- JfS us Issued October 2, 1912. U. S. DEPARTMENT OF AGRICULTURE, BUREAU OF ANIMAL INDUSTRY. BULLETIN 150. A. D. MELVIN, CHIEF OF BUREAU. HE BACTERIOLOGY OF CHEDDAR CHEESE. OUTHERN f RS!TY OF -CALIFORNIA, LIBRARY, *~QS ANGELES, CALIF. BY E. G. HASTINGS, Bacteriologist, Wisconsin Agricultural Experiment Station , ALICE C. EVANS, Bacteriologist, Dairy Division, Bureau of Animal Industry, AND E. B. HART, Chemist, Wisconsin Agricultural Experiment Station. WASHINGTON: GOVERNMENT PRINTING OFFICE. 1912 Issued October 2, 1912. U. S. DEPARTMENT OF AGRICULTURE, BbREAU OF ANIMAL INDUSTRY. BULLETIN 150. A. D. MELVIN, CHIEF OH BUREAU. THE BACTERIOLOGY OF CHEDDAR CHEESE. BY E. G. HASTINGS, Bacteriologist, Wisconsin Agricultural Experiment Station, ALICE C. EVANS, Bacteriologist, Dairy Division, Bureau of Animal Industry^ AND E. B. HART, Chemist, Wisconsin Agricultural Experiment Station. WASHINGTON: GOVERNMENT PRINTING OFFICE. 1912 BUREAU OF ANIMAL INDUSTRY. Chief: A. D. MELVIN. . Assistant Chief: A. M. FABBINGTON. Chief Clerk: CHARLES C. CARBOLL. Animal Husbandry Division: GEOBGE M. ROMMEL, chief. Biochemic Division: M. DORSET, chief. Dairy Division: B. H. RAWL, chief. Field Inspection Division: R. A. RAMSAY, chief. Meat Inspection Division: RICE P. STEDDOM, chief. Pathological Division: JOHN R. MOHLEB, chief. Quarantine Division: RICHABD W. HICKMAN, chief. Zoological Division: B. H. RANSOM, chief. Experiment Station: E. C. SCHBOEDEB, superintendent Editor: JAMES M. PICKENS. DAIRY DIVISION. B. H. RAWL, chief. HELMEB RABILD, in charge of Dairy Farming Investigations. S. C. THOMPSON, in charge of Dairy Manufacturing Investigations. L. A. ROGEBS, in charge or Research Laboratories. EBNEST KELLY, in charge of Market Milk Investigations. ROBERT MCADAM, in charge of Renovated Butter Inspection. 2 ADDITIONAL COPIES of this publication 2\. may be procured from the SUPERINTEND- ENT or DOCUMENTS, Government Printing Office, Washington, D. C., at 10 cents per copy LETTER OF TRANSMITTAL. U. S. DEPARTMENT or AGRICULTURE, BUREAU OF ANIMAL INDUSTRY, Washington, D. C., May 24, SIR: I have the honor to transmit for publication in the bulletin series of the bureau the accompanying manuscript entitled " The Bacteriology of Cheddar Cheese," by Messrs. E. G. Hastings and E. B. Hart, of the Wisconsin Agricultural Experiment Station, and Miss Alice C. Evans, of the Dairy Division of this bureau. Cooperative work on the factors concerned in the ripening of Cheddar cheese has for several years been carried on at Madison, Wis., between the Dairy Division and the Wisconsin Experiment Station, and this bulletin is one of a series in which the results of the work are detailed. Previous experiments have already been de- scribed in Bulletin 122 of the Bureau of Animal Industry, entitled " Factors Controlling the Moisture Content of Cheese Curds," and in Bulletins 7 and 11 of the Wisconsin Station. In connection with the present study credit is due to Mr. L. D. Bushnell, now with the Kansas Agricultural Experiment Station, and Mr. Alfred Larsen, of the North Dakota State Hygiene Labora- tory, who were formerly bacteriologists in the Dairy Division and while thus engaged prepared a portion of the data presented in this bulletin. Kespectfully, A. D. MELVIN, Chief of Bureau. Hon. JAMES WILSON, Secretary of Agriculture. 3 CONTENTS. Page. Introduction 7 The bacterial flora of milk 8 Differences in composition of cheese 9 The enzym content of cheese 10 Biological problems 10 Bacteriological methods 11 The role of Bacterium lactis acidi in Cheddar cheese 13 Effect of curdling on distribution of bacteria in milk 15 Effect of acid on the expulsion of the whey 18 Effect of acid on the texture of the curd 18 The role of acid in the ripening of cheese 18 The protective action of acid 20 The rate of bacterial growth in cheese 21 Methods of examination 21 Results of the work 22 The enzymic action of lactic bacteria 25 Other groups of bacteria in Cheddar cheese 31 Determinations by milk-dilution and plate-culture methods 31 Determinations by microscopic examination of the cheese 37 The disappearance of bacterial cells in cheese 39 Detailed study of lactic bacilli 40 Acidity produced 41 Forms of lactic acid produced 41 Type of curd produced 42 Fermentation of sugars 43 Form of colonies 43 Thermal death point 44 Morphology 44 Conditions for growth in cheese 45 Solvent effect on milk proteins 47 Coccus forms in Cheddar cheese 48 Chromogenic cocci 50 Liquefying organisms 50 The sequence in development of bacterial groups in Cheddar cheese 51 Summary 52 5 ILLUSTRATIONS. PLATE. Page. PLATE I. Fig. 1. The action of rennet extract on casein suspended in agar in the presence and in the absence of acid-forming bacteria. Fig. 2. The effect of acid on cheese 20 TEXT FIGURES. FIGURE 1. Increase of acidity in raw milk preserved with 3 per cent toluol 29 2. Increase of acidity in heated milk preserved with 3 per cent toluol. 29 3. Typical microscopic fields from cheese No. 54 at different stages in the ripening process 38 4. Typical microscopic fields from cheese No. 91 at different stages in the ripening process 39 6 THE BACTERIOLOGY OF CHEDDAR CHEESE. INTRODUCTION. The role of microorganisms in the preparation and ripening of the various kinds of cheese is a problem that has attracted the at- tention of bacteriologists since the beginning of the science and since the establishment of the importance of microorganisms in all de- composition processes. From the same raw materials cow's milk, salt, and rennet, which is an extract of the fourth stomach of the young calf a great number of varieties of cheese are prepared. These cheeses differ not only in texture, but more especially in flavor and aroma. From what is known of the importance of microorgan- isms in the preparation of the products of the fermentation indus- tries it is evident to one who gives consideration to the question that undoubtedly microorganisms are as essential in the making of cheese as they are known to be in the preparation of wine, beer, and other products. In the making of wine and beer the desired changes are produced by a single form of life, the true yeasts, and in the preparation of any desired type of product attention need only be directed as far as the causal organism is concerned in order to insure the presence of the particular variety of yeast that has been found by experience to form a product that has the desired properties as well as to insure the absence of harmful forms. In the preparation of still other products of the fermentation industries not only a single form of life is essential to produce the desired changes, but two or more are needed, one of which may work on a certain constituent of the raw material, the other on another, or the one may serve to make conditions favorable for the growth of the second, usually by the preparation of suitable food. It is evident that as the number of kinds of microorganisms that are needed to produce the desired changes in any product increases, the complexity of the problem of demonstrating the role of each of them is also greatly increased, and it may become most difficult to prove even whether any particular organism is absolutely essential in the preparation of the product, to say nothing of demonstrating its exact role. It is not to be supposed that a single form of life is 7 8 BACTEKIOLOGY OF CHEDDAR CHEESE. responsible for the complex chemical changes that occur in the ripen- ing of any particular variety of cheese, but rather that a considerable number will be involved. The fermentation industries include those in which microorganisms are essential for the production of the desired decomposition changes. In all such industries the quality of the product will depend on the raw material and the organisms that are used in the decomposition of that material. The preparation of cheese has not until recent years been classed as a fermentation industry, although it is one in which microorganisms play a dominant role. Here, as in all other similar industries, if control of the quality of the product is to be obtained, knowledge must be gained concerning the essential biological agents. In this paper are presented a summary of the present knowledge of the bacteriology of Cheddar cheese and the results of a detailed study of a number of cheeses. THE BACTERIAL FLORA OF MILK. In the case of many of the products of the fermentation industries the essential microorganisms are contained in the raw materials, and the development of the methods of preparation of the products have been wholly empirical. Especially is this true of certain kinds of cheese, the methods of manufacture of which are in some cases cen- turies old. Even in the case of cheese to which at some stage in manufacture material containing certain microorganisms is added the methods of manufacture antedate the science of bacteriology. The sources from which microorganisms enter the milk are much the same in all parts of the world. Any natural source, such as the interior of the udder, the dust from the animal, or even the utensils, will always furnish certain definite types of microorganisms. By this is not meant that the flora of milk will always include these definite types and no others, but that certain forms will always be present. That this is true is shown by the fact that a sample of ordinary market milk, if stored at temperatures ranging from 60 to 90 F., will undergo a definite sequence of decomposition changes with almost unerring certainty. In this sequence three main groups of microorganisms are prominent; first, the acid-forming bacteria that change the milk into an acid, semisolid mass, favorable for the development of the mold that is characteristic of milk Oidium lactis. This mold, by its gradual destruction of the acid and the establish- ment of an alkaline reaction, makes it possible for the putrefactive organisms to develop. When the environment is changed this sequence of microbial life in the milk is also changed. For example, milk may be kept at such low temperatures that the acid-forming bacteria can not grow and the putrefactive bacteria appear first. DIFFERENCES IN COMPOSITION OF CHEESE. 9 It is thus proper to speak of a characteristic milk flora. By some it is believed that this flora is so constant that fermentations that depart from the normal are to be looked upon not as due to the in- troduction of other forms of microorganisms, but most often to changes in the properties of the organisms or to changes in environ- ment that favor the particular organisms which produce the abnor- mal fermentation. Slight changes in the composition of the milk may also have a determining influence. As has been stated, the microorganisms necessary for the prepara- tion of most kinds of cheese are contained in the milk. Whether one form or the other is to develop in the cheese depends upon the en- vironment established by the cheese maker. The manufacture of cheese is thus a problem in the ecology of microorganisms. DIFFERENCES IN COMPOSITION OF CHEESE. The differences due to the methods of manufacture result in differ- ences in composition of the cheese at the time it begins to undergo the ripening process, which is to determine whether the cheese is to be classed as one kind or another. The differences in composition of the fresh cheese are very largely in the amount of moisture pres- ent. Since the water that is left in the cheese is whey, carrying in solution sugar and other constituents of milk, the effect of differ- ences in moisture is not only in influencing the texture, but also the composition of the cheese. The difference in moisture content between Cheddar and Camembert cheese is approximately 25 per cent. If it is supposed that the moisture of the cheese will have about the same sugar content as milk, this means that the sugar con- tent of the Camembert cheese will be about 1 per cent greater than Cheddar; and since the sugar is all fermented, differences in sugar content ultimately result in differences in acidity. One of the basal differences between Cheddar and Swiss cheese is that during the making of the first that is, the operations before pressing a large amount of acid is formed in the curd, while in the case of Swiss no such acid production occurs. Van Slyke and Hart, 1 Boekhout and Ott de Vries, 2 and Van Dam 3 have shown that acid is held chemically by the proteins of cheese, while sugar is not. Thus if in the making of one cheese acid is formed, and in another not formed, the result will be a somewhat different composition, even though the moisture content of the two cheeses is initially the same. 1 Van Slyke, L. L., and Hart, E. B. A study of some of the salts formed by casein and paracaseln with acids : Their relations to American Cheddar cheese. New York Agricul- tural Experiment Station Bulletin 214. Geneva, July, 1902. See p. 60. Boekhout, F. W. J., and Ott de Vries, J. J. Uber den Kasefehler " Kurz " (Kort). Centralblatt fUr Bakteriologie, Parasitenkunde und Infektionskrankheiten, Abteilung 2, vol. 24, No. 5/7, pp. 122-129. Jena, Aug. 2, 1909. Van Dam, W. Uber die Konsistenz der Kasemasse bei Edamkasen. Centralblatt fur Bakteriologie, Parasitenkunde und Infektionskrankheiten, Abteilung 2, vol. 32, No. 1/2, pp. 7-40. Jena, Dec. 5. 1911. 50978 Bull. 15012 2 10 BACTERIOLOGY OF CHEDDAR CHEESE. Another difference in composition of cheese is caused by variations in the amount of salt and in the time at which it is added. This dif- ference is an important one in the case of Cheddar and Swiss cheese. These differences in the initial composition are sufficient to exert a great influence on the types of life that are to grow in and on the cheese. THE ENZYM CONTENT OF CHEESE. The role of the inherent enzyms of milk and those of the rennet extract in the ripening of cheese have been emphasized by the work of Babcock and Russell and of Von Freudenreich and Jensen. There remains no doubt concerning the importance of certain enzyms, es- pecially those of rennet extract, in the ripening of cheese. The enzymic content of all cheese must be much the same qualitatively, since they are made from the same raw materials. Again, it is dif- ficult to see how one enzym can be active in one cheese and not in other kinds, since the conditions are quite similar as regards reaction and other factors. Accepting the theory of the specificity of enzyms, it must be admitted that the products of the activity -of any enzym will be the same in kind no matter what the conditions under which it may be working. Hence it does not seem that the enzyms of milk or rennet can be factors determining the kind of cheese to be made by any method from the ordinary raw materials, but that the deter- mining factors must be biological. BIOLOGICAL PROBLEMS. The methods of manufacture of cheese result in an environment that causes certain of the organisms constantly present in milk to develop in a definite sequence. The empirical methods of the cheese- maker result in a cheese that approximates, if it is not identical with, the normal of its kind. Each of the great commercial varieties of cheese thus presents a distinct problem for the bacteriologist, who should be able to demon- strate the constant presence of the essential groups of bacteria. It can not be expected here any more than in most decomposition proc- esses that a single specific organism will be concerned, as in the causation of disease, but rather that groups of organisms will be present, the basis of grouping being largely biochemical. Neither can it be expected that one group will grow for a period, then dis- appear and be followed by a second, but rather that the sequence of development will be confused. The work of Thorn on Camembert cheese furnishes an excellent illustration. It has been shown that a definite balance between the various forms of life concerned is essential for the normal ripening BACTERIOLOGICAL METHODS. 11 of this cheese. If through methods of manufacture or through conditions of ripening this balance is destroyed, the cheese will not be typical. The biological factors concerned in the ripening of Camembert cheese are the acid-forming bacteria, the Camembert mold (Penicil- lium camemberti) , or its white form (Penicillium camemberti var. rogeri) ; Oidium lactis; and bacteria which together with Oidium lactis form a reddish slime on the surface of the cheese. If condi- tions favor the growth of Oidium lactis at the expense of the penicil- lium, the cheese will not be normal; if the mold is too luxuriant, texture is obtained but flavor is not. It is thus evident in the case of Camembert cheese too wide variations in the balance between the different forms of life can not occur if a cheese normal in texture and flavor is to be obtained. A large amount of work has been done on Camembert cheese and the necessity for the presence of the dif- ferent forms established, but their exact role has not been shown and never can be until the chemist devises means of following in detail the complex chemical changes. It is not only necessary that the constant presence of any organism in cheese be demonstrated in order to prove its importance in the ripening, but it must also be present in sufficient numbers so that it is certain that growth has taken place in the cheese. The simple presence of any form is no evidence of its activity. Such an error was made by Duclaux, one of the first bacteriologists to occupy him- self with cheese problems. Since there are such a multitude of forms of bacteria in milk, and hence in cheese, any particular method of examination of the cheese is likely to bring some one group of organisms to the front. The methods used by Duclaux in his exami-: nation of Cantal cheese were fitted to favor the development of the spore-bearing, liquefying bacteria. Their constant presence, together with the fact that they produced in pure culture in milk compounds which he had already demonstrated in the ripe cheese, led him to consider them an important factor in the ripening of this cheese. It has been demonstrated since that the conditions in hard cheese do not permit this class of bacteria to develop. BACTERIOLOGICAL METHODS. The modern bacteriological methods are not such as will demon- strate the presence of all kinds of bacteria in milk, cheese, or any other substance in which a considerable number of kinds of bacteria are present, even though all may find conditions that will permit of growth on the medium employed. It is usually easy to demonstrate the presence of the form that occurs in greatest numbers, since in the more lightly inoculated culture plates this form will be present 12 BACTERIOLOGY OF CHEDDAR CHEESE. in pure culture or else so freed from competition with the other forms that its growth will not be prevented. For those forms present in less numbers this condition does not obtain, and usually special methods must be employed to demonstrate their presence, such as the use of differentiating media or enrichment cultures. Anyone who has much experience in plating milk on lactose media has noted that in certain cases the plates thickly seeded show colonies of but a single organism, usually the ordinary lactic organism Bacterium lactis acidi, while on the more thinly seeded plates from the same sample other forms may appear. In the thickly seeded plates the lactic organisms, on account of favorable conditions, have grown most rapidly and by the acid produced have prevented the develop- ment of other forms, while in the more thinly seeded plates the colonies may be separated by such a distance that the products of one colony do not reach the others, each colony then grows as though it were the only colony on the plate. Hence forms appear on thinly seeded plates that do not on those crowded with colonies. If the organism that finds most favorable conditions greatly exceeds in numbers some other form the latter may never appear on the cul- ture or be so inconstant as not to attract the attention of the bac- teriologist. A ratio of 1,000 to 1 or even 500 to 1 may prevent the detection, by use of the ordinary plate-culture methods, of the organisms present in smaller numbers. This statement may serve to explain the results obtained by previous investigators of Cheddar cheese. Before 1896 no consecutive bacteriological examinations had been made of a ripening Cheddar cheese. The first detailed work on this variety of cheese with which the writers are acquainted is that of Russell. 1 This work was largely concerned with a quantitative exam- ination of the cheese during the ripening process by means of gelatin plates. It established the fact that acid-forming organisms make up 99 per cent and over of the bacteria thus determined. It may be inferred that the acid-forming bacteria belonged to the Bacterium lactis acidi group. The work of Lloyd on English Cheddar cheese led to similar results. The most extensive work on the bacterial flora of Cheddar cheese is that of Harding and Prucha, 2 who iso- lated the different types of organisms appearing on cultures made from 9 normal Cheddar cheeses during the entire period of ripening. The more than 300 cultures thus isolated were studied in detail and reduced to 33 groups. Of these 33 groups 4 belonged to the Bacte- rium lactis acidi group, the only one which was always found, and it practically always included over 99 per cent of the total germ 1 Russell, II. L. The rise and fall of bacteria In Cheddar cheese. Wisconsin Agricul- tural Experiment Station, Thirteenth Annual Report, pp. 95-111. Madison, 1896. 2 Harding, II. A., and Prucha, M. J. The bacterial flora of Cheddar cheese. New York Agricultural Experiment Station, Technical Bulletin 8. Geneva, Dec., 1908. THE ROLE OF BACTERIUM LACTIS ACIDI. 13 content. The 10 other groups which these investigators classed as important on account of frequency of occurrence were not found in all of the cheeses, and it may be inferred that they do not represent essential factors in the ripening of Cheddar cheese. In speaking of one cheese the authors say : The results from this cheese accord with the idea that aside from the lactic group there is no single group or at least no single species of bacteria abso- lutely essential to the ripening process. The work of previous investigators has shown that the liquefying, the gas-forming, and the 4nert bacteria are not essential factors in the ripening of Cheddar cheese, since all or any one of these groups may be absent and yet the cheese may ripen in a normal manner. It is true that all of these groups are usually represented in Cheddar cheese, since they are present in milk, but the numbers are small, and in the case of the liquefying organisms there is no evidence that growth ever occurs during the ripening process. Yeasts can not be classed as an important factor since they may be absent from a normal cheese. The work of previous investigators may be summarized in the statement that the group of bacteria represented by Bacterium lactis acidi is the only one that up to the present has been shown to be of constant occurrence in Cheddar cheese. This fact- together with the enormous numbers, amounting to millions and at times over a billion per gram of the fresh cheese, leaves no doubt of the importance of this group of organisms in Cheddar cheese, and undoubtedly in all cheese that undergoes a ripening process. THE ROLE OF BACTERIUM LACTIS ACIDI IN CHEDDAR CHEESE. The empirical methods of the Cheddar cheesemaker demand a milk so far advanced in the acid fermentation that during the few hours between time of curdling and placing the curd in the press acid de- velopment is rapid. The increasing acidity, as has long been known, favors the curdling of the milk by the rennet. Indeed the tests most frequently used in cheesemaking to determine the " ripeness " or acidity of the milk are those in which the time of curdling is noted, when a definite amount of rennet is added to milk at a definite tem- perature (tests of Monrad and Marschall). The Cheddar maker desires milk that has just passed through the " period of incubation." By this expression is meant the time during which no apparent in- crease in acidity results from the growth of acid-forming bacteria. This has been supposed to be due to the fact that no acid was formed by the rapidly growing organisms. More recently it has been pointed out by Kahn * that this is not the true explanation but that until the 1 Rahn, Otto. The fermenting capacity of the average individual cell (Bacterium lactis acidi). Science, new series, vol. 33, No. 849, pp. 539-540. New York, Apr. 7, 1911. 14 BACTEKIOLOGY OF CHEDDAE CHEESE, bacteria have increased to a great extent the amount of acid formed is so small as to escape detection. Whatever the explanation, it is true that the acid-forming bacteria may increase until there are millions per cubic centimeter and yet the acidity show no change whatever, as has been shown by Koning 1 and by Burri and Kiirsteiner. 2 At last the acidity begins to develop with increasing rapidity until it reaches a point where it begins to exert an inhibitive effect upon the growth of the organism. In Table 1 are given some data illustrating the rate of acid development in milk kept at 95 F. It will be noted in both samples that the initial rate of increase in acid is small, that it reaches a maximum that may be over 0.15 per cent per hour and then declines. Both samples of milk were slightly too acid for cheesemaking. TABLE 1. Rate of development of acid in milk kept at 95 F. Hours. 1 2 3 5 24 Sample 1: Acidity Per cent. 0.24 Per cent. 0.30 .06 .24 .04 Per cent. 0.40 .10 .34 .10 Per cent. 0.47 .07 .5 .16 Per cent. 0.71 .12 .74 .12 Per cent. 1.00 .015 .99 .012 Increase per hour .> Sample 2: Acidity .20 Increase per hour This rapid increase in acidity is the result of the growth of the acid- forming bacteria in the favorable environment. In Table 2 are given the results of the bacteriological examination of two samples of milk kept at 95 F. for a number of hours. It. will be noted that the increase in numbers of bacteria, like that of the acidity, goes on with increasing rapidity until a maximum is reached, after which the growth is less and less rapid. TABLE 2. Increase of bacteria per cubic centimeter in milk kept at .9,7 F. [Number of bacteria expressed in millions.] Hours. 2 4 6 8 10 Sample 1 59 83 231 927 1,064 1,167 Sample 2 4 157 826 1,545 1,982 1 Koning, C. J. Der Silurojrrad dor Milch. [Pharmaceutisch Weekblnd, 1004] Milch- wirtshaftliches Zentralblatt, vol. 1, No. 7, pp. 289-305, July; No. 8, pp. 337~356, AiiKust. Leipzig, 1905. See p. 294. - Burrl, R., nnd Kiirsteiner, J. UntprsuchunRon iibor dio Relfung der Kiisereimilch. Landwlrtschaftliches Jabrbuch der Schweiz, vol. 24, pp. 437-4G6. Bern, 1910. EFFECT OF CURDLING. 15 It is essential that the acid development should be rapid during the time between the curdling of the milk and pressing the curd. The modern Cheddar maker insures this by the addition of large numbers of acid-forming bacteria in the form of a starter. EFFECT OF CURDLING ON DISTRIBUTION OF BACTERIA IN MILK. The solid bodies present in the milk will be held by the curd in the same manner as the formation of aluminum or ferric hydrate in water enmeshes the solid bodies presentj or the coagulating of albumen in a solution removes turbidity therefrom, as in the clearing of bac- teriological media and of wine. In order to illustrate the effect of curdling on the distribution of bacteria several samples of milk were subjected to a quantitative ex- amination. Rennet solution was then added and the curd cut. As soon as possible a sample of the whey was likewise examined. In Table 3 are given the data obtained from a number of such examina- tions. It will be noted that in all cases a unit volume of the whey contained less bacteria than the milk before curdling. From the average figures of the determinations approximately 77 per cent of the bacteria were retained in the curd in the trials made in the laboratory. TABLE 3. Number of bacteria per cubic, centimeter in milk and in whey im- mediately after curdling Laboratory experiments. [Number of bacteria expressed in thousands.] Trial No. 1 2 3 4 5 6 Milk... 128,000 34,000 11,000 2,900 2,050 730 155,000 75fi 23,000 773 59,000 776 Whey A number of similar examinations were made under practical con- ditions in the cheese room, the sample of milk being taken from the cheese vat and the whey after cutting the curd in the usual manner. The data are given in Table 4. It will be noted that the results are similar to those obtained in the laboratory. Approximately 73 per cent of the bacteria in the milk were retained in the curd. TABLE 4. Number of bacteria pet- cubic centimeter in milk y dilution cultures in milk. Age. Cheese No. 1. Cheese No. 64. Cheese No. 58. Cheese No. 85. Cheese No. 91. Cheese No. 92. Cheese No. 96. Bact. lactis ac*di. Acid- pro- duo- ing ba- cilli. Bact. lactis acidi. Acid- pro- duc- ing ba- cilli. Bact. lactis acidi. Acid- pro- duc- ing ba- cilli. Bact. lactis acidi. Acid- pro- duc- ing ba- cilli. Bact. lactis acidi. Acid- pro- duc- ing ba- cilli. Bact. lactis acidi. Add- pro- duc- ing ba- cilli. Bact. lactis acidi. Acid- pro- duc- ing ba- cilli. Days. 4 8 11 14 22 30 37 45 66 77 86 100 113 124 143 155 165 176 187 208 P.ct. 90.9 P.ct. 9.1 P.ct. P.ct. P.ct. P.ct. P.ct. P.ct. P.ct. 99.9 90.9 P.ct. 0.1 9.1 P.ct. 99.9 99 99.9 P.ct. 0.1 1 .1 P.ct. P.ct. 90.9 9.1 99 1 99 1 90.9 9.1 99.99 90.9 .01 9.1 99.9 99.9 .1 .1 99.99 99.9 0.01 .1 60 50 50 50 9.1 50 99 9.1 99 99 50 60 50 50 1 90.9 50 1 90.9 1 1 50 50 50 50 99 99 50 1 50 90.9 50 50 9.1 50 50 90.9 90.9 50 90.9 50 90.9 50 90.9 50 90.9 50 9.1 9.1 60 9.1 60 9.1 60 9.1 50 9.1 50 90.9 99.9 90.9 99 90.9 90.9 90.9 99 99 60 1 9.1 .1 9.1 1 9.1 9.1 9.1 1 1 50 99 50 50 90.9 99 9.1 1 60 50 50 50 9.1 90.9 50 50 90.9 90.9 90.9 60 50 50 60 9.1 9.1 9.1 50 50 90.9 9.1 90.9 90.9 9.1 9.1 9.1 9.1 90.9 90.9 50 60 1 99 1 99 1 1 50 99 99 50 50 60 60 .1 50 99.9 99 1 1 99 1 99 1 99 99 Since some of the lactic bacilli develop on lactose agar plates, it was thought that if all of the colonies on a certain portion of each plate were inoculated into milk one should obtain information con- cerning the sequence of development of the different groups of lactic bacteria in the cheese and also concerning the ratio existing between them. For this purpose an area showing well-isolated colonies has been circumscribed, and every colony within the area has been inoculated into milk. It has been inferred that all tubes that curdled within 48 hours at 37 C. contained Bacterium lactis acidi, while those that curdled between the second and tenth day contained the lactic bacilli. Enough control work was done to show that this method of differen- tiation is sufficiently accurate for the purpose in hand. The tubes that did not curdle in 10 days were examined microscopically to determine the organism present. Six cheeses have been thus examined at frequent intervals during the period of ripening. The data are given in Table 18. It will be noted that the results are confirmatory of those obtained by the dilu- tion method in milk, although not so striking, since many of the lactic bacilli do not develop on the plate cultures. The period at which the lactic bacilli appear is later than in the previous exami- nations (Table 16), which demonstrated their presence in consider- able numbers within the first week of the ripening period. The pre- vious examinations were made with dilution cultures in milk. In 36 BACTERIOLOGY OF CHEDDAR CHEESE. such the lactic bacilli may make themselves evident when they are present in very small numbers as compared with the Bacterium lactis acidi. With the plate-culture method, unless they are present in considerable numbers, their presence in the cheese is not likely to be detected. This error in the method has been previously pointed out. In the plate cultures they are likely to be missed unless they are pres- ent in a ratio of 1 to 10 of Bacterium lactis acidi. The same cheeses have also been examined by the dilution method in milk. The results are given in Table 19. It will be seen that they are confirmatory of the previous analyses. Tlie great delicacy of the dilution method as a means of detecting the lactic bacilli in the presence of Bacterium lactis acidi is shown in the results given in Table 19. On the examination of cheese 308C at two days the percentage of Bacterium lactis acidi is given as 99.9999 and that of lactic b.acilli as 0.0001. By this is meant that Bacterium lactis acidi was detected in dilutions approximately one million times greater than the lactic bacilli. For example, the acidity of the flask inoculated with one ten-thousandth gram of cheese was 1.64 per cent, thus indicating the presence of lactic bacilli, while the flask inoculated with one ten-billionth gram of the cheese had an acidity of 0.87 per cent, indicating Bacterium lactis acidi. TABLE 18. The relative proportions of Bacterium lactis acidi and lactic bacilli in cheese as determined ~by plating on lactose agar. Age. Cheese No. 307C. Cheese No. 308C. Cheese No. 309C. Cheese No. 310C. Cheese No. 311C. Cheese No. 312C. Bact. lactis acidi. Lactic bacilli. Bact. lactis acif. Lactic bacilli. Bact. lactis acidi. Lactic bacilli. Bact. lactis acidi. Lactic bacilli. Bact. lactis acidi. Lactic bacilli. Bact. lactis acidi. Lactic bacilli. Milk before ren- net was adaed When put in ipress P.ct. 100 100 100 100 100 100 100 P.ct. P.ct. 100 100 100 100 100 86 P.ct. 14 P.ct. 100 100 100 100 P.ct. P.ct. 100 100 100 P.ct. P.ct. 100 100 100 100 100 100 P.ct. P.ct. 100 100 100 100 P.ct. When taken from press 2 days 3 days 4 days 90 100 10 100 100 6 days 6 days 100 100 86 90 100 80 100 14 10 20 7 days ... .... 100 100 100 100 100 100 100 100 100 90 100 100 70 10 30 100 100 100 100 9 days 11 days ... 100 86 14 14 days 100 33 66 100 90 10 18 days . 21 days... . 22 days 100 28 days .. 100 86 14 90 10 100 33 days 36 days .. 100 100 100 00 90 10 10 42 days 89 11 49 days 29 40 71 60 56 days 71 29 64 days RESULTS OP PLATE CULTUEES. 37 TABLE 19. The ratio between the numbers of Bacterium lactis acidl and the lactic bacilli at different stages in the ripening of Cheddar cheese, as deter- mined by milk-dilution method. Age. Cheese No. 307C. Cheese No. 308C. Cheese No. 309C. Cheese No. 310C. Cheese No. 311C. Cheese No. 312C. Bad. lactis acidi. Acid- pro- duc- ing ba- cilli. Bact. lactis acidi. Acid- produc- ing bacilli. Bact. lactis acidi. Acid- ro- ue- ing ba- cilli. Bact. lactis acidi. Acid- pro- duc- ing ba- cilli. Bact. lactis acidi. Acid- pro- due- ing ba- cilli. Bact. lactis acidi. Acid- pro- duc- ing ba- cilli. Milk before rennet was added Perct. Perct. Perct. Perct. Perct. 99.99 Perct. 0.01 Perct. Perct. Perct. 99 99.99 Perct. 1 .01 Perct. 99 Perct. 1 Just before put in press When taken from press. . 2 days 99.999 99.99 99.9999 99 99.9 "go.'g"" 90.9 90.9 50 90.9 50 99 90.9 .001 .01 .0001 1 .1 "9.Y" 9.1 9.1 50 9.1 50 1 9.1 99.9 99.9 99 .1 .1 1 99.99 99.99 99.9 99.99 99.99 0.01 .01 .1 .01 .01 99 99 1 1 99.9 50 99 99.99 0.1 50 1 .01 99 90.9 99.99 1 9.1 .01 3 days 4 days .... 99.9 99 99.9 99 50 50 90.9 50 90.9 50 50 .1 1 .1 1 50 50 9.1 50 9.1 50 50 99 1 5 days 6 days ... 99.9 90.9 .1 9.1 7days 90.9 50 99 99 50 90.9 50 9.1 50 1 1 50 9.1 50 99.9 99 .1 1 90.9 99 9.1 1 11 days .... 90.9 9.1 14 days 90.9 9.1 50 60 18 days 21 days 28 days .. 33 days 35 davs. 50 50 50 50 50 99 50 1 42 days 90.9 9.1 It must not be inferred that the figures given in the tables repre- senting the results obtained by the dilution method indicate the ex- act number of the different groups of lactic organisms or the exact ratio existing between them. The sudden increase or decrease of the ratio is due to the inherent errors of the dilution method. It would be an endless task to show by this method, or any other, in fact, the exact proportion between the different types of organisms in the cheese at various stages in the ripening period. From the data presented there would seem to remain no doubt that the lactic bacilli develop later than the Bacterium lactis acidi group, making their appearance within a week or 10 days after the cheese is made, gradually increasing in numbers and probably attaining their maximum during the first month and then gradually decreasing in numbers. The number found per gram of the cheese ranges from a few million to one billion. Usually they do not attain such great numbers as do the ordinary lactic bacteria. Again, their numbers may equal the lactic bacteria, as in cheese No. 1, Table 16. DETERMINATIONS BY MICROSCOPIC EXAMINATION OF THE CHEESE. It was also thought that the gradually changing flora should mani- fest itself in the appearance of microscopic preparations made from the cheese. At the various periods of sampling smear preparations 38 BACTEEIOLOGY OF CHEDDAR CHEESE. were made from the emulsions and stained with Gram-Weigert's stain as described on page 32. The preparations show in a general way the same change in flora as has already been made evident by the analyses presented. It is not to be expected that the microscopic examination would give re- sults as striking as the cultural, since the latter measures the living cells, the former only those cells that have not lost their staining properties. 2, DAYS 57 DAYS I \Z DAYS 1 89 DAYS PIG. 3. Typical microscopic fields from cheese No. 54 at different stages in the ripening process. \ Camera lucida drawings.) Figures 3 and 4 have been prepared from camera-lucida drawings of typical microscopical fields of several slides made from 2 cheeses. The results of the cultural examination have been presented in Tables 15 and 16. Figure 3 shows that at 2 and 57 days the organ- isms are almost, if not wholly, of B.acterium lactis acidi group; at 112 days there is a preponderance of Bacterium lactis acidi and a few lactic bacilli, while at 18? days but few Bacterium lactis acidi cells RESULTS OP MICROSCOPIC EXAMINATIONS. 39 remain, the lactic bacilli having become more evident. Figure 4 in- dicates likewise that at 29 and 78 days the organisms are wholly of the Bacterium lactis acidi group, while at 121 and 185 days a decrease is seen in Bacterium lactis acidi and an increase, in lactic bacilli. It will be noted that rod-shaped organisms do not appear nearly as soon as would be indicated by the cultural analyses, but that they do at last appear, especially when the cells of the lactic bacteria have greatly decreased in numbers. This may again be taken as evidence 29 DAYS 7G DAYS 131 DAYS IS5DAY5 PIG. 4. Typical microscopic fields from cheese No. 91 at different periods in the ripening process. (Camera lucida drawings.) that the lactic bacilli develop subsequent to the Bacterium lactis acidi group and that they tend to disappear less rapidly. DISAPPEARANCE OF BACTERIAL CELLS IN CHEESE. The drawings presented in figures 3 and 4 are evidence of the grad- ual disappearance and disintegration of the bacterial cells in cheese. It is a well-known fact that the enzyms elaborated by bacteria in pure cultures continue to act long after the death of the cells. There 40 BACTERIOLOGY OP CHEDDAR CHEESE. are many reasons for believing that there is no enzym action until cell disintegration begins. It has been previously shown that the Bacterium lactis acidi group of bacteria elaborate enzyms that are able to increase the acidity of milk. As previously indicated, it may be surmised that still other types of enzyms are formed by this group of bacteria. The direct influence of the immense number of acid- forming bacteria, amounting to billions per gram, must be of great importance in cheese ripening; the indirect action through their enzyms may be still greater. It is certainly true that in normal Cheddar cheese the period of development of the ordinary lactic bacteria the organisms which heretofore have been believed to be the only ones of importance in cheese because of their constant presence in great numbers is fol- lowed by the development of another group of organisms, which, while they ferment milk sugar, producing lactic acid, must have some other source of carbon in cheese on account of the total disap- pearance of the sugar before their maximum period of development. The influence of the first group has been pointed out early in this paper. The role of the second group can not at this time be de- termined, but because of their constant presence in numbers, approxi- mating if not equaling those of the first group, their influence on the ripening of the cheese can not be a minor one. DETAILED STUDY OF LACTIC BACILLI. The work of Von Freudenreich and numerous later investigators indicates that the lactic bacilli represent a group with certain com- mon characteristics, such as morphology, optimum temperature for growth, and to some extent in the production of compounds from the sugar fermented. In all fields of bacteriology it is difficult at the present time to draw the boundary lines of any group of bacteria. The placing of the lactic bacilli found in cheese in a single group may be incorrect, but because of the fact that all the cultures studied have certain characters in common, it seems best to discuss them as a single group, although more detailed study might divide them into a number of groups. A comparative study has been made of 20 pure cultures isolated in the course of the work! The main facts of this study are here presented. All the cultures were isolated from cheese with the exception of No. 160, which was obtained from milk. Cul- tures 58A and 58B represent different colonies from plates made to insure the purity of the cultures to be used in the detailed study. The differences noted in the study of these two cultures originally from the same culture illustrate the differences one may expect to obtain in cultural work. The action of the different cultures in milk is given in Table 20, in which they have been arranged according to the degree of acidity produced in milk. DETAILED STUDY OF LACTIC BACILLI. 41 ACIDITY PRODUCED. It will be noted from the data in Table 20 that both the rate of acid development, and hence the time required to curdle milk, as well as the maximum amount of acid produced, varies widely, the acidity varying from 0.91 per cent to 2.31 per cent. The acidity produced by each culture increased after the tenth day. Since the flasks were protected by tin-foil caps, this increase can not have been due to evaporation. White and Avery, 1 in studying cultures from fermented milks, noted the same differences in acid production. They established two groups, one producing an acidity in milk of about 1 per cent, the other of 3 per cent, in 10 days. Such a division of the cultures studied could not be made. Rogers 2 states that a typical culture of the lactic bacilli from fermented milk produces nearly 3 per cent of acid in three days at 37 C. Von Freudenreich and Thoni 3 studied cultures from Em- mental cheese that produced from 0.2 to 1.26 per cent of acid in whey to which peptone had been added. Out of the several hundred titrations made of milk inoculated with cheese emulsions but 15 showed an acidity above 2 per cent. When raw milk is incubated at 37 C., the acidity will often, if not in the majority of cases, exceed 2 per cent and at times may reach 3.5 to 4 per cent. It would thus seem that certain lactic bacilli pres- ent in milk do not find favorable conditions for development in Cheddar cheese. FORMS OF LACTIC ACID PRODUCED. A number of investigators have determined the rotary power of the lactic acid formed by the lactic, bacilli. Bertrand and Weis- weiller* concluded that the acid was a mixture of the levo and dextro forms, with a slight predominance of the latter. 1 White, Benjamin, and Avery, Oswald T. Observations on certain lactic acid bacteria of the so-called Bulgaricus type. Centralblatt filr Bakteriologle, Parasltenkunde und Infektionskrankheiten, Abteilung 2, vol. 25, No. 5/9, pp. 161-178. Jena, Nov. 30, 1909. 2 Rogers, L. A. Fermented milks. United States Department of Agriculture, Bureau of Animal Industry, Twenty-sixth Annual Report (1909), pp. 133-161. Washington, 1911. Reprinted as Bureau of Animal Industry Circular 171, Washington, 1911. 3 Von Freudenreich, Edward, and ThSni, J. Sur 1'action de dlffe'rents ferments lac- tiques sur la maturation du fromage. Revue Ge'ngrale du Lait, vol 4, No. 8, pp. 169-181, Jan. 30; No. 9, pp. 200-209, Feb. 15; No. 10, pp. 225-232, Feb. 28; No. 11, pp. 247-259, Mar. 15. Lierre, 1905. * Bertrand, Gabriel, and Weisweiller, Gustav. Action du ferment bulgare sur le lalt. Annales de Institut Pasteur, vol. 20, No. 12, pp. 977-990. Paris, Dec. 25, 1906. 42 BACTEBIOLOGY OF CHEDDAE CHEESE. TABLE 20. Action of lactic bacilli in milk. Cul- ture No. Acidity pro- duced in milk. Rotation of acid. Time of cur- dling. Cul- ture No. Acidity pro- duced in milk. Rotation of acid. Time of cur- dling. 10 days. 45 days. 10 days. 45 days. 5 152 52 165.1 163 58A 58B 128 116 120 104 Pact. 0.324 .643 .68 .69 Per ct. 0.91 1.12 1.05 1.10 1.26 Days. 11 116.2 92 160 165 113 71 91 85 116.1 96 Per ct. 1.2 1.24 1.37 1.41 1.57 1.6 1.68 1.73 1.75 1.89 Per ct. 1.44 1.4 1.6 1.57 1.88 1.75 Inactive... Day*. Inactive 4 4 4 4 4 4 4 9 Dextro 9 Dextro .89 .86 .89 .81 1.02 1.17 1.38 1.21 1.2 1.36 Inactive+levo do 1.89 1.96 2.31 Dextro+ inactive Inactive 4 do 3 1.46 Dextro+ inactive 4 Heinemann and Hefferan 1 report the formation of an inactive acid, while White and Avery 2 found that the organisms that pro- duced a large amount of acid, 3 per cent, formed an inactive acid, whereas the cultures that formed acid slowly and in smaller amounts showed a levo-rotary power at the end of 24 hours, but in older cultures there were approximately equal amounts of levo and of inactive acids. The rotary power of 9 of the cultures studied was determined by Dr. J. M. Currie. Four of these produced inactive acid ; 1 produced inactive and levo acid, with a predominance of the inactive form; 2 produced a mixture of dextro and inactive acids, with a predomi- nance of the dextro-rotatory form; and the remaining 2 produced pure dextro acid. One culture isolated from milk, of which no other study was made, produced pure levo acid. With 11 cultures isolated from other sources than milk or cheese, only the pure dextro or pure inactive acids were found. This type of organism, then, can produce inactive acid, or active acid of either modification, or a mixture of the inactive acid with either of the active acids. All of these forms were found in cultures isolated from Cheddar cheese, with the exception of the pure levo acid. TYPE OF CURD PRODUCED. In most of the more active cultures the reduction of litmus and the return of color proceeded in exactly the same manner as in a litmus- milk culture of Bacterium lactis acidi. On the other hand, curdling took place with only a slight or partial reduction of the litmus in some cultures. 1 Heinemann, P. G., and Hefferan, Mary. A study of Bacillus Bulgarlcus. Journal of Infectious Diseases, vol 0, No. 3, pp. 304-318. Chicago, June 12, 1909. 2 White, Benjamin, and Avery, Oswald T. Observations on certain lactic acid bacteria of the so-called Bulgarlcus type. Centralblatt filr Bakterlologle, Parasltenkunde und Infektlonskrankhelten, Abtellung 2, vol. 25, No. 5/9, pp. 161-178. Jena, Nov. 30, 1909. DETAILED STUDY OF LACTIC BACILLI. 43 There was a slight gas formation in most of the cultures, evidence of which was found in the slight furrows or tiny holes which some- times appeared in the curd. These gas holes were, however, not always found in curds from the same culture. Although most of the organisms grew in lactose-agar shake cultures, no gas formation was noted in any case. FERMENTATION OF SUGARS. Sixteen of the cultures were tested as to their power to ferment sugars. The cultures were incubated eight days at 37 C. The test for growth and fermentation of the sugar was the reddening of litmus paper. The results are given in Table 21. All of the strains tested except one, No. 128, grew in glucose bouillon. Many of the strains made no growth or only a feeble growth (indicated by the sign ) in maltose and sucrose bouillon, although all except No. 128 grew in one or the other, or both, of these sugars. TABLE 21. Growth of lactic bacilli in culture media. Cul- ture No. Growth in sugar bouillon. Growth on lac- tose agar. Growth on or- dinary gelatin. Cul- ture No. Growth in sugar bouillon. Growth on lac- tose agar. Growth on or- dinary gelatin. Glu- cose. Mal- tose. Su- crose. Glu- cose. Mal- tose. Su- crose. 5 152 52 165.1 163 58A 58B fl28 116 120 104 + + + -H + + + 1 + + -H + + + + + + 116.2 92 . 160 165 113 71 91 85 116.1 96 + + + + + + + + + + + + + + + + + + + + + + + + + + ++++++ 1 + + + H- + + H- + + + + + + + + + + + + indicates feeble growth. FORM OF COLONIES. The growth upon lactose agar and ordinary gelatin was deter- mined in plate cultures on the same media that was used for the bacteriological analysis of cheese. Growth was obtained in lactose- agar plate cultures from every strain except No. 128. There was a good growth in most of the cultures, although with some only a few colonies developed. When the plates were incubated for several days the colonies continued to increase in size, sometimes attaining a diameter of 1 mm. The surface colonies are round and the deep colonies have the common flattened elliptical form. They can not be distinguished from the colonies of Bacterium lactis acidi. This is probably one reason why previous investigators have failed to find this type of organism in Cheddar cheese. Growth upon plates of ordinary gelatin was obtained from 8 of the cultures, This, however, was not constant for some strains 44 BACTEKIOLOGY OF CHEDDAR CHEESE. which developed from one inoculation failed to grow from another inoculation. The variable growth of this type of organism in plate cultures can account for some of the inconsistent results obtained in the quantitative analysis of cheese by means of lactose-agar and gela- tin plates. The readiness with which some of these cultures of lactic bacilli isolated from cheese grow upon ordinary media is rather surprising, in view of what has been stated by many authors concerning this property. One of the characteristics generally given for this group of organisms is their meager growth upon ordinary media. The five cultures studied by Von Freudenreich failed to grow on ordinary gelatin or on gelatin to which an extract of cheese had been added. Cohendy * and Lohnis 2 found these organisms hard to cultivate be- cause of their reluctance to grow upon the ordinary nutrient media. White and Avery state that when freshly isolated from their natural environment milk they do not develop on any of the usual nutrient media, even though sugar be present. Heineman and Hefteran, on the other hand, state that they grow well in milk, in media prepared from milk, or if glucose is added to the ordinary media. All of the cultures studied were isolated by means of lactose-agar plates with the exception of No. 128, which was obtained in pure culture in the dilution cultures in milk. THERMAL DEATH POINT. The resistance of the cultures to heat was determined on 3-day -old milk cultures which had been diluted and shaken with water and then drawn into sterile capillary tubes. The tubes were held for 30 seconds in water at various temperatures. Most of the cultures were killed at some temperature between 62 and 67 C. Two cultures were killed between 60 and 62.5 C., and two cultures were killed at about TO C. MORPHOLOGY. The bacilli are nonmotile and do not bear spores. In a single mi- croscopic field of a slide prepared from a pure culture the organisms may vary in length from 2.5 microns to 20 or 30 microns or more. Sometimes the filaments are very long. In one of the pure cultures a filament was found 75 microns in length. In one of the mixed cul- tures inoculated from cheese there was found a filament which was about 9GO microns in length, extending four times across the field of 1 Cohendy, Michel. Essals d'accHmation mlcroblenne perslstante dans la cavlte" Intes- tinale. Comptes Rendus Hebdomadaires des Stances de la Socl^te" de Blologle, vol. 60 (annee 58, tome 1), No. 7, pp. 364-366. Paris, Feb. 23, 1906. L<5hnl8, F. Versuch elner Grupplerung der Milchsaurebakterien. Centralblatt ftir Bakterlologle, Parasltenkunde und Infektlonskrankheiten, Abteilung 2, vol. 18, No. 4/6, pp. 97-149. Jena, Mar. 14, 1907. DETAILED STUDY OF LACTIC BACILLI. 45 the microscope. The filaments are apparently made up of a number of individual cells which for some reason do not show the cell divi- sions, for frequently chains of rods are found instead of filaments. Occasionally in a mixed, culture from a cheese inoculation filaments were found very much curled, or curled at one end, but attempts to isolate a culture which would regularly produce curled filaments always resulted in securing a culture with the usual morphology. No curled filaments were ever observed in pure cultures. The width of the cells in a pure culture seems to be fairly constant; in some cultures the individual cells are all slender, in other cultures they are all com- paratively thick. In different cultures the width varies from 0.4 to 1.3 microns. No branching forms have ever been observed. When stained with methylene blue the ends of the cells may take the stain much more deeply than the remainder of the cell, or the deeply stained spots may be scattered irregularly in the filament. Occasionally the deeply stained spots appear as tiny nodules on the rod. These characteristic staining properties are less frequently ex- hibited when stained by the Gram-Weigert method. In a weakened culture filaments are sometimes found with some of the cells Gram positive, some of them Gram negative, and other cells taking the stain partially. Although there is such a wide difference between the cultures in regard to the production of acidity in milk, the other characteristics do not differentiate a culture producing a low acidity from one which produces a high acidity. The morphology, however, seems to bear some relation to the acidity produced, those producing a high acidity being more slender than those producing a low acidity. CONDITIONS FOR GROWTH IN CHEESE. It has been pointed out that in their development in cheese the lactic bacilli follow the Bacterium lactis acidi group, and that the greater part of the growth must occur after the disappearance of the sugar. Since lactic acid is the principal by-product of the fermenta- tion of the sugar, it might seem probable that the lactic bacilli would make use of this as a source of carbon and of energy. Analyses of cheese show no decrease in lactic acid 1 at the period of development of the lactic bacilli. This indicates that this group of bacteria does not act on the lactic acid. The action of the pepsin of the rennet extract activated by the lactic acid results in the formation of peptones from the paracasein. The favorable effect of peptone on the lactic bacilli is shown in the 1 Suzuki, S. K. ; Hastings, E. G. ; and Hart, E B. The production of volatile fatty acids and esters In Cheddar cheese and their relation to the development of flavor. Wis- consin Agricultural Experiment Station, Research Bulletin 11. Madison, June, 1910. See p. 135. 46 BACTERIOLOGY OP CHEDDAK CHEESE. following experiment. Small flasks containing 100 c. c. of sterile milk were inoculated with cultures Nos. 5, 92, 91, and 96. To one flask of each set there had been added before sterilization 0.5 gram of peptone, and to another flask 1 gram. The control flask received no peptone. In Table 22 the acidity at 10 days and 45 days is given. The results show clearly that milk to which peptone has been added is a better medium than plain milk for the growth of this group of organisms. This was especially true in the case of culture No. 5, which usually curdled milk in about 11 days, but which curdled the milk to which peptone had been added in two days more rapidly than the most active cultures curdle plain milk. The time of curdling was shortened by the peptone 3 days in the case of culture No. 91, and 1 day in cultures Nos. 92 and 96. In every case the final acidity at 42 days was considerably greater than in plain milk, and in every case but one (culture No. 91, at 42 days) there was a greater development of acidity in the milk containing 1 gram than in the milk containing 0.5 gram of peptone. Incidentally, this experiment shows that the cessation of growth in milk when a certain percentage of acidity is reached, which is a fairly constant percentage for each particular culture, is not brought about by the antiseptic action of the acid, but by a lack of suitable nitrogenous food. TABLE 22. Percentage of acidity produced by lactic bacilli in milk to which peptone has been added. Culture No. Acidity in 10 days. Acidity in 42 days. Plain milk. Milk with 0.5 gram peptone. Milk with 1 gram peptone. Plain milk. Milk with 0.5 gram peptone. Milk with 1 gram peptone. 5 92 91 9C Per cent. 0.324 1.24 1.C8 1.58 Per cent. 1.19 1.4 1.9 1.87 Per cent. 1.32 1.58 2.1 1.96 Percent.. 0.855 1.4 Per cent. 1.35 1.56 2.42 2.13 Per cent. 2.34 1.77 2.29 2.25 1.93 The action of the enzyms present in the cheese curd brings about an increasing amount of soluble nitrogenous compounds so that after one month of ripening about 18 per cent of the total nitrogen is in the form of water-soluble compounds. 1 This action must render the curd a medium favorable to the growth of the lactic bacilli, and particularly to those strains similar to culture No. 5, which grow so slowly in milk, or fail to grow therein, as frequently happens. The dependence of this group of organisms upon enzyms from other sources is shown by growing them together with other types 1 Van Slyke, L. L., and Hart, E. B. Conditions affecting chemical changes In cheese- ripening. New York Agricultural Experiment Station, Bulletin 23G. Geneva, July, 1903. See p. 160. DETAILED STUDY OP LACTIC BACILLI. 47 of bacteria. Marshall and Farrand * have shown that when Bac- terium lactis acidi grows in milk together with certain other organ- isms there is an associative action which accelerates the production of acid and the proliferation of cells. This quickening action was found to occur in the case of 57 per cent of the cultures grown in association with Bacterium lactis acidi. A similar action, but more, pronounced, takes place when the lactic bacilli grow in milk together with certain other types of organisms. Suspensions in water were made of a 48-hour milk culture of the lactic bacillus No. 104 and also of 48-hour glucose bouillon cultures of two different strains of liquefying bacteria, which were isolated from poor cheese. This particular type of organism has not been found in good cheese. Small flasks of milk were inoculated with various dilutions of these suspensions, as given in Table 23. Since the results from the two experiments were similar, the figures for only one of them are given. TABLE 23. Associative action of lactic bacilli and a liquefying organism. Flask No. Inoculation with liquefler. Inoculation with lactic bacilli. Acidity in 2 days. Acidity in 3 days. Acidity in 7 days. I II III IV V VI VII 1 1,000 No inoculation. Per cent. 0.17 .18 .26 .40 .36 .42 .31 Per cent. 0.17 .59 .85 .96 .91 .99 .62 Per cent. 0.34 1.44 1.53 1.44 1.42 1 1,000 1:10,000,000 .. . 1 1000 1:100,000 1 1,000 1:1,000 1 100,000 1:1,000 1 10,000,000 1:1,OCO No inoculation 1:1,000 1.36 It will be seen that in 2 days the production of acidity was accel- erated in the cultures which received an inoculation of the liquefier. This was more pronounced in 3 days, but less pronounced in 7 days, when the flask which received no inoculation of the liquefier showed only a small amount of acidity less than the flasks inoculated with both cultures. Other experiments were made with cultures of coccus forms iso- lated from cheese. The liquefying action of these was not pro- nounced, yet they exerted a stimulating effect on the lactic bacilli. Reference to the coccus forms found in cheese will be made later. SOLVENT EFFECT ON MILK PROTEINS. Von Freudenreich was the first to demonstrate the fact that lactic bacilli exert a digestive effect. The same action 2 was shown for cul- 1 Marshall, Charles E., and Farrand, Bell. Bacterial associations In the souring of milk. Michigan Agricultural Experiment Station, Special Bulletin 42. East Lansing, Mar., 1908. 2 Hastings, E. G., Hammer, B. W., and Hoffman, C. Studies on the bacterial and leucocyte content of milk. Wisconsin Agricultural Experiment Station. Research Bulletin 6. Madison, June, 1909. See p. 202. 48 BACTERIOLOGY OP CHEDDAR CHEESE. tures isolated from milk and has also been demonstrated by more recent investigators. The solvent effect of 8 of the cultures studied has been determined by the anatyses of milk cultures after 3 months' incubation. .The results are given in Table 24. TABLE 24. The solvent effect of lactic iKicilli on milk proteins. Culture. Soluble N. in 100 c. c. of milk. Increase in soluble N inlOOc.c. of milk. Sterile milk Gram. 0.064 .072 .076 .080 .081 .090 .092 .096 .104 Or am. o.'oos .012 .016 .017 .026 .028 .032 .040 Percent 12.5 18.7 25.0 . 26.5 40.6 43.7 50.0 62.5 Ii6 113 152 120 116.1 116.2 71 52 COCCUS FORMS IN CHEDDAR CHEESE. Several investigators have mentioned coccus forms in relation to cheese ripening. Von Freudenreich and Thoni x found regularly a liquefying micrococcus in quite large numbers in fresh Emmental cheese. They made a number of experimental cheeses, using this type of organism alone as a starter or together with lactic starters. They concluded that when present in too great numbers the liquefy- ing micrococci produced bitterness, but that they disappeared quite rapidly in practical cheesemaking. Gorini 2 isolated a coccus with the property of peptonizing casein in an acid medium from Grana, Emmental, and Edam cheese. He found these forms not only in the fresh cheese, but in cheese several months old. He thought that they we're not all of the same type, and he related them to the normal flora of the udder. In a more recent publication 3 Gorini states that later investigations have confirmed his opinion that acid-producing peptonizing ferments are important in the ripening of cooked cheeses and that the fundamental flora of Grana and other cooked cheeses is composed of two types of bacteria; (1) lactic acid bacteria, and (2) acid-producing, peptonizing ferments. In the latter class he in- cludes bacillus forms with those properties and divers types of cocci. No reference to coccus forms having been found in considerable 1 Von Freudenreich, Edward, and ThSnl, J. Sur lea bacte"ries du lalt normal et lours rapports avec la maturation des fromages. Revue Ge'ne'rale du Lait, vol. 2, No. 11, pp. 241-247, Mar. 15 ; No. 12, pp. 271-280, Mar. 30, Lierre, 1903. * Gorini, C. Sur la presence de bactgries productrices d'acidite" et de pre"sure dans les fromages en maturation. Rue Ge'ne'rale du Lait, vol. 3, No. 22, pp. 505-510. Lierre, Aug. 31, 1004. 8 Gorini, C. Studi sulla fabbricazlone del fromaggl Grana, ecc. Italy-Ministero dl Agricoltura, Industriae Commercia. Bollettino. Anno 9, vol. 1, ser. C, No. 6, pp. 9-17. Rome, June, 1010. COCCUS FOKMS IN CHEDDAR CHEESE. 49 numbers has been made in the literature on Cheddar cheese. Hard- ing and Prucha * report the presence of acid-producing, liquefying coccus forms in 9 out of 10 cheeses studied. They state that these forms occurred sufficiently often to suggest that they- might play some part in the ripening changes, but that they made little headway in the cheese, and their number, as compared to the total germ con- tent of the cheese, was relatively insignificant. Coccus forms which produce a small amount of acidity were oc- casionally found to be the predominating type of organism in the cheeses studied, as shown by the milk cultures from high dilutions of the cheese. Unfortunately, with the dilution method of analysis this type can be differentiated only when it predominates, for if it occurs in a culture with the other cheese organisms it can not be distinguished with certainty from the Bacterium lactis acidi type in a microscopic preparation, and the titration of the milk gives no information, since its production of acidity is less than either the lactic bacilli or Bacterium lactis acidi. However, this type has been found to predominate at some time or other in 11 of the 13 cheeses examined by this method. In 4 of the 11 cheeses the maximum number of coccus forms found was 100 ; 000,000 per gram, in 4 other cheeses 1,000,000,000, and in the 3 remaining 10,000,000,000 per gram. The time at which they pre- dominated varied from the 14th to the 161st day. This would indi- cate that they increase early in the ripening period and maintain such numbers for a considerable period. The coccus forms have more or less of a liquefying action on gelatin ; a few show a decided action, some none at all, while the majority produce a minute de- pression in the gelatin around the colony. Their action is not com- parable with that of those organisms usually classed as liquefying bacteria. They produce small crystals in milk that enables one to differentiate them from Bacterium lactis acidi. The nature of these crystals has not yet been determined. The various cultures produced acidities in milk varying from 0.35 to 0.80 per cent. As has been previously mentioned, all of the colonies from a cir- cumscribed area on the lactose-agar plates prepared from the cheeses last examined were inoculated into milk. The formation of the crystals was used to distinguish the coccus forms. In some cheeses, especially those with a low germ content, the cocci have made up from 10 to 40 per cent of the bacteria as determined by lactose-agar plate cultures. In other cheeses they have not been detected in many of the examinations made, and when found were in minor numbers. 1 Harding, H. A., and Prucha, M. J. The bacterial flora of Cheddar cheese. New York Agricultural Experiment Station, Technical Bulletin 8. Geneva, Dec., 1908. See p. 184. 50 BACTERIOLOGY OF CHEDDAR CHEESE. It should be mentioned, however, that this method of detecting the various kinds of organisms in cheese is subject to considerable error, especially when certain forms are present in much smaller numbers than other forms, as is the case with the coccus forms as compared with the lactic bacteria of both groups in many of the cheeses examined. The error can be reduced by making subcultures of a greater number of colonies. By the extension of this method or by the use of some differentiating medium they may be shown to make up a considerable part of the flora of normal Cheddar cheese. CHROMOGENIC COCCI. The chromogenic cocci can be distinguished on the plate cultures when they are present in appreciable numbers. In a portion of the work the number of colonies of chromogens appearing on gelatin plates was determined. The results seemed to indicate a relative increase late in the ripening period. As has previously been pointed out, it is impossible to determine, in the case of any group of organ- isms for which no means of differentiation from other groups has been found : whether growth is taking place in cheese or not. It is characteristic of the chromogenic cocci that they persist and possibly grow under conditions which rapidly destroy less resistant types. They are present in butter, in which food material is limited, and in which the moisture represents a saturated solution of sodium chlorid. Efforts were made to determine their number in cheese by plating on gelatin containing 3 or 4 per cent of sodium chlorid. The results were quite satisfactory as far as the chromogenic types were concerned, but since many other coccus forms did not grow thereon its use was given up. In some cheeses very few chromogenic forms have been found, while in others of similar quality they have been present quite con- sistently in considerable numbers. In cheese which contained more than the usual amount of salt they made up a relatively greater pro- portion of the flora than in other cheese. LIQUEFYING ORGANISMS. Attention has been directed to those organisms that show a pro- nounced liquefying action on gelatin and casein. The results have been confirmatory of a statement made earlier that they can not be considered of importance in the ripening of Cheddar cheese since they are not consistently present in sufficient numbers to exert any effect. SEQUENCE IN DEVELOPMENT OF BACTERIAL GROUPS. 51 THE SEQUENCE IN DEVELOPMENT OF BACTERIAL GROUPS IN CHEDDAR CHEESE. In the first part of this bulletin it was stated that the normal ripening of each kind of cheeese is to be looked upon as a problem in the ecology of microorganisms, and that the only group of organ- isms which has been found by previous investigators in every Cheddar cheese in great numbers is the Bacterium, lactis acidi group. The work herein reported proves that another group of bacteria, the so-called B. bulgaricus group, develops after the first, and that it reaches approximately equal numbers. Each of these groups of organisms produces certain changes in the cheese mass, consuming the same class of substances and giving out the same by-products in every cheese which ripens normally. The second group takes up the work of decomposition at a certain stage of the ripening, and brings about its own peculiar changes which prepare the cheese mass for possibly still other types of organisms, and so on to the end of the ripening. If a certain sequence of groups of microorganisms is essential for the preparation of a certain product from raw material, and if the various members of the sequence find favorable conditions for growth in the raw material, the resulting product will depend on the first member of the normal sequence developing to the necessary degree before the second appears. If the first is overwhelmed by the addi- tion of great numbers of the second, the decomposition changes will not be normal. The presence of great numbers of organisms of the first member of the sequence will not cause a disturbance in the normal decomposition changes. Thus, in the manufacture of Ched- dar cheese, cultures of Bacterium lactis acidi are added. The other essential groups of microorganisms are present in the milk in small numbers, but as conditions become favorable they de- velop in the cheese, and a typical Cheddar cheese results. If heavy inoculations of lactic bacilli are made in milk which con- tains a small number of Bacterium lactis acidi the normal ecological balance will be destroyed, and the result will not be a normal product. Experiments have been made along this line by the use of milk which has been pasteurized in such a manner that the result has been the great reduction of all groups of bacteria therein, but the total destruc- tion of none. In its decomposition such milk is similar to raw milk, which contains but few bacteria. If cheese is prepared from such milk after the addition of a culture of Bacterium lactis acidi, it may ripen in a quite normal manner. The development of the flavor is usually slower than in a cheese made from raw milk. If to this same milk is added a culture of lactic bacilli instead of Bacterium lactis acidi, the ripening is not nearly so normal. The result is an increased rate of ripening and the production of an abnormal flavor. 52 BACTERIOLOGY OF CHEDDAE CHEESE. This work also indicates that it is often useless to attempt to establish the role of any organism in cheese ripening by the addition of cultures to the milk to be used, since thereby the natural equi- librium is destroyed, and the results obtained indicate that the addi- tion has injured the product, and hence the conclusion is drawn that the organism added is not only not essential, but even harmful, although the organism may be an essential factor in the decomposi- tion changes when developing in its normal sequence. The constant presence in large numbers is the only certain proof of the importance of an organism or group of organisms in the ripening of any cheese. SUMMARY. 1. From the same raw materials various kinds of cheese are pre- pared, which differ especially in flavor. The factors that determine whether a cheese to be prepared from a given mass of milk, rennet, and salt is to be of one kind or another are to be found in the methods of the cheese maker, who is able to vary in one way or another the composition of the cheese, with the result that conditions are estab- lished that favor or retard the growth of the groups of micro- organisms, which must be the determining factors between different kinds of cheese. k 2. The only group of bacteria found constantly in -great numbers in Cheddar cheese by previous investigators is the Bacterium lactis acidi group. The functions of this group in Cheddar cheese are, through their chief by-product, lactic acid (a) To favor the curdling of milk by rennet. (5) The bacteria of the milk are held in great part in the curd. Through the acid they influence the shrinking of the curd and expul- sion of the whey. (