 5, 1906, and Centralblatt f. Bakt. II. Abt. 1907, XVIII., p. 211. 2 Olav Skar, " Skandinavsk Veterinaertidsskrift," 1913, p. 51. 3 " Milcbwirtschaftliches Zentralblatt," 1912, p. 269. * " Skandinavisk Veterinaertidsskrift," 1916, p. 155. The author has been able to confirm BartheVs results, and has found that milk with a low bacterial count which is kept free from atmospheric oxygen by passing a current of hydrogen through it reduces methylene blue in forty-five minutes at 40°C., no matter whether raw or sterilised. (On the addition of a little formaldehyde decolorisation was complete in ten minutes.) As pure lactose solutions were not found to decolorise methylene blue under similar experimental conditions, the reducing action of milk itself cannot be due to the lactose. 168 DAIRY BACTERIOLOGY explanation, however, hardly covers the case of the obligate anaerobic bacteria, but, as far as the aerobic organisms are concerned, the reducing action of the milk itself is, no doubt, a contributing factor, so that in the reductase test the degree of aeration of the milk is a condition which must not be overlooked. Milk will already begin to absorb oxygen freely as it comes in a fine stream from the udder, and its oxygen content will naturally be increased on pouring from vessel to vessel, and especially during any process of aeration to which it may be submitted. The dissolved oxygen will gradually be consumed as the micro- organisms increase in number ; the less the milk is shaken, and the deeper the vessels in which it is kept, the quicker will the oxygen content fall off. The temperature at which the milk is kept is also an important factor in this connection, for not only Fig. 70. — ^Apparatus for Reductase and Fermenting Test to take 200 Samples. will the bacteria develop more rapidly at higher temperatures, but the individual cells will consume more oxygen at higher than at lower temperatures ^. In order to equalise these conditions, it will be advisable always to shake the milk well before subjecting it to the reductase test. It will be seen that the theory of the reductase test is not so simple as was formerly supposed ; nevertheless, numerous experiments with mixed milk of commerce have shown that the reduction time, as determined by the reductase test, furnishes just as satisfactory a njeasure of the bacterial contents as the troublesome method of plate counts, which, as a matter of fact, is by no means less subject to error than the reductase test. Moreover, the differences between the reducing powers of the different species of bacteria are not greater than the differences displayed in this respect by the members of the same species 1 This is clearly shown by G. Lind's work, " Reduktaseproven sammen- ignet med Bakterietaellingsmetoden " (" Maelkeritidende," 1915, p. 921). THE GRADING OF MILK 169 when subjected to different conditions. The more favourable the conditions for - bacterial development, the shorter will be the reduction time. It follows that not only does the reductase test give an estimate of the number of organisms present, but the result will be influenced to some extent according to the vitality of the organisms, which is a factor as important as any other in determining the keeping power of the milk. As the reductase test will reveal any appreciable bacterial increase before this becomes apparent through the presence of lactic acid, it is not only the most convenient, but also the most sensitive, method for the grading of milk, whether for retail direct or for the making of dairy products. On the other hand, it must be admitted that milk from individual cows or milk that has been subjected to unusual treatment may give divergent results. The translator ^ has thus found that milk which has been kept for a long time at low temperatures appears to be better than it really is, in the reductase test carried out at 38° C. , because the majority of the bacteria in it are greatly weakened at the temperature in question. Such milk is decolorised quicker at 28° C. A comparison between the reduction times of the same milk at 28° and 38° C. can thus furnish information as to how this milk has been ta-eated. According to the author's proposal, however, the test should be carried out at 38° to 39° C, as this will, in the majority of cases, give the shortest reduction time. On the other hand, it is a wrong principle to employ a still higher temperature, as was originally done, for then the development of all the most common milk bacteria will be hindered. The methods of sampling and the apparatus required for the reductase test are the same as have already been described under the fermentation test. Particular care must be taken to measure out exactly 40 c.c. of mUk, either by means of a graduation mark on the tube or by taking the sample in a measure holding exactly 40 c.c. when filled to the rim. As the preparations of methylene blue on the market are very different in their properties, and the solutions made from them are not permanent, it is necessary to use a fresh solution of definite strength, made from a standard pre- paration. The tabloids prepared for the purpose ^ are readily soluble in warm water ; each tabloid makes 200 c.c. of solution, 1 c.c. of which is required for each test with 40 c.c. of mUk. The colour is mixed with the milk by rolling the tube in the hands, then pressing the mouth against a clean portion of the palm of the 1 "Analyst," 1918, 43, p. 1. 2 By Messrs. Blauenfeldt and Tvede, of Copenhagen, who also supply all requisites for the reductase test, including complete outfits suitable for dairies dealing with milk from 100 to 400 suppliers. 170 DAIRY BACTERIOLOGY hand and shaking vigorously. For each tube a different portion of the palm should be used, and, 'when completely wetted, the hand should be carefully washed before proceeding any further. In actual practice, the colour is added when all the samples have been taken and the tubes are in position in the stand. After placing in the water bath, the samples should be examined at frequent intervals during the first twenty minutes, after which they need only be examined every quarter or half hour. As mentioned above, the author has found it best to employ the same temperature as in the fermentation test ; in this way there is the additional advantage that the two tests may be combined. The combined reductase and fermenting test ^ gives information regard- ing both the number and the nature of the organisms in the milk. The joint investigations of Barthel and the author ^ have shown that it is possible by means of the reductase test to grade milk and creain into four classes, as follows : — Class 1. — Good milk, not decolorised in five and a half hours, containing, as a rule, less than J million bacteria per cubic centi- metre *. Class 2. — Milk of fair ^.verage quahty, decolorised in less than five and a half hours but not less than two hours, containing, as a rule, J to 4 million bacteria per cubic centimetre. Class 3. — Bad milk, decolorised in less than two hours, but not less than twenty minutes, containing, as a rule, 4 to 20 million bacteria per cubic centimetre. Class 4. — Very bad milk, decolorised in twenty minutes or less, containing, as a rule, over 20 million bacteria per cubic centimetre. If samples of retail milk from different dairies are to be com- pared, they must, of course, be examined simultaneously ; thus it would be unfair to sample one dairy on a cold morning and another on a warm afternoon. Unpasteurised milk, as sold in large towns, should retain its colour in the test for at least two hours, and the pasteurised milk for at least five and a half hours. Most of the milk retailed by the large dairies in Copen- hagen fulfil these requirements *. The pasteurised milk supplied 1 " Maelkeritidende," 1909, p. 359. 2 " Milchwirtsohaftliches Zentralblatt," 1912, No. 14. 3 As the counts were found by the plating method, they were really under-estimated ; milk bacteria usually occur in pairs and not infrequently in long chains, or large clumps, which are not broken up on shaking, and in such cases only one colony is obtained. The counts should at least be doubled, and in some cases they should be trebled or quadrupled. * Orla Jensen, " Maanedsskrift for Sundhedspleje," 1909, p. 239. The translator has found the reductase and fermenting test to be of great use in the control of milk as it arrives from the farms, and Uiat the conditions of treatment of the milk on various farms were found to correspond with conclusions which had previously been drawn from the behaviour of the samples in this test. THE GRADING OF MILK • 171 by these dairies is, generally speaking, not so good as might be expected ; this matter deserves attention, for pasteurised milk which is rich in bacteria can only be regarded as a highly objection- able product. It should, therefore, be forbidden to sell pasteurised milk as raw milk. In the dairies it is impossible to lay down a definite line of demarcation between first and second grade milk without unduly lowering the standard, for obviously milk cannot be expected to conform to the same standard on a close summer's day as on a frosty day. The author has therefore proposed the adoption of the average reduction time of all the samples tested at one time as the standard for that particular batch of samples. Only milk which is better than the average will be placed in class 1. Milk showing a reduction time equal to or less than the average, but not less than two hours, will be placed in class 2, and other cases can be dealt with as detailed above. As it may be inconvenient to have the samples examined for. longer than twelve hours, the reduction time of any samples not finished in this time may be set down as twelve hours in calculating the average ; such cases will only occur on cold winter days. All reduction times may be estimated to the nearest quarter of an hour, the reduction time of milk in class 4 being set down as a quarter of an hour. If it is desired to combine the grading according to taste and smell and according to the results of the fermenting test, with the grading according to the reductase test, the following system may be adopted : — Samples placed in classes 1, 2 or 3 according to the reductase test are degraded by one class if the taste and smell are decidedly bad, and samples thus placed in classes 2 or 3 are further degraded by one class if the results of the fermentation test show 62, 63 or S3. If the average reduction time should be under five and a half hours, class 1 milk which is decolorised before this time, and which is bad according to the fermenting test, should also be placed in class 2. It will be seen that, excepting in the case just mentioned, the results of the fermenting test are not taken into account in the cases of samples placed in classes 1 and 4, the reasons for this being as foUows : — Milk which contains relatively few bacteria will generally show a bad result in the fermenting test as it will be particularly poor in true lactic acid bacteria ; moreover, the results observed in the combined reductase and fermenting test in such cases may often be worse than would be shown in the fermenting test alone, as methylene blue exerts a certain toxic effect on bacteria, especially the true lactic acid bacteria, and the fewer the bacteria the more of the poison will each cell have to reduce. On the other hand, the fermenting results are not affected by the small amount of methylene blue 172 • DAIRY BACTERIOLOGY used in the reductase test, in the case of milk containing a greater number of bacteria. Conversely, milk which is very rich in bacteria generally shows good results in the fermenting test, for even if it may contain millions of gas-producing bacteria, it will, generally contain still greater numbers of true lactic acid bacteria, and therefore become sour so quickly that the former type will not be able to gain predominance. No error will be committed in ignoring the results of the fermenting test in the cases mentioned, for the small numbers of bacteria in the best milk, whatever their nature, will not be able to exert any influence on the mixed milk of the dairy, while the worst milk will already have been placed in the lowest class, and cannot therefore be degraded any further. In the combined test, the fermentation need not be observed until after the lapse of twenty to twenty-four hours as the reductase test gives a far more accurate estimate of the number of bacteria present than the fermenting test after twelve hours. It should he clearly understood that while the indications afforded by the fer- menting test are purely qualitative, those afforded by the reductase test are purely quantitative, and it is only by combining the two tests that any real insight will be obtained into the nature of the bacterial contents of the milk. In large dairies it will only be possible to take an average sample of each supplier's milk. If the number of suppliers is not large, there is no reason why the morning and evening milk should not be tested separately provided that the cans are properly marked according to the respective meals as they always should be. When the milk only comes to the dairy in the morning, it wiU generally be found that the morning milk will be better than the evening milk, though if the milk comes from a long distance, the reverse may be the case during warm weather if the morning milk has not been cooled. In such cases it will also be necessary to cool the morning milk. The more frequent the test the juster will be the impression formed as to the relative goodness of the milk from different suppliers. Large dairies which of course should keep a well-equipped laboratory under the guidance of a chemist, who has been trained in bacteriology, will do best to test each supplier's milk daily. In the co-operative dairies, samples from all the suppliers should be tested once a week, and one of the members of the association should be present in turn, as an impartial witness. The co-operation of expert milk tasters is no longer absolu,tely necessary in view of the more objective methods which are now at our disposal ; the examination of samples in the reductase test requires no particular scientific knowledge, and may be performed by any reliable boy or girl. Samples which need not be subjected to the fermenting test should be removed from THE GRADING OF MILK 173 the water bath in the evening ; next morning the blown or very spongy samples may quickly be picked out. The following table has been drawn up in order to illustrate the system : — ! Remarks on Taste and Smell. Time for decolorisation. Fermenta- tion test. Classification according to Exact. In round numbers. Reductase test alone. Also taste and smell. Also taste and smell and fer- mentation test. 1 2 3 4 5 6 7 8 9 10 11 Acid Flat or slight turnip taste Bitter 19 min. 3 hr. 31 min. 24 min. 5 hr. 30 min. 5 min. over 12 hr. 1 hr. 57 min. 7 hr. 5 min. 52 min. 5 hr. 17 min. 1 hr. 40 min. ihr. 3|hr,* Shr 5f hr. ihr. 12 hr. 2 hr. 7 hr. Ihr 5Jhr. IJhr. 9i CiS, IV. II. III. I. IV. 1. II. I. III. I. III. IV. II. III. I. IV. I. II. I. III. 11. IV. IV. III. III. I. IV. I. III. I. III. II. IV. The average reduction time wa« 3J hours. The samples were placed ; — 3 in Class I., 1 in Class 11., 4 in Class III., 3 in Class IV. 1 * In practice round figures only need be noted. To facilitate the calculation of the average, J may be written as |. When the fermentation result is denoted by two letters, this denotes an intermediate form. The average of the four weekly tests places the milk in the class which has to be reckoned with during the corresponding month. The figure is rounded off to the nearest unit, fractions of | or over being counted as 1. The grading of mUk has been dealt with at length because it furnishes just basis for payment according to quality, the only really effective means available for improving the quality of milk. If such a system were established there would be some hope that the farmers would exert themselves to ensure the clean treatment and proper cooling of their milk, just as the more progressive of the Danish farmers have succeeded in increasing the fat content of their milk since the system of pay- ment according to " fat units " was adopted by most of the co- operative dairies in Denmark. The two systems may easUy be combined by allowing a small increase in the price per fat unit for first-class milk, and making a corresponding deduction in the case of third-class milk. Double the amount should be deducted for fourth-class milk. There would be no surer means of guarding against butter and cheese defects, and there could be no better re- commendation for the dairy products than the fact that they had been made from milk sufficiently clean and fresh to be palatable 174 DAIRY BACTERIOLOGY to the most critical. This reform would also have far-reaching results contributing indirectly to the welfare of coming generations ; for once the principles of hygiene have gained a footing in the cow- shed they will gain admission everywhere else. Healthy and clean cows, good milk, healthy children. INDEX Acid, acetic, 1, 16, 30, 128 butyric, 42, 135, 139 lactic, 30, 31, 32, 49, 128, 129 optical isomers, 32 propionic, 41, 139 Acidity of cream, determination of, 107 of milk, determination of, 161 of starter, determination of, 117 of whey, determination of, 131 Acids, fatty, 122, 134, 144 Actinomyces bovis, 29 chromogena, 126 Actinomycetes, 29 Actinomycosis, 70 Aeration of cream, 112 of milk, 78, 168 of starter milk, 116 Aerobic bacteria, 10 Aerogenes bacteria, 39, 153 mastitis, 151 Aftef-fermentation of cheese, 139 Albumin, 81, 96, 97, 113, 130, 149 Alcohol test, 162 Alcoholic fermentation, 50, 102 Alizarol test, 162 Alkali-forming bacteria, 63, 65, 75 Amino acids, 4, 140 Ammonia formation in cheese, 134, 135, 136, 146, 147 Ammonium caseinate, 119, 134, 135 Amylase, 13 Anaerobic bacteria, 10, 19, 42 Anthrax, bacterium of, 10, 71 Anti-toxins, 14, 97 Appetitost, 149 Aroma bacteria in butter, 108, 110, 116 in cheese, 142 Aspergillus repens, 87 Autoclave, 17 Average reduction time, 171 BAciLLtrs, 27, 29 Bacillus, abortus, 71 acidi lactici, 32 acidophilus, 3^ anthracis, 10, 47 Ufidus, 33, 101 botulinus, 48 bulgaricus. See " Thermobacterium bulgaricum." butyricus immobilis, 43 mobilis, 43 Chauvaei, 43 coli, 40, 45, 65 cyaneofuscus, 45, 154 Delbrilcki. See " Thermobacterium cereale." firmitatis, 155 mesentericiis, 47 I Bacillus, mycoides, 47, 67, 71, 101 putrificas, 47 rudensis, 154 suhtilis, 46, 47 tetani, 48 typhosus. See ^^ Bacterium typho- Bacteria, 1, 27, 29 classification of, 26, 29 in air, 61 in butter, 20, 120 in cheese, 20, 137 in earth, 47, 61 in manure, 61, 63, 70, 158, 163 in mUk, 19, 20, 62, 64, 71, 69, 163, 166, 170 in salt, 126 in straw, 61 in udder, 61, 63 in water, 21, 40, 44, 68, 74, 75, 123 lactic acid, classification of, 32 pathogenic in milk, 69, 71, 97 staining of, 24, 25 Bactericidal substances, 55, 89, 113 of milk, 64, 97 Bacteriological methods, 16 Bacterium abortus, 71 acidi propionici, 41 bifidum, 33, 101 carotce, 74 casei a. See " Streptobacterium caseiy casei y. See " Betabacterium breve." casei S. See " Betabacterium longum." casei t. See " Thermobacterium helveticumJ''' casei fusci, 155 limburgensis, 146, 148 caucasicum, 35 cloacae, 40, 104 coli, 40, 45 cyaneofuscus, 45 enteriditis, 40 erylhrogenes, 46 fiuorescens liquefaciens, 44, 46, 74, 160 lactis, 32 acidi. See " Streptococcus lact'iK.'' aerogenes, 40 innocuum, 63, 75, 107 longi, 36 acidi, 32 minimum mammce, 69 pneumonice, 40 prodigiosum, 46 pyocyaneum, 26, 44 pyogenes, 69 sapolacticum, 75 176 INDEX Bactermm syncyaneum, 26, 45 synxantum, 46 typhosum, 27, 40, 71 vulgare, 27, 46 Zopfii, 45, 75 Betabacterium, 33, 67 6rej;e, 34 coMctwiciim, 34 longum, 34 Belacoccus, 33, 37, 38, 65 Biological characteristics, 15 Biorisation, 83 Bitter taste in butter, 125 in cheese, 129, 155 in milk, 75 Blood in milk, 69, 159 Blown cheese, 139, 151 mUk, 164 Blue milk bacteria, 73 Boiling test, 162 Bottling of mUk, 91 stoppers for, 91 Brie cheese, 133 Buchner, 12 Budde's process, 89 Burnt taste, 79, 118, 125 Burri's Indian ink method, 25 tubes, 19, 65 Butter, aroma of, 108, 110, 116 defects, original, 124 secondary, 125 flora, abnormal, 124 normal, 120 making of, 106 milk, 98, 118 rancidity of, 51, 52, 121, 127 Butyric acid bacteria, 42, 139, 149, 152, 160 Cabbage taste in cheese, 156 Calcium lactate, fermentation of, 41, 42, 135 Camembert cheese, 133, 136, 147 Capsule formation by bacteria, 7 Carbol fuchsine, 25 Carbon, sources of, 9 Carbonic acid, 122, 151, 156 Casein, 13, 14, 97, 99, 129, 131 Catalase, 14, 33, 90 test, 159 CeU walls, 5 Cells, 4 Cellulose, fermentation of, 43 Certified mUk, 60, 62 Cheddar cheese, 43, 130, 141, 154 Cheese curd, consistency of, 131, 132 making of, 129 defects, 73, 129, 151 flora, abnormal, 15 1 normal, 135 in milk, 72, 164 poisoning by, 156 rennet, 129, 166 ripening of, 128, 132 course of, 132 depth of, 134 extent of, 134 salt in, 131, 132, 139 Cheesy milk, 72, 164 sour butter, 126 Chlahydospores, 4 Cholera bacteria, 28, 71, 82 Ghymosin, 14, 81, 129, 138, 143, 165 Gilia, 26, 46 Cladosporium, 51 butyri, 51, 123 heriarum, 51, 56, 155 Classification of bacteria, 28 of lactic acid bacteria, 32 Cleaning, 54, 58, 59 separator, 90 Clostridium, 3, 4 Coagulating milk, 72 Coagiilation of milk proteins, 81, 129 Cocci, peptonising, 45 Coccus, 27 Coccus liquefaciens. See " Streptococcus liquefaciens." Coli bacteria, 39, 45, 65, 125, 152, 153 Colonies, 20, 23 Colostrum, 69, 71, 151, 162 Colour defects in butter, 126 in cheese, 45, 153 in milk, 73 producing organisms, 44, 45, 46, 73 Condensing of milk, 86 Conidia, 4 Cooked taste in butter, 125 in cream, 81 in milk, 81 Cooling of cream, 81, HI of milk, 65, 77, 91, 95 Copper in cheese, 153 in milk, 75 Counts, bacterial, of air, 61 of butter, 20 of cheese, 20 of earth, 61 of manure, 61 of mUk, 19, 20, 62, 64, 170 of straw, 61 of water, 21 Cow-sheds, fioor covering of, 58, 59 Cows, diarrhoea of, 57, 72, 151, 158, 163 diseases of, 57, 69, 71 Cracks in cheese, 73, 153 Cream, cooling of, 81, 111 fat in, 110 homogenieation of, 80 pasteurisation of, 109, HI, 120 pathogenic germs in, 109 souring of, 36, 106 whipped, 67 Cultivation methods, 16, 17, 22 Cultures, lactic acid, 108, 117 dry, 109 mould, 145, 148 pure, 21, 24, 145, 148 single cell, 21, 24 Curd, acidity of, 131 consistency of, 131, 132 making of, 131 Danish dairy cheese, 133, 136, 142 Swiss cheese, 140 Defects of butter, 122, 124 of cheese, 161 of milk, 68, 71 Diarrhoea of cows, 67, 72, 151, 158, 163 INDEX 177 Diastase, 13, 47 Diet of cows, 57, 68 Dietetic preparations, 100 Diffusion slices, 58 Diphtheria bacteria, 29, 71, 82 Dirt in milk, 60, 61, 90, 157 Disease germs in milk, 29, 71, 82 Diseases of cows, 69, 71 Disinfecting, 55 Dry cultures, 109 Dutch cheese, 36, 73 Dysentery bacteria, 82 Earthy smell in butter, 126 Edam cheese, 36, 73, 133, 136, 141, 154 Emmental cheese, 34, 41, 42, 131, 133, 136, 142, 152 Endoenzymes, 12, 137, 142 Enrichment method, 22 Enzymes, 12, 15, 97, 129, 137, 140, 142, 167 Erepsin, 14, 137, 140, 142 Exoenzymes, 12, 140, 142 Eyes in cheese, abnormal, 139 normal, 41, 42, 138, 152 Fat butter, hydrolysis of, 121, 134 oxidation of, 121 Fat hydrolysis by microorganisms, 44 in butter, 122 in cheese, 134, 144 in cream, 110 in cheese, 134 in milk, 96 Feeding of cows, 57, 68 Fermentation processes, 11, 30, 41, 42, 44, 106, 119, 128, 139 Fermenting milk, 72 test, 163, 170 combined with reductase test, 166 Ferments, 12 Fishy taste in butter, 126 FlageUffi, 26, 46 Flash pasteurisation, 82 Flies, 60 Floor of cow-sheds, 58, 59 Fluorescent bacteria, 44, 65 Fodder, influence of, on mUk, 68 Foot and mouth disease, 82 Freudenreich flask, 17 Fuohsine, 25 Galaotase, 90, 129 Gassy milk, 72 Gentian violet, 25 Gioddu, 100 Gldsler, 139 Globulin, 130 Grorgonzola cheese, 143 Gouda cheese, 141, 155 Grading of milk, 157, 170 Gram's staining, 25 Grass taste in butter, 125 in milk, 74 Green Alpine cheese, 133, 136, 146, 149 Growth and reproduction, 2 Gruydre cheese, 34, 136 Hansen's distinction of yeasts, 49 single cell culture, 24 Harrach cheese, 147 Hay bacteria, 46, 83 Hard cheeses, 132, 149 Harz cheese, 150 Heat action on bacteria, 19, 80 on enzymes, 12 Holder pasteurisation, 82, 93 Holes in cheese, abnormal, 139 normal, 41, 42, 138, 152 Homogenisation, 79, 80, 85 Household pasteurisation apparatus, 83, 97 Hydrogen peroxide, 14, 89, 90, 93, 159 Ice milk, 60, 78, 94 Immunity, 15 Incubation period, 1 1 Incubator, 17, 18 Infants, nutrition of, 88, 96, 97 Infection, 56 by milk, 69, 71, 82 Invertase, 13 Involution forms, 16 Iron in cheese, 153 in mUk, 98 Isiguy butter, 110 Jobsensen's moist chamber, 23 Kaeldbb milk, 102 Kefir, 51, 100, 103 Knapost cheese, 149 Knopist cheese, 147 Koch, 17 Kumys, 100, 102 Lactase, 13 Lactic acid, 30, 31, 32, 49, 128, 129 optical isomers, 32 bacteria, pseudo, 30, 38, 39, 118 true, 32, 114, 160, 171 fermentation in cheese, 129, 153 psuedo, 30, 163 true, 30, 106, 114, 128 micrococci, 38 rod bacteria, 33 streptococci, 35 Lactobacillus, 33 Lactose, 96, 135 Leben, 100, 102 Lecithin, 98, 126 Leucocyte test, Tromsdorff's, 158 Leucocytes in milk, 93, 158, 167 Leuconostoc, 7, 38 Limburg cheese, 133, 136, 146 Lipase, 13 Liquefying of cheese, 153 organisms, 14, 153 Lofotrich bacteria, 26 Long mUk, 7, 8, 36, 72, 100 whey, 72 Low temperature pasteurisation, 82, 93 Malt taste in butter, 118 Maltase, 13 Manure, bacteria in, 61, 63, 70, 158, 163 preservation of, 119 Mastitis, 69, 72, 151, 159 Mazun, 100 Meat poisoning, 46 12 in INDEX See " Streptococcus Medicines passing into milk, 68 Metallic taste in butter, 125 in milk, 75 Methyl violet, 25 Methylene blue, 25, 166 Microbacterium, 33, 38 Micrococcus, 27, 65 Micrococcus candicans, 160 casei amari. Kquefaciens." casei liquefaciens. See " Tetracoccus liquefaciens." chromoflavus, 155 flavus, 155 Treudenreichii, 72 rubri casei, 155 Microorganisms, 1 cultivation of, 16, 17, 22 mode of growth, 3 multicellular, I size of, 2 spore formation by, 3 unicellular, 1 Midday milk, 79 Milk, acidity of, 161 aeration of, 78, 168 bacterial counts of, 62, 170 bottling, 90 certified, 60, 62 coloured, 73 coagulation of, 72, 87 condensing of, 86 cooling of, 65, 77, 81, 91, 94, 95 defects, primary, 68 secondary, 71 dirt in, 60, 61, 90, 157 dried, 88 fermented, 100 fermenting, 72 flora, abnormal, 68 normal, 63 from cows long in milk, 69, 151, 162 gassy, 72, 164 grading of, 157, 170 homogenisation of, 79, 80, 85 keeping of, 65, 77 pails, 60 pasteurisation of, 70, 81, 93, 94, 162 pathogenic germs in, 69, 71, 80, 97 poisoning of, by bactsria, 83 powder, 88 preparations, 100 preservation of, 77 ropy, 100 separated. See " Separated milk." skim. See " Separated milk." slimy, 100 souring of, 66, 67 stale, 67 sterilised, 79 strainers, 60 town supplies, 90, 95, 170 sugar, 96, 135 unclean, sour-tasting, 73 Milking, 59 machines, 59 Moist chamber, 23 Monilia nigra, 51, 155 species of, 51 Monolrich bacteria, 26 Morphology, 4 Mould spots on butter, 125 on cheese, 155 on walls, 56 Moulds, 2, 49, 51, 118, 121, 122, 125, 134, 143, 145, 148, 155 Mouldy cheeses, 143, 146 Mucin, 5 Muoor, species of, 4, 56 Mucorinse, 29 Mycelium, 2 Mycoderma, species of, 50, 104 Mycoderma ccreviaim, 50 Mycomycetse, 29 Nielsen's steriliser, 80 Nitrates, reduction of, 33 Nitrogen, /sources of, 9 Norwegian Gammelost, 133, 149 Kaeldermaelk, 102 Nucleus, 5 Nutrient media, 7, 17, 19 Nutrition of animals, 96 of bacteria, 7, 15, 16, 17 of infants, 88, 96, 97 OlDIUM, 4, 51 Oidium aurantiacum, 155 camemberti, 147 lactis, 6, 51, 67, 107, 122, 123, 125, 144, 147, 148, 149, 150 Oily taste in butter, 118, 125 Optimum temperature, 12 Orla-Jensen's household pasteuriser, 97 Oxidases, 14, 167 Oxygen, sources of, 9 Paracasein, 13, 129 Parmesan cheese, 140, 153 Pasteur 32 Pasteurismg of cream, 81, 109, 111, 120 of milk, 81, 93, 162 of milk for starter, 113 of separated milk, 70, 84 of water, ] 22 Pathogenic germs in butter and cream, 109 in milk, 69, 71, 80, 97 Payment according to quality, 61, 173 Penicillium, 52, 144, 145, 148 brevicaule, 53, 74, 156 camemberti, 53, 147 candidum,, 53, 147 casei, 53, 155 glaucum, 2, 6, 52, 122, 123 rogueforti, 62, 145, 147 Pectins, 43, 57 Pepsin, 14, 129 Peptones, 13 Peptoniaation, 13, 129, 133, 137, 140 Perhydrase milk, 90 Peritrich bacteria, 26 Petri dishes, 19 Petruschky flasks, 17, 20 Pleciridium, 3, 4 fostidum, 47, 147 INDEX 179 Poisoning by cheese, 156 by meat, 46 Poisons in milk, 68, 83 Potato bacteria, 46, 83 Preservation of butter, 120 of milk, 77, 89, 128 of stable manure, 119 Preservatives, 89, 120 Pressler, 138 Propionic acid bacteria, 41 fermentation, 41, 42 Proteins, hydrolysis of, 44, 48, 81, 129, 133, 136, 143, 146 Proteolytic enzymes, 13, 129, 133, 137, 140, 143 Proteus bacteria, 40, 45, 65, 104, 125 Protoplaism, 4 Pultost, 149 Pure cultures, 21 Pus in milk, 69 Putrefaction, 11, 40, 143, 147 Putrefactive bacteria, 44, 125 Quality, payment according to. 61. 173 Eancidity, 51, 52, 121 Ray fungi, 29 Red milk bacteria, 73 Reductase and fermenting test, 95, 170 test, 166, 170 Reductases, 14 in milk, 167 Rennet, 14, 81, 129, 138, 143, 165 and fermentation test, 166 curd cheeses, hard, 135 soft, 145 test, Marshall's, 161 Schaffer's, 81, 161 Reproduction, 3 Retting, 43 Ripening of cheese, 128, 132, 134 Romadour cheese, 146 Ropy milk, 7, 8, 36, 72 whey, 36, 73 Roquefort cheese, 134, 136, 143 Rosolic acid test, 162 Rotting, 11, 57 Russian Steppe cheese, 140 Rust spots in cheese, 155 Saochabomycetbs, 49, 65, 87 Salt, bacteria on, 126 mUk, 68 stones, 140 Salting of butter, 120 of cheese, 131, 132, 139 Saltpetre, 152 Salt stones in cheese, 140 Saroina, 27, 38, 160 Scarlatina, 71 Scarlet fever, 71 Schabzeiger cheese, 133, 136, 146, 149 Sohotte, 86, 137 Sediment in milk, 157, 159 Self-souring of cream, 106 of milk, 64, 67, 72 Separated milk, 84, 89, 118, 149 Separator, cleaning, 91 Single cell cultures, 24 Skim milk. See Separated Milk. Skuta, 106 Slime formation, 38, 42 Slimy mUk, 72 whey, 36, 73 Smeared cheeses, 146 Smell and taste of milk, 72, 172 Soapy taste, 75 Soft cheeses, 132, 145, 150 Soil bacteria, 11, 38, 43, 44, 47 formation, 11, 43 Sour cheesy smell in butter, 126 Sour mUk cheeses, hard, 1 49 soft, 150 Souring defects, 118 of cream, 36, 106 of mUk, 66, 67, 112 of separated milk, 112, 118 of starter milk, 112 Soxhlet's nursery pasteuriser, 83 Species, 15 Spirillum, 28, 29 Spongy cheese, 139, 151 mUk, 165 Sporangia, 4 Spores, 3, 4, 49, 80, 83 endogenous, 3 exogenous, 3 germination of, 3 Stab cultures, 10, 21, 38 Stabbing of cheese, 144 Stable floors, 58, 59 smell in butter, 125 in milk, 74 Staining of bacteria, 24, 25 Staphylococcus, 27 Starter for cream, 108, 112, 113 Sterilised milk, 79, 167 Sterilising, 19, 79, 1 67 Stilton cheese, 143 Storch's reaction, 89, 93 Streak cultures, 21 Streptobacterium, 33, 34, 35, 67 casei, 34 plantarum, 34 Streptococci, 27, 33, 35, 63, 65, 107, 120, 141, 159 Streptococcus acidi lactici, 32 agalactice. See " Streptococcus mas- titidis" brassicoe, 38 casei amari. See " Streptococcus liquefadens." cremoris, 35, 36, 73, 102, 112, 114, 115, 120 fcecium, 36, 65, 85, 102 glycerinaceus, 85, 102 Jtollandicus, 36 lacticus. See " Streptococcus cre- moris." lactis, 27, 36, 37, 65, 66, 102, 114, 118, 143 liquefaciens, 37, 72, 75, 140, 153, 155 masiitidis, 36, 69 pyogenes, 36 tUrmopUlus, 35, 36, 65, 85, 114, 138 Streptococci, heat-resisting, 85 pathogenic, 159 180 INDEX Spirillum, 28 Storch's reaction, 14, 89, 93 Stribolt's anaerobic cultivation, 22 Sugar beet slices, 58 in condensed milk, 86 Sugars, fermentation of, 23 Sulphur bacteria, 29, 75 Swedish manor farm cheese, 140 Swine erysipelas bacterium, 10 Swiss cheese making, 86 Swiss skim milk cheese, 133, 136 Symbiosis, 102, 103, 109, 146 Tallowy taste in butter, 118, 122, 125 in cheese, 155 in mUk, 75 Taste and smell of milk, 157 Tears in cheese, 139 Tetanus bacillus, 10 Tetraoocci, 33, 38, 72, 140, 142 Tetracocciis liqiiefaciens, 39, 140, 143, 146 Thermobactermm, 33, 67, 85 bulgaricum, 30, 31, 34, 101 116 cereale, 33 hdveticum, 34, 50, 116, 143 jugurt, 34 Thermophilic bacteria, 47, 83, 85 Thermostat, 18 Thick butter, 124 ^ Thread bacteria, 29 Tilsit cheese, 140 Torula amara, 50, 75, 155 Torulw, 49, 60, 67, 123, 126, 153 Town's milk supplies, 90, 95, 170 Toxins, 14 Train oil taste in butter, 126 Trommsdorf's leucocyte test, 158 True lactic acid bacteria, 30, 72 Trypsin, 14, 129, 140, 142 Tubercle bacteria, 70, 82, 97, 109 Tuberculosis, 69, 71, 82 Turnip taste in butter, 125 in milk, 74 tops, 67 Typhoid bacteria, 27, 40, 71, 82 Tyrothrix bacteria, 135, 140 Udder, bacteria in, 61, 63 inflammation of, 69 tuberculosis, 69 Ultra-violet rays, 90 Unclean taste in butter, 125 in cheese, 155 in milk, 73 Urda, 106 Vactjoles, 6 Variability, 15 Variants, 15 Variety, 15 Vibrio, 28, 29 Vitamiues, 98 Volatile acids in butter, 121 in cheese, 136 Washlno of butter, 111, 118, 124 Water, bacteria of, 21, 40, 44, 74, 76, 123 Wet butter, 124 Whey, 86, 119, 131 champagne, 106 long, ropy or slimy, 36, 73 sparkling, 106 Yeast, 2, 3, 49, 67, 105, 118, 121, 123 taste in butter, 125 Yeasts, fermentation of sugars by, 50 Yellow milk, bacteria of, 46 Yoghurt, 100, 138 Zooqlosa, 7 Zygosaccharomycetes, 87 Zymase, 12 THE WHITEPKIARS PRESS, LTD., lONDOH AND XONBRIDOE.