. S. DEPARTMENT OF AGRIC 
 
 BURLY U OF ANIMAL INDUSTRY. BULLETIN No. 82 
 
 A. D. MtLVIN, CHIEF OF BUREAU. 
 
 SOUTHERN BRANCH, 
 
 UNIVERSITY OF CALIFORNIA, 
 
 LISRA 
 
 GI IN CHEESE RIPENING 
 
 CAMEMBERT AND ROQUEFORT. 
 
 BY 
 
 CHARLES THOM, PH. D., 
 
 Mycologist in Cheese Investigations > Dairy Division, Bureau of 
 Annual Industry. 
 
 SJ9AIUfl 
 
 WASHINGTON: 
 
 r.oVI-RNMF.NT PRINTING ()l-l 
 
 1M
 
 IfLKEAL OF ANIMAL 
 
 A. D. MF.LVIX, IX V. s. 
 
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 /Clerk: K. B.JoNBS, LL. M., M. D. 
 
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 Librarian: BEATRICE C. OBEKLY. 
 
 LABORATORIES. 
 
 Biochemic Division: MARION DORSET, M. I)., chief. 
 
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 EXPERIMENT STATION. 
 
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 Packing Company. 
 
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 Agar Packing Company. 
 
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 Standish & Co. 
 
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 mond Brothers. 
 
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 & Co. 
 
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 "o. 
 
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 Pevey & Dexter Co.
 
 U. S. DEPARTMENT OF AGRICULTURE, 
 
 BUREAU OF ANIMAL INDUSTRY. BULLETIN No. 82. 
 
 A. D. MELVIN, CHIEF OF BUREAU. . 
 
 FUNGI IN CHEESE RIPENING 
 
 CAMEMBERT AND ROQUEFORT. 
 
 BY 
 
 CHARLES THOM, PH. D., 
 
 Mycologist in Cheese Investigations, Dairy Division, Bureau of 
 Animal Industry. 
 
 WASHINGTON: 
 
 GOVERNMENT PRINTING OFFICE. 
 1906.
 
 LETTER OF TRANSMITTAL 
 
 U. S. DEPARTMENT OF AGRICULTURE, 
 
 BUREAU OF ANIMAL INDUSTRY, 
 
 Washington, D. C., February 6, 1906. 
 
 SIR: I have the honor to transmit herewith the manuscript of an 
 article entitled "Fungi in Cheese Ripening: Camembert and Roque- 
 fort," by Charles Thorn, Ph. D., and to recommend its publication as 
 Bulletin No. 82 of the series of this Bureau. This is the second paper 
 dealing with the cooperative experiments in soft -cheese making 
 undertaken by the Dairy Division of this Bureau in conjunction with 
 the Storrs (Conn.) Agricultural Experiment Station, the first paper 
 having been published as Bulletin No. 71 of this Bureau. 
 
 These experiments have been carried on at the Storrs Station under 
 the general direction of Prof. L. A. Clinton, the station director, and 
 under the personal supervision of Dr. H. W. Conn, the station bac- 
 teriologist, in accordance with the plan outlined in the introduction 
 to Bulletin No. 71. 
 
 While there are many problems'yet to be investigated with refer- 
 ence to the manufacture in this country of soft cheeses of the best 
 European types, this article indicates that good headway is being 
 made in that direction, and it is believed that the information here 
 presented is of considerable scientific and economic value. 
 Respectfully, 
 
 A. D. MELVIN, 
 
 Chief of Bureau . 
 Hon. JAMES WILSON, 
 
 Secretary of Agriculture.
 
 CONTENTS. 
 
 Introduction i 5 
 
 Canietnhcrt cheese 5 
 
 Resumd of previous paper 5 
 
 Culture media and methods 6 
 
 Effect of a fungus upon a culture medium 8 
 
 Literature of cheese fungi 8 
 
 Biological analysis of a cheese 9 
 
 The flora of Camembert cheese .'. 10 
 
 Outline of the work 11 
 
 Relation of molds to acidity 12 
 
 The breaking down of casein , 14 
 
 Liquefaction of gelatin 15 
 
 Raulin 's fluid 16 
 
 Casein 16 
 
 Sterile milk and curd 17 
 
 Does the mycelium penetrate the cheese i 17 
 
 Camembert Penicillium upon cheese 18 
 
 Comparative studies of fungous digestion 18 
 
 Flavors 21 
 
 Temperature f 23 
 
 Humidity 24 
 
 Inoculating material 25 
 
 Inoculation with Penicillium : . . . 26 
 
 Vitality of spores 27 
 
 Contaminations 27 
 
 Roquefort cheese 28 
 
 Cheeses related to Roquefort 29 
 
 American Brie and Isigny 30 
 
 Molds referred to in this paper 31 
 
 The Camembcrt mold (Penicillium camemberti) 32 
 
 Technical characterization of the Camembert mold 33 
 
 The Roquefort mold (Penicillium roqueforti) 34 
 
 Technical characterization of the Roquefort mold 35 
 
 Oidium laclis 3(5 
 
 Summary 38 
 
 Camembert cheese 38 
 
 Roquefort cheese 38 
 
 Other varieties of .cheese 39 
 
 Bibliography 40 
 
 LLUSTRATIONS 
 
 Fio. 1. Camembert Penicillium (P. camemberti) 32 
 
 2. Roquefort Penicillium (P. roqueforti) 35 
 
 3. Oidium lactis 37 
 
 3
 
 FUNGI IN CHEESE RIPENING: CAMEMBERT AND 
 
 ROQUEFORT. 
 
 INTRODUCTION. 
 
 It has been shown in a previous bulletin that certain fungi are the 
 active agents indispensable to the ripening of Camembert cheese. 
 The general results and the data upon which they rest are there dis- 
 cussed, but the more special mycological studies, involving several 
 lines of work, remained to be brought out in greater detail. These fall 
 naturally under two heads: (1) The physiological studies of the func- 
 tions of particular species in the ripening processes of Camembert, 
 Roquefort, and certain related types of cheese; (2) the classification 
 and description of these and other forms occurring in dairy work. 
 This paper includes only the work done under the first head. The 
 description of the fungi occurring in dairy work is reserved for another 
 paper. 
 
 Aside from such obligations as are mentioned in the discussion of 
 special topics, the author wishes to acknowledge the assistance of Dr. 
 B. B. Turner, Prof. W. A. Stocking, Mr. A. W. Bosworth, and Mr. 
 T. W. Issajeff, members of the experiment station staff, in numerous 
 cases where the work of each presupposes the results of the other,, and 
 especially to acknowledge the constant assistance of the supervisor of 
 the investigation, Dr. H. W. Conn, with whom the cheese problems 
 have been fully discussed at every stage. 
 
 CAMEMBERT CHEESE. 
 RESUMES OF PREVIOUS PAPER. 
 
 The biological conditions and the physical changes encountered in 
 the production of a Camembert cheese from market milk may be 
 restated from our former bulletin 10 as a basis for defining the special 
 problems of the mycologist. 
 
 Milk as ordinarily received contains bacteria of many species and 
 the germinating spores of numerous fungi from the stable and from 
 the food of the cattle. When such milk is curdled for cheese making, 
 
 a Tin 1 figure references are to bibliography at end of bulletin.
 
 6 FUNGI IN CHEESE RIPENING. 
 
 representatives of all of these species are inclosed in the mass of coagu- 
 lum. Freshly made cheese from this curd, then, may contain any 
 species of mold or bacterium found in the locality which is capable of 
 living in milk or its products. The first step in the ripening of a Ca- 
 membert cheese is the production of lactic acid. The lactic bacteria 
 very soon increase their rate of multiplication so enormously as to be- 
 come entirely dominant. The acid produced by these forms soon 
 reaches a percentage sufficiently high to restrict the further growth 
 of nearly every other species of bacteria, and even to eliminate the 
 organisms themselves. In a time varying from a few hours to three 
 or four days, according to the proportional numbers of these antago- 
 nistic species at the start, further bacterial growth seems to be entirely 
 stopped. Bacterial development can not begin again until this acidity 
 is reduced below the critical point for the species involved, and even 
 then, since the acid is neutralized on the outside first, for most species 
 it begins at the surface and works slowly inward. The uncertainties 
 due to the presence of many species of bacteria in the milk are in this 
 way avoided by the natural, simple, and almost universally successful 
 process of souring. 
 
 The further ripening of a Camembert cheese is attended by a 
 gradual reduction of this acidity until the ripe cheese is usually alka- 
 line to litmus. At the same time the mold action in the mass of curd 
 produces chemical changes which in from three to five weeks reduce 
 the previously insoluble mass to a high percentage of solubility in 
 water. In the later stages of this breaking down compounds are 
 formed which give the characteristic odors and flavors to this type of 
 cheese. Associated with these chemical changes there is a progressive 
 physical change from the firm curd to a soft, buttery, or even 'semi- 
 liquid texture, characteristic of ripe cheese. The biological problems 
 then were, hi general, the determination of what organisms cause 
 
 (1) The changes in the acidity of the curd. 
 
 (2) The breaking down of the casein, with the associated changes in 
 the physical character of the cheese. 
 
 (3) The production of the flavors. 
 
 (4) The recognition and control of deleterious species. 
 
 CULTURE MEDIA AND METHODS. 
 
 The common dairy fungi grow readily upon any of the standard cul- 
 ture media. Among the media used have been peptone agar, whey 
 gelatin, sugar gelatin with or without the addition of litmus, milk 
 agar, gelatin and agar made with Raulin's fluid, potato agar, potato 
 plugs, and sterilized milk and curd. Special studies have involved 
 other preparations. The fact that these fungi grow readily upon all 
 the common media has led to the selection of two preparations for con- 
 stant use, and the careful study upon these of all species found. For
 
 CAMEMBERT AND ROQUEFORT. 7 
 
 this purpose the sugar gelatin, described by Conn 2 for the qualitative 
 bacteriological analysis of milk, and potato agar have been used. 
 
 The sugar-gelatin formula produces an accurately titrated medium 
 in which every effort is made to secure a uniform composition. 
 Although absolute uniformity in chemical and physical properties is 
 never obtained, the reaction of many species of fungi, when grown 
 upon successive lots of gelatin made after this formula, have been so 
 reliable as to commend its use for determining physiological charac- 
 ters. It seems clearly shown, therefore, that slight variations in the 
 composition of the medium do not produce great differences in the 
 species studied in this paper. In the discussion of the relation of a 
 mold to this gelatin it must be borne in mind that the same results 
 might not follow the use of any other formula. 
 
 The other medium, the potato agar, was selected because of its use 
 in many mycological laboratories. In this medium uniform compo- 
 sition can hardly be claimed. The following process has been used in 
 this work: The potatoes are carefully washed, pared, and sliced, then 
 slowly heated for about two hours in approximately two volumes of 
 water. At the close of the heating the water is allowed to boil. The 
 whole is then filtered through cloth, and commonly through cotton 
 also, water being added to make up the losses of evaporation and fil- 
 tering. To this is added 1 per cent of shredded agar. It is then 
 heated for from twenty to thirty minutes in the autoclave to 120 C. 
 or higher, when it may at once be put into tubes for use, or, if 
 cloudy, it may be very quickly filtered through absorbent cotton, 
 after which it should be quite clear. The uncertainties in the com- 
 position of this medium result from the differences in the potato ex- 
 tract itself and from the fact that the difficulties in filtering this 
 extract take out a varying amount, which is replaced with water. 
 Titration shows that this medium is nearly neutral (4-6 acid on Ful- 
 ler's scale) in cases tested to phenolphthalein ; consequently it is used 
 without neutralizing. Culture and study of the same species upon 
 successive lots of this medium show that these differences in compo- 
 sition have little if any effect upon the morphology of the species 
 studied. 
 
 P6tri-dish cultures have been used continually because they admit 
 of direct study under the microscope. Slanted test tubes were found 
 useful for stock cultures and for gross studies of physiological effects, 
 but they are of little value for comparative work. It is useless to 
 attempt to get a correct idea of the normal gross structure of these 
 molds from fluid mounts. The extremely delicate hyphffi are so tan- 
 gled in such preparations as to give but very little idea of their ordi- 
 nary appearance, while the chains of conidia break up immediately 
 when placed in any fluid. Such mounts are useful and necessary to 
 get at details of cell structure and cell relations, but in comparative
 
 8 FUNGI IN CHEESE RIPENING. 
 
 studies of species of such a genus as Penicillium their value is only that 
 of a useful accessory. The primary source of comparative data must 
 be direct study of the growing colony, undisturbed upon the culture 
 medium, with the best lenses that admit of such use. 
 
 This method of study recognizes that morphology is the basis of 
 fungus determination, but takes into consideration 
 
 (1) That morphology must not only include the minutest details 
 of cell structure and cell relations such as are undisturbed in fluid 
 mounts, but also the appearance and character of the colony. 
 
 (2) That the morphology of the colony i. e., the size of conidio- 
 phore and fructification, relation of these to substratum, appearance, 
 and relations of aerial and submerged mycelium is different upon 
 various substrata, but has been found to be characteristic for each 
 particular substratum. 
 
 (3) That a description of morphology to be of value must, there- 
 fore, specify the formula of the medium used and the conditions. 
 
 Dilution cultures have been necessary usually to obtain the colonies 
 pure, but the direct transfer of large numbers of spores upon a plati- 
 num needle to the surface of gelatine or agar plates which have been 
 allowed to cool has been found to give equally reliable results, and to 
 have many advantages for the study of species once obtained in pure 
 culture. This is often spoken of as inoculation of cold-poured plates. 
 Litmus solution may be used with either gelatin or agar, and gives 
 striking evidence of differences in species and the rate of their physio- 
 logical action. Bacterial contamination has been usually restrained 
 by the addition of from 2 to 4 drops of normal lactic acid to 8 or 
 10 c. c. of medium. 
 
 EFFECT OF A FUNGUS UPON A CULTURE MEDIUM. 
 
 In studying the relation of a fungus to a culture medium we find (1) 
 that the fungus absorbs food from the surrounding medium; (2) that 
 it may secrete or excrete substances into the medium which may 
 transform its chemical composition and its appearance. The amount 
 of food absorded by the fungus is small, and for our purposes may be 
 practically ignored, but the changes induced by indirect action 
 secretions from the mycelium are great and far-reaching. To this 
 latter group belong the changes in acidity, digestive effects, and fla- 
 vors produced by fungi. 
 
 LITERATURE OF CHEESE FUNGI. 
 
 A review of the literature at the outset showed that no work on the 
 fungous flora of the various types of soft cheese had been published in 
 English. Epstein, 3 at Prague, studied the ripening of Camembert and 
 Brie cheeses. He attributes the breaking down of the curd in French 
 Brie to the action of Penicillium album, but denies the participation
 
 CAMEMBERT AND ROQUEFORT. 9 
 
 of molds in the ripening of Camembert. Johan-Olsen, 4 in Sweden, has 
 published a brief review of the fungi related to the ripening of Gamme- 
 lost, barely mentioning work done upon Camembert. Constantin and 
 Ray, 5 in France, have described the appearance upon the cheese of the 
 species of Penicillium involved in the ripening of the French Brie. 
 Roger, 6 also in France, has attributed a single phase of Camembert 
 cheese ripening to the activity of Penicillium candidum, for which he 
 gives no description. Of these references, that of Epstein and that of 
 Constantin and Ray describe the mold found upon the French Brie 
 sufficiently clearly to aid in its recognition. A popular article, signed 
 Margaret, 7 in the Creamery Journal of October, 1904, gives in entirely 
 untechnical language a very satisfactory description of the appearance 
 upon cheese of the penicillium concerned in the ripening of Camem- 
 bert. The general insufficiency of the literature available made a 
 first-hand study of the types of cheese found in American markets the 
 only source from which definite information could be secured. 
 
 BIOLOGICAL ANALYSIS OF A CHEESE. 
 
 In the biological analysis of a market cheese it is carefully un- 
 wrapped to avoid contamination as far as possible. Series of dilution 
 cultures on neutral and acid media are made at once from each part 
 of its surface which shows any variation in appearance. In this way 
 all the surface molds and bacteria are secured in one set of plates. 
 Afterwards this surface is examined in detail, usually with a lens, the 
 appearance of the different areas being noted, and direct transfers 
 from each area made to cold agar or gelatin plates. The cheese is 
 then cut with a sterilized scalpel and cultures are made from various 
 portions of the interior. Usually the transfers were made from the 
 center and from the area just inside the rind. Any part showing spe- 
 cial appearances is reserved for a separate series of cultures. 
 
 Most of the brands of Camembert cheese found in our markets, as 
 well as some sent by Roger, have been examined in this way. For 
 comparison, similar studies have been made from several specimens of 
 Roquefort cheese bought in different markets, and from individual 
 specimens of Gorgonzola and Stilton. Single studies for molds have 
 been made from Lirnburger, Port du Salut, Brinse, and from several 
 brands of prepared cheese found in the market. From these cultures 
 all species of bacteria found have been isolated and handed over to the 
 bacteriologists. Each variety of mold occurring upon these cheeses 
 has been isolated and studied. It has been possible in this way to 
 show that a comparatively small number of species characteristically 
 occur upon soft cheese. Although this list may be greatly extended 
 by including forms which are occasionally found, it is rather surpris- 
 ing that a restricted group of species occurs with much regularity in 
 studies of cheese from so widely different countries. 
 21156 No. 8206 2
 
 10 FUNGI IN CHEESE RIPENING. 
 
 To study the origin and distribution of these molds several labora- 
 tories and cheese factories have been visited and cultures taken. Cor- 
 respondents in distant States have kindly sent cultures of molds 
 occurring in their work. Among those who have sent material are 
 Dr. C. E. Marshall, Agricultural College, Mich.; Mr. E. G. Hastings, 
 Madison, Wis. ; Prof. F. C. Harrison, Guelph, Ontario; Dr. H. A. 
 Harding, Geneva, N. Y., and Prof. P. H. Rolfs, Miami, Fla. Thus, in 
 addition to a large number of cultures from the dairy laboratories of 
 the stations at Storrs and at Middletown, we have accumulated from 
 various sources a considerable number of species representing the 
 characteristic molds occurring in dairy work, as well as many forms 
 collected in the field and from laboratories not associated with dairy 
 investigation. 
 
 THE FLORA OF CAMEMBERT CHEESE. 
 
 Although a considerable variety of molds appeared in cultures from 
 Camembert cheeses, a list of possibly twenty species would include 
 those which were often found. Among these there are perhaps six 
 species of Penicillium, two or three of Aspergillus, Oidium lactis, Clado- 
 sporium herbarum, one or two of Mucor, one or more of Fusarium, 
 Monilia Candida, and two species perhaps related to it, with the inci- 
 dental occurrence of Acrostalagmus cinnabarinus, a Cephalosporium, 
 various species of Alternaria, and Stysanus. Besides these, yeasts in 
 large numbers and considerable variety are found in many cases. 
 
 The comparison of the results of culture with comparative studies 
 of the surfaces of different brands of cheese showed that a single spe- 
 cies of Penicillium was present upon every Camembert cheese exam- 
 ined. In partially ripened cheeses this mold often covered the larger 
 part of the surface. We shall call this the "Camembert Penicillium" 
 or the " Camembert mold." This species develops a large and charac- 
 teristic growth of aerial mycelium in addition to a densely felted mass 
 of threads which penetrate the surface of the cheese for 1 or 2 mm. and 
 largely constitute the rind. In all except a few very old cheeses 
 which were almost covered with red slime of bacterial origin it was 
 readily seen to be the dominant species upon the surface. 
 
 Similarly, cultural data showed Oidium (Oospora) lactis to be abun- 
 dant upon every brand of Camembert. This mold is practically in- 
 distinguishable upon the surface by its characters, except under very 
 favorable conditions, and at best its recognition, even with a hand lens, 
 is not often certain. Mycelium of this fungus develops only in very 
 moist substrata, and is usually entirely submerged. Only part of its 
 chains of conidia even rise above the surface. In old and very ripe 
 cheese, when the rind is covered with yeasts and bacteria, it is often 
 difficult under the microscope to find the spores of Oidium. In such 
 cases, unless one is familiar with the peculiar smell associated with its
 
 CAMEMBERT AND EOQUEFORT. 11 
 
 action, he must depend entirely upon the culture for evidence of its 
 presence. 
 
 No other species of mold has been found upon every cheese exam- 
 ined, although no market cheese has failed to show contamination 
 with at least one or two of the other fungi listed above. In other 
 words, comparative biological examination of imported Camembert 
 cheeses established the fact that these two species of mold were pres- 
 ent upon them all, however abundantly they might be contaminated 
 with other forms. The examination of hundreds of cheeses in the city 
 markets has shown the presence of the same two molds upon all the 
 brands of Camembert offered for sale. Such analyses clearly estab- 
 lished the presence of these molds upon the ripe cheese, but gave no 
 information either as to whether they were necessary or what func- 
 tion, if any, they might have. Experiments were therefore devised 
 to test the relationship of these molds to the ripening processes out- 
 lined above. The constant occurrence of other molds upon the cheese 
 brings up the question, How and to what extent do the latter affect 
 the ripening process ? The experiments, therefore, have been made to 
 include as many species as possible. Where detailed chemical analy- 
 ses had to be made the work has necessarily been restricted to a few 
 forms. 
 
 For this purpose, in addition to the Camembert Penicillium and 
 Oidium lactis, the Penicillium found in Roquefort cheese ("der Edel- 
 pilz" of German authors) has been generally used. For convenience 
 it is called the " Roquefort Penicillium " or " Roquefort mold." One of 
 the Mucors, probably Mucor or Chlamydomucor racemosus, is so com- 
 monly found that it has often been included. A pure white mold 
 closely related to the Camembert Penicillium has given some inter- 
 esting contrasts. When reference is made to any of the numerous 
 undetermined green species of Penicillium, they will be indicated by 
 the letter or number under which they appear in the record book of 
 cultures, and under which the origin and subsequent cultural history 
 of all species studied has been kept. 
 
 OUTLINE OF THE WORK. 
 
 These studies involve two classes of data, first, those experiments 
 requiring quantitative analyses, which have been conducted in 
 cooperation with Mr. A. W. Bosworth, chemist to this investigation, 
 the results of which series of analyses will appear in his report; second, 
 experiments which show the physiological characters of the fungi by 
 physical changes in the appearance, texture, or color of the medium 
 used, or by the production of flavors. 
 
 The results may be anticipated here by noting that these two classes 
 of data did not prove mutually interdependent, but that analysis may 
 show in general the right stage of chemical changes called for in a ripe
 
 12 FUNGI IN CHEESE RIPENING. 
 
 cheese without the necessary texture and flavor; and, conversely, the 
 practically necessary texture and flavor may be obtained in a cheese 
 differing considerably in its chemical characteristics from the standard 
 market article. In our practical experiments we sought first for 
 proper appearance, texture, and flavor of the cheeses; then, without 
 disturbing these, endeavored so to control the processes of ripening as 
 to satisfy the standard of chemical composition established from the 
 study of market cheeses. 
 
 RELATION OF MOLDS TO ACIDITY. 
 
 The development of lactic acid has been shown to be of primary im- 
 portance in the control of deleterious bacteria. In our previous paper 
 it has also been seen that after doing its work this acidity gradually 
 disappears in the ripening process. The disappearance of the acid 
 has been attributed by Roger, 6 by Epstein, 8 and by Maze 9 to the activ- 
 ity of molds, and interpreted as preparing the way for the action of 
 peptonizing bacteria. This view of the relation of molds to cheese 
 ripening has been widely quoted as their only function in the process. 
 
 The acid exerts practically no selective action upon any of the molds 
 studied. Stoll has recently shown that species of Penicillium grow 
 readily in media containing a much higher percentage of acid than 
 ever occurs in cheese work. The use of acid in fungous cultures to re- 
 strain bacteria is practically universal, but the action of the different 
 species of mold upon the acid is very different. This is strikingly 
 shown by the introduction of a solution of litmus into the culture 
 media used. Litmus gelatin or litmus agar may be a deep blue if used 
 at 15 acid on Fuller's scale, as is usual for bacterial studies, or a clear 
 bright red if 2 to 4 drops of normal lactic or other acid are added to 
 10 c. c. of medium. No mold cultivated in this work has failed to 
 show some definite relation to acidity indicated by litmus reaction. 
 Some fungi, as soon as they develop visible colonies, begin to change 
 red (acid) media to blue (alkaline), and consistently maintain this 
 character. Many others, when grown in blue gelatin (designating by 
 blue gelatin 15 points acid to phenolphthalein=10 points alkaline to 
 litmus on Fuller's scale), begin by changing the blue to red. This 
 change may vary from the faintest tinge of red in only that part of the 
 medium directly in contact with the threads of the young colony to 
 deep red over large areas. Oidium lactis and Roquefort Penicillium 
 produce at times a very slight pink, which barely traces the outer limits 
 of the young colonies before the blue reaction begins to appear. At 
 other times the red, if appearing at all, has been so evanescent as to be 
 overlooked. It has been suggested that this slight appearance of 
 acidity might be due to the excretion of carbon dioxide in respiration, 
 which, although continuous, is afterwards masked by many times 
 larger changes in other substances.
 
 CAMEMBERT AND ROQUEFORT. 13 
 
 The Camembert Penicillium, and several of the very common 
 green species of Penicillium, when grown upon blue gelatin, at first 
 turn all the substratum in contact with the growing colonies to a 
 bright red. Some species produce areas of red beyond the limits of 
 the mycelium. These effects are most clearly seen by examining the 
 colony from the under side. Later a spot of blue appears in the cen- 
 ter of the colony below and gradually extends outward until com- 
 monly the entire mass of culture medium has become blue. This 
 often involves a change of reaction in agar or gelatin 2 to 3 cm. 
 beyond the colony. It is thus clear that there must be either the 
 secretion or the excretion by the mycelium into the medium of a 
 substance capable of changing this reaction or the absorption from 
 the medium of some substance, thus changing its reaction. The 
 exact nature of this change has riot been determined. Increase in the 
 percentage of acidity or of alkalinity retards the change of reaction. 
 In certain experiments phenolphthalein was introduced into red 
 litmus media and several species of Penicillium and Oidium lactis 
 were grown upon it. With the Camembert Penicillium the entire 
 mass of agar became blue in a few days, and remained so for nearly 
 three weeks. Then the characteristic pink color for the alkaline 
 reaction of phenolphthalein appeared on the under side of the 
 colony. This was tested by opening the colony with a platinum 
 needle and introducing a very small drop of normal acid, when the 
 pink area was changed first to blue and then to red. As the acid 
 diffused outward from the center the wave of blue traveled outward, 
 being replaced constantly by red until all trace of the phenolphthalein 
 reaction was gone. The other species used did not give this reaction. 
 There are forms including some species of Penicillium, Asperyillus 
 niger, Moniliafructigena, and others, which produce the acid reaction 
 in litmus media without any change to blue. Several species of Peni- 
 cillium rapidly produce the purplish color which is characteristic of 
 the turning point of litmus at which their further development 
 occurs. Apparently these bring acid or alkaline media to that point 
 without further change. It would appear, then, that the relations of 
 these molds to acidity, as indicated by the litmus reaction, is reason- 
 ably uniform. To determine whether the litmus reaction would be 
 reliable upon a medium closely allied to cheese, test tubes of sepa- 
 rated milk were prepared, blue litmus added, and the tubes sterilized. 
 Eleven species of Penicillium were inoculated into these tubes and 
 observations made every day. Of the eleven species, four, including 
 the Camembert Penicillium, produced a layer of red milk for a few 
 millimeters below the colonies, which later was changed back to blue. 
 The other species either intensified the blue or produced no change. 
 
 The suggestion has been made. that neutralization of acid is due 
 to the production of ammonia. A series of cultures were made in
 
 14 FUNGI IN CHEESE RIPENING. 
 
 cooperation with Mr. A. W. Bosworth to test the production of ammo- 
 nia compounds by mold action. The species used were the Roquefort 
 Penicillium, the Camembert Penicillium, Penicillium sp. (record No. 
 310), Oidium lactis, Oidium sp. (record B), and Aspergillus niger. 
 These were grown upon potato ager, to which litmus and lactic acid 
 were added. The Aspergillus culture remained bright red; all the 
 others became deep blue. Upon analysis the Aspergillus niger was 
 found to have produced the largest amount of ammonia. Study of 
 the figures showed that the ammonia alone was not sufficient to neu- 
 tralize the acid used in any case. It is clear, then, that the lactic acid 
 must have been neutralized by some other basic products of digestion 
 rather than by ammonia. If the acid were absorbed and dissociated 
 after absorption the area of blue would be restricted to the neighbor- 
 hood of the hyphas, or the diffusion of the acid for considerable dis- 
 tances would produce purple tones instead of sharply marked areas 
 of red and blue. The data seem to indicate that chemical decompo- 
 sition or neutralization of acid must be the action of some product 
 excreted by the fungus, probably an enzyme. 
 
 It ha$ thus been shown by many experiments that the Camembert 
 Penicillium and Oidium lactis are two of many species capable of reduc- 
 ing the acidity of the media upon which they grow. Many other species 
 of the same genus produce this effect more quickly than the Camembert 
 Penicillium and some act at about the same rate. The reduction of 
 the acidity of the cheese may clearly be attributed to these molds; 
 but the study of the relations of many other molds to acid indicates 
 that any of a large number of species might be equally or more useful 
 for the accomplishment of this step in cheese ripening. If, there- 
 fore, these particular molds are essential to Camembert cheese ripen- 
 ing, their special function must be sought in other steps of the 
 process. 
 
 THE BREAKING DOWN OF CASEIN. 
 
 The changes in firm sour curd which result in the production of the 
 soft, buttery, or semiliquid texture of the Camembert cheese present 
 some very complex problems. These may be grouped as (1) the 
 purely chemical questions, which involve qualitative and quantitative 
 analyses of the material at every stage; (2) The biological and 
 physical questions, which deal with the agents and conditions which 
 produce these results and with the gross appearances of the final 
 products, whose descriptions do not depend upon detailed chemical 
 analysis. 
 
 (1) The chemist describes the general course and extent of these 
 processes * as a change in which the insoluble or but slightly soluble 
 compounds of casein found in sour curd are rendered almost com- 
 pletely soluble in water. The details of the process and the data will 
 appear later in the report of the chemist.
 
 CAMEMBERT AND ROQUEFORT. 15 
 
 (2) To determine what relation the molds might have to this change 
 involved a great many cultures on different media. In some experi- 
 ments the number of species used was large and the results acquired 
 in that way a comparative value, but in the more complicated trials 
 the work was limited to those mentioned above. 
 
 It is practically impossible to produce a normal cheese in such a 
 way as to avoid contamination with bacteria or molds. It is difficult, 
 therefore, to study directly upon cheese the relations of organisms to 
 the steps of cheese ripening. Even were this possible, the complexity 
 of the changes encountered would make the interpretation of the phe- 
 nomena difficult. The activities of these molds have, therefore, been 
 studied in pure culture upon a series of media which would give infor- 
 mation as to steps of the process. While these cultural studies were 
 proceeding, many cheeses were made and inoculated with the Camem- 
 bert and Roquefort Penicillia. The measure of success obtained from 
 cheese inoculated with the Camembert Penicillium gave good, practi- 
 cal ground for its continued study. These detail studies may be dis- 
 cussed best separately. 
 
 LIQUEFACTION OF GELATIN. 
 
 The liquefaction of gelatin media has been much used as an index of 
 digestive activity. All species obtained have been grown upon neu- 
 tral and acid sugar gelatin and the effects noted carefully. 
 
 The difference in action between the molds important in this inves- 
 tigation are striking. The Mucor produces a slow but rather com- 
 plete liquefaction; Oidium lactis will gradually soften the gelatin so 
 that the center of the colony is liquefied; a pigment-producing Peni- 
 cillium (recorded simply as O) will liquefy all the gelatin in contact 
 with it so quickly that it becomes in a week a floating colony in a 
 watery pool twice its own diameter. Several other species of Peni- 
 cillium have the same effect. The Roquefort Penicillium softens gela- 
 tin somewhat, but never produces a watery liquefaction. The 
 Camembert Penicillium often produces a slight liquefaction under the 
 center of the colony, but never extends that liquid area to half the 
 total size of the colony. This seems to indicate that the Penicillium O 
 and its allies would produce a rapid digestion, that the Mucor would 
 be somewhat slower, that the Camembert mold might have some diges- 
 tive effect and the Roquefort mold very little, if any, value. The test 
 of the ability to liquefy the gelatin used gives, therefore, only indefi- 
 nite or negative results as to any advantageous relation of these par- 
 ticular species to cheese ripening. 
 
 Comparative study of numerous cultures of many species of fungi 
 upon gelatin gives, however, some very interesting suggestions. In 
 many species which liquefy litmus gelatin rapidly, the area of liquefac- 
 tion is surrounded by a blue (alkaline) band. For example, in one
 
 16 FUNGI IN CHEESE RIPENING. 
 
 experiment with Penicillium 392 at its most active period of growth a 
 colony 15 mm. in diameter was surrounded by a liquefied area 4 to 8 
 mm. wide. This area was in turn surrounded by a band of intense 
 blue shading gradually in a width of perhaps 10 mm. into unchanged 
 red litmus gelatin. The medium which had been liquefied was almosi 
 colorless. 
 
 Several suggestions may be drawn from many such observations. 
 The change in acidity of the medium, as has been noted above, may 
 be effected at a distance of 2 to 3 cm. from the colony. This change 
 of litmus reaction advances faster than the area of liquefaction of 
 the gelatin. The breadth of the area of liquefaction shows that the 
 action of the fungus is not a digestion by contact, but the secretion 
 into the medium of diffusible agents, that is, enzymes. In most of 
 these species liquefaction occurs only in areas having alkaline reac- 
 tion. No general relation between acidity and digestion is estab- 
 lished. The substantial uniformity of the results of repeated cultures 
 of the same species of fungi upon gelatin made after the formula used 
 established its usefulness as a test of the ability of an organism to per- 
 form this particular digestion. It will be shown later that the ability 
 to'liquefy this variety of gelatin is not to be regarded as a general test 
 of the ability of a species to produce active proteolytic enzymes. 
 
 RAULIN'S FLUID. 
 
 To test the ability of these species to grow in a medium entirely lack- 
 ing in proteid, Raulin's fluid was used as given by Smith and Swingle, 10 
 but modified by leaving out the potassium silicate and zinc sulphate. 
 Sterilized flasks of this solution were inoculated with Mucor, Oidium 
 lactis, Camembert Penicillium, and Roquefort Penicillium. All four 
 grew. The Oidium lactis and Mucor did not appear to develop in an 
 entirely normal way. Both species of Penicillium grew richly and 
 fruited normally. The culture of the Camembert mold, after growing 
 several weeks, was examined chemically and digestive experiments 
 conducted by Mr. Bosworth demonstrated the presence of a proteolytic 
 enzyme. In this way it was shown that this fungus could not only 
 construct proteid from inorganic compounds of nitrogen, but would 
 produce proteolytic enzymes in such a solution. Enzyme studies 
 were not made for the other species used in this experiment. 
 
 CASEIN. 
 
 For a medium at the opposite extreme, the chemists prepared pure 
 casein. This was weighed into 2-gram lots, moistened, sterilized in 
 the autoclave, and inoculated with five species of mold. All grew and 
 fruited luxuriantly. This experiment showed only that the species 
 used were able to break down casein and to grow normally upon the 
 products of this digestion without the addition of other nutrients.
 
 CAMEMBERT AND ROQUEFORT. 17 
 
 STERILE MILK AND CURD. 
 
 Sterilized milk and sterilized curd offer a substratum related to 
 cheese. Sterilized milk in quantities varying from 40 c. c. to 150 c. c. 
 in test tubes and Erlenmeyer flasks has often been used. Nearly all 
 species of Penicillium grow luxuriantly, forming a felted mass of 
 mycelium often 2 to 4 mm. in thickness upon the surface of the milk. 
 With the absorption of the milk in such cultures of the Camembert 
 and Roquefort species the mass of mycelium buckles and bends, 
 tubercles of mycelium arise on the under side of the mass and grow 
 downward, keeping the mold in connection with the fluid. In this 
 way a culture may continue to grow T for several months until it forms 
 tough, irregular masses of felted hyphae, filling the test tube for an 
 inch or more downward from the original surface of the milk. The 
 milk below the colony soon becomes transparent, giving reactions for 
 digestion, with a residue of curd at the bottom, which in the course of 
 time may be almost completely dissolved. With the Oidium lactis, 
 on the contrary, the colonies largely sink below the surface, so that the 
 milk may be quite well filled with mycelium upon which chains of 
 spores are only produced in quantity at or just below the surface. 
 Similar experiments with 100 grams of sterilized curd in flasks, inocu- 
 lated with the Camembert and Roquefort molds, 'have shown that 
 either species is able to change the chemical composition until the 
 derivatives of casein are amost completely water soluble. Such cul- 
 tures were plated to show their freedom from contamination by bac- 
 teria before analysis. The resulting products give the standard reac- 
 tions for digestion. These experiments show that either of these 
 molds is capable of producing digestive changes comparable in their 
 completeness, rapidity, and general nature to those shown by analysis 
 to have occurred in the ripening of Camembert cheeses. 
 
 DOES THE MYCELIUM PENETRATE THE CHEESE? 
 
 It must be noted carefully that this action of the Camembert mold 
 goes on without the complete penetration of the substratum by the 
 mycelium of the mold. That this is true is readily seen in milk cul- 
 tures, where the limits of the development of the mycelium are sharp 
 and clear. The same fact has been demonstrated for cheese by hun- 
 dreds of sections and careful cultural studies many times repeated. 
 The mycelium forms a dense mat upon the surface of the fluid or the 
 mass of curd, or the newly made cheese. It follows the irregularities 
 of the surface and is not found to enter well-packed curd to any extent. 
 It is very difficult to prove that hyphte of this mold actually appear in 
 curd of uniform texture below 1 or 2 mm. When found deeper, careful 
 search usually shows a cracking of the surface, so that the mycelium 
 may follow the opening already made. In no case of many hundreds 
 21156 No. 8206 3
 
 18 FUNGI IN CHEESE RIPENING. 
 
 of cheeses studied and experiments performed has the mold been 
 found to fruit in cavities not opening broadly upon the surface. This 
 is in marked contrast to the habit of the Penicillium instrumental in 
 the ripening of Roquefort cheese, which penetrates the channels of the 
 substratum and fruits in every cavity large enough to accommodate a 
 conidiophore. The Roquefort mold will make every cavity in a 
 cracker or piece of bread green with spores, while the Camembert 
 mold will fruit upon the surface of the bread or cracker with only 
 vegetative mycelium inside the bread. 
 
 Definite experiments to prove that this digestive power on the part 
 of the Penicillium is due to the secretion of one or more enzymes have 
 given characteristic reactions for digestion many times. Without 
 here discussing these chemical reactions, it has been shown that the 
 chemical action of the fungus is carried on at distances from the 
 mycelium which preclude direct action. The enzyme must therefore 
 be secreted and diffuse outward from the mycelium into the sub- 
 stratum. This explains why the Camembert cheese begins to ripen 
 just under the surface and the process progresses inward from all 
 sides until the cheese is entirely ripe. Before this process is complete 
 the center is simply sour curd. A good illustration of this action is 
 seen in cheeses which are ripened without turning. In such cases the 
 development of mold and enzyme on the lower surface is prevented, 
 and as a consequence ripening is delayed on that surface. 
 
 CAMEMBERT PENICILLIUM UPON CHEESE. 
 
 Many cheeses have been made and inoculated with this mold in con- 
 junction with pure cultures of lactic starter. Little difficulty is found 
 in this, since, if an abundance of spores are put upon the cheese when 
 made, this mold seems capable of taking and maintaining the lead of 
 all others. A cheese made in this way and ripened for from three to 
 four weeks will finally be rendered creamy, or, under some conditions, 
 waxy throughout, in color white within, in flavor almost neutral, 
 having no particular character good or bad and hence, to one fond 
 of Camembert cheese, tasteless and insipid. The important fea- 
 tures of this ripening process are, then, the completeness of its action 
 and the entire absence of any objectionable character in its flavor. 
 Biological analysis has shown that the center of such a ripened cheese 
 may be practically a pure culture of lactic organisms. The texture is, 
 therefore, obtainable by the use of the Penicillium alone. 
 
 COMPARATIVE STUDIES OF FUNGOUS DIGESTION. 
 
 Comparative tests of digestive action have been made for a number 
 of molds. The Roquefort Penicillium has been used in parallel cul- 
 tures with the Camembert Penicillium in many determinations. It
 
 CA1VJEMBERT AND ROQUEFORT. 
 
 19 
 
 has shown equal or greater ability to digest milk and curd. A typ- 
 ical example of several series consisted of the cultivation of 1 1 species 
 of Penicillium upon sterilized milk in large test tubes. Observation 
 of results after seven days showed digestion by 7 of these species. In 
 5 of them the amount of action exceeded that of the Camembert Peni- 
 cillium, and some of them appeared to digest milk at least twice as 
 rapidly as did that species in the first week. 
 
 In another series milk agar was made by dissolving 1 to 2 per cent 
 of the agar in water at 130 C. and pouring together equal quantities of 
 the hot agar and hot sterilized milk. If poured into Petri dishes at 
 once this medium was smooth and clear, but if acidified or sterilized 
 after mixing, flakes of precipitate appeared. The flaky precipitate in 
 the acidified cultures was found very useful as an indication of diges- 
 tion. In cultures upon the surface of such plates where digestive 
 action was strong the flakes would entirely disappear. Twenty- 
 three species of mold were tested upon milk agar in this way. Of 
 these, 8 produced a distinctly stronger digestion than the Camembert 
 Penicillium; 5 produced digestion approximately equaling that spe- 
 cies, and 10 produced less digestion. These cultures were mostly 
 made in duplicate, and both results in all but two cases agreed fully. 
 Oidium lactis produced comparatively little effect upon this medium. 
 
 TABLE I. -Reaction of certain species of molds. 
 
 Species. 
 
 Litmus. 
 
 Liquefaction 
 or gelatin. 
 
 Rate of 
 digestion 
 of curd. 
 
 Rate of diges- 
 tion of milk. 
 
 5 to 10 C. 
 
 Camembert P 
 
 Red, then blue 
 
 Partial 
 
 Medium 
 
 Medium 
 
 Grow, slow fruit- 
 
 Roquefort P 
 
 Blue 
 
 Softening 
 
 Rapid 
 
 Rapid 
 
 ing. 
 Characteristic 
 
 Oidium 
 
 Blue 
 
 Incomplete. . 
 
 Slow 
 
 Slow 
 
 growth. 
 Characteristic. 
 
 Mucor 12 
 
 Blue 
 
 Incomplete 
 
 Slow 
 
 
 Poor growth. 
 
 Mucor 191 
 
 Blue 
 
 Incomplete. . . 
 
 Slow. 
 
 
 
 O 
 
 Blue 
 
 Rapid 
 
 Rapid 
 
 Rapid 
 
 
 300 
 
 Blue . 
 
 Partial 
 
 Medium 
 
 Rapid 
 
 Retarded. 
 
 132. 
 
 Red, then slowly 
 
 Slight 
 
 to rapid. 
 Medium.. 
 
 
 
 310 
 
 blue. 
 Red, then blue. 
 
 Slight 
 
 Slow to 
 
 Slight 
 
 Slow growth. 
 
 68 
 
 Monilia Candida 
 
 Red, then blue 
 Blue 
 
 Partial 
 Rapid 
 
 medium. 
 Slow 
 Rapid . . 
 
 Medium 
 Rapid 
 
 Slow fruiting. 
 
 198. 
 P. brevicaule 
 
 Blue 
 
 Rapid 
 
 Rapid .. 
 
 Rapid 
 
 
 392 
 
 Blue 
 
 Rapid 
 
 Slight 
 
 Rather slow . . 
 
 
 240 
 
 Blue 
 
 Rapid 
 
 Rapid 
 
 
 
 Aspergillux niger 
 
 Red 
 
 Rapid . . 
 
 
 Slow 
 
 
 135 
 
 Blue 
 
 Rapid 
 
 
 Rapid 
 
 
 136 
 
 
 
 
 
 Characteristic. 
 
 
 
 ening. 
 
 
 
 
 Two species of Penicillia, 68 and 310, found closely associated upon 
 cheese with the Camembert Penicillium, produced little digestion. 
 The Roquefort Penicillium and several other molds often found upon 
 Camembert cheese appeared to act much more rapidly than the Ca- 
 membert mold itself.
 
 20 FUNGI IN CHEESE RIPENING. 
 
 All of these series of cultures under different conditions have many 
 times shown the same results and prove that the ability to digest curd 
 is common to many species of fungi. The species we have been led to 
 call the Camembert Penicillium possesses this character in common 
 with numerous other molds, many of which act more rapidly than 
 this one. 
 
 After the ability of several molds to digest curd is established, the 
 relation of any particular mold to cheese ripening must be determined 
 by the character of the products of that digestion and the flavors asso- 
 ciated with it. No pure culture upon a medium previously sterilized 
 by heat has given a taste resembling that of Camembert cheese. 
 Cheese made and kept in an atmosphere of chloroform, which pre- 
 vented mold and bacterial development, refused to ripen. Numerous 
 cheeses made and not inoculated with molds have uniformly failed to 
 develop the texture and flavor of Camembert cheese, although such 
 cheeses have usually become covered with molds of various species. 
 The type of cheese made and sold in this country as Isigny and Brie, 
 and sometimes labeled Camembert, which always shows Oidium lactis 
 associated with bacteria, differs entirely m appearance, texture, odor, 
 and flavor from Camembert; yet Oidium lactis is capable of neutraliz- 
 ing the acid of the cheese much more rapidly than the Camembert 
 Penicillium. Nevertheless the center of such a cheese remains acid 
 for a longer time than is required to ripen a Camembert cheese, while 
 the texture of Camembert is not produced. The necessity for the pres- 
 ence of another agent in this ripening is clearly established. 
 
 More than 2,000 cheeses have been made and ripened at this station 
 with the Camembert mold under varying conditions. Hundreds of 
 these cheeses have shown repeatedly that cheese so made will assume 
 in ripening the texture of the best imported article. The Camembert 
 Penicillium, therefore, is seen to be able to neutralize the acid of the 
 freshly made cheese and to produce the texture desired, but not the 
 flavor. It remains to determine whether other molds may not be 
 equally useful in this process. For comparison cheeses have been 
 made and inoculated with the Roquefort Penicillium with undeter- 
 mined species of Penicillium appearing on the record as O, 300, 310, 68, 
 132. Of these species one, 310, when cultivated upon every medium 
 used except the cheese duplicated the reactions of the Camembert 
 mold completely. Its morphology is scarcely distinguishable. It 
 differs only in that it remains pure white during its entire cycle of de- 
 velopment, while the Camembert species turns gray-green in age. The 
 close relationship apparent, together with a promising test, led to its 
 use upon over 100 cheeses. The breaking down resulting from its 
 action was widely different. These cheeses were drier, waxy, with a 
 mealy crumbling layer just under the rind. The physical character 
 of the results and the flavor produced were so different that the
 
 CAMEMBERT AND ROQUEFORT. 21 
 
 cheeses were entirely worthless. This mold was originally isolated 
 from a market Camembert cheese, where it was found mixed with 
 others. 
 
 The presence of the Roquefort Penicillium may be seen by the spots 
 of green it produces and may be detected by a sharp, bitter, perhaps 
 astringent, taste. The texture of the cheese produced is different, and 
 the flavor when it is present in any large amount is so strong as to be 
 very objectionable to many. When present in small amounts upon a 
 cheese it gives a certain sharpness or piquancy to it, such as has been 
 found often in certain brands of imported cheese, and is sought for by 
 some buyers. 
 
 The species marked O and 300 secrete a bright yellow pigment into 
 the cheese, which colors every area with which it comes in contact. A 
 cheese was inoculated with No. 300 and examined when 8 weeks old. 
 It had produced no trace of the texture of Camembert. The center of 
 the cheese remained practically sour curd, while the portion for per- 
 haps one-fourth of an inch under the colony was decomposed. 
 
 The species marked 68 has been obtained from cheese from widely 
 different sources. In cultures upon milk and milk agar it produced 
 little change. A cheese inoculated with it remained largely sour curd 
 for two months. The species marked 132 is a very common green 
 form, appearing in dairy and other cultures. It has given no satisfac- 
 tory results when grown upon cheese. In this way related species 
 found in cheese work have been tested in their effects upon cheese and 
 shown not to produce digestion comparable in physical character to 
 that demanded in a Camembert cheese and constantly obtained by 
 the use of the Camembert Penicillium. There seems to be no further 
 question that this species of Penicillium, among all the molds so far 
 studied, is the only agent capable of producing the characteristic 
 texture of thp best type of Camembert cheese, with no objectionable 
 flavors or colors. 
 
 FLAVORS. 
 
 All attempts to produce the flavor of Camembert cheese in pure cul- 
 tures upon milk and curd with particular organisms have failed. Here 
 again we have had to depend upon the use of cheeses so that direct, posi- 
 tive proofs have not been possible. The value of the indirect or cir- 
 cumstantial evidence offered must depend upon the completeness with 
 which all factors have been considered. It has been previously 
 shown that a cheese may be ripened to the texture of the best Camem- 
 bert by the action of lactic bacteria and the Camembert Penicillium, 
 but that it will lack flavor. A series of difficulties are met here. 
 The typical flavor does not begin to appear until ripening is well 
 along. This would indicate that the flavor-producing agent or agents 
 must act upon already partially ripened cheese to produce the par-
 
 22 FUNGI IN CHEESE RIPENING. 
 
 ticular end products which give this flavor. But coincident with this 
 change the acidity of the curd has become so far reduced that bac- 
 terial development may now occur on the surface at least, and as a 
 matter of observation few cheeses begin to show flavor until cultures 
 from their surface show swarms of bacteria of various species. It has 
 not been practically possible to change these conditions sufficiently to 
 make cheeses bearing only pure cultures upon the surface. The prob- 
 lem becomes, then, one of comparative study and the elimination of 
 the unnecessary factors one by one, rather than the direct produc- 
 tion of the flavor sought in a single conclusive experiment. 
 
 Some organism or organisms must be sought for to produce the 
 flavor. The appearance of the flavor of the imported article in cer- 
 tain experimental cheeses at this stage of the investigation led to 
 their immediate study. This showed that Oidium lactis was abundant 
 upon these cheeses and emphasized the fact that it had always ap- 
 peared in cultures from market cheeses. Oidium had been excluded 
 from many experiments in cheese making because it had been found to 
 be associated with odors that seemed undesirable, as well as because of 
 the conclusion of Epstein from his researches, that the presence of 
 Oidium is uniformly deleterious. The inoculation with spores of 
 Oidium of a half-ripened cheese entirely lacking flavor produced the 
 flavor distinctly in a single week, but since bacterial action seemed 
 always associated with this, further evidence was necessary. Roger 
 and Epstein have attributed the ripening of Camembert to the action 
 of certain bacteria without distinguishing that the production of the 
 texture of the cheese is accomplished by a different agent from the 
 production of flavor. In their descriptions ripened Camembert is 
 always referred to as slightly reddish in color, and the appearance of 
 this color is regarded as an indication of the progress of ripening. In 
 cheeses selected and forwarded by M. Roger this red color was very 
 prominent and the red layer was found to consist of myriads of bac- 
 teria of a few species. Cultures from these cheeses showed that 
 Oidium lactis was also present in abundance. Numerous tests have 
 been made with the bacteria found associated with the various 
 brands of Camembert cheese hitherto without producing the flavor in 
 any case independently of the molds. The comparative study of 
 many cheeses from the market and from our own cellars seems to 
 show that cheeses may have the typical Camembert flavor without 
 the development of any specific surface growth of bacteria. The 
 character of the bacterial growth upon the surface appears, therefore, 
 to be incidental or accidental, though its presence may be necessary 
 to exclude air, as maintained by Maze 9 in a recent paper. 
 
 Cheeses of good flavor have been produced here and also purchased 
 in the market, which indicate that particular surface appearances are 
 not essential to the typical flavor. Similarly the introduction into
 
 CAMEMBERT AND ROQUEFORT. 23 
 
 new cheeses of species of bacteria found in cultures from the interior 
 of good cheeses has produced either no effect whatever or disagreeable 
 flavors. Thus far, therefore, no species of bacterium has been found 
 capable of producing the Camembert flavor. Although the flavor 
 question is manifestly still unsettled, we may offer the following sum- 
 mary of the data at hand upon relation of molds to flavor in Camem- 
 bert cheese : 
 
 (1) Oidium lactis has been found in every brand of Camembert 
 cheese studied. 
 
 (2) It has never been found upon a ripened Camembert cheese which 
 lacked the flavor. 
 
 (3) The flavor has never been found in a cheese without the Oidium. 
 
 (4) Every other species with which the flavor seemed obtainable has 
 been eliminated from one or more experiments without loss of flavor. 
 
 (5) Bacteria or other molds do in many cases modify the flavor of 
 Camembert cheese, but do not seem to be able to produce it inde- 
 pendently of the mold. There thus arise characteristic secondary 
 flavors which are associated with the output of certain factories and 
 which command special markets. These varieties are usually more 
 highly flavored than what we have regarded as typical. 
 
 The essential relation of the Camembert Penicillium and Oidium 
 lactis to the production of Camembert cheese is, therefore, well estab- 
 lished. Several mycological questions remain: What are the opti- 
 mum conditions of temperature and moisture for the use of these 
 molds in cheese ripening? What are the most practicable means of 
 cultivating material for inoculation? How can the proper inocula- 
 tion with these molds be most effectually secured ? What other fungi 
 occur as contaminating species and how can they be controlled? 
 
 TEMPERATURE. 
 
 Since the higher temperatures of the ripening cellar lead more rap- 
 idly to the development of bacteria, it is necessary to determine the 
 lowest temperature which will permit mold growth and also enzyme 
 action. The different species respond quite differently to tempera- 
 ture. In one experiment eight species were inoculated into slanted 
 tubes of gelatin and put in a refrigerator where the temperature 
 varied from 5 to 10 C. Of these the Camembert Penicillium and 
 two nearly related species, Nos. 68 and 310, grew, but fruited very 
 slowly, showing an inhibiting effect. The Roquefort Penicillium 
 grew and fruited normally, as also did Oidium lactis. The species of 
 Mucor used developed very slowly and fruited only slightly. Two of 
 the very common green species of Penicillium grew richly. Oidium 
 lactis grows abundantly in the Brie and Isigny cellars visited. In 
 these the temperature was 50 to 55 F. (11 to 12 C.). Numerous 
 experiments in the ripening cellar show that the Camembert Penicil-
 
 24 FUNGI IN CHEESE RIPENING. 
 
 Hum does not grow its best in a room cooler than 60 F. (15 C.), and 
 that to obtain rapid development the room should be slightly warmer. 
 Until this mold is well established, therefore, it is distinctly an advan- 
 tage to grow it at a temperature of 65 to 70 F. Repeated experi- 
 ments have shown that lowering the temperature to 52 to 55 F. 
 checks the rate of ripening very materially. A difference of less than 
 10 degrees between two rooms will often make as much as two weeks' 
 difference in the ripening period of cheeses from the same lot in the 
 two rooms. A temperature as low as 54 to 55 F. ; as given in an 
 article in the Creamery Journal previously referred to, appears to pro- 
 long the ripening period without contributing any compensating 
 advantages. A half-ripened cheese was cut, the progress of the 
 softening of the curd was noted, and the cheese put in a refrigerator, 
 where it was held for four weeks at 48 F. It was then found to be 
 completely ripened and perhaps a little old in one place, but the 
 changes noted at the end of this period would have been produced 
 within a single week at 60 F. The cold-storage possibilities sug- 
 gested by this experiment will be further studied. 
 
 Some experiments were made to show the resistance of spores to 
 heat. The spores of the Camembert and Roquefort Penicillia were 
 inoculated into gelatin and placed in an incubator. Heating for an 
 hour and fifteen minutes at 56 C. killed all spores of the Camembert 
 species. Only a few spores of this mold grew after one hour at the 
 same temperature, while some spores of the Roquefort Penicillium 
 grew after two and one-half hours. 
 
 HUMIDITY. 
 
 The use of very moist cellars and caves in the ripening of this class 
 of cheeses is practically universal. The richest development of mold 
 is seen in rooms where the atmosphere is saturated or nearly so. This 
 appears to be exceptionally true for species like the Camembert Peni- 
 cillium, which is peculiarly a milk fungus, and in which there is a 
 large development of thin-walled aerial mycelium. So dependent is 
 the Camembert mold upon abundance of moisture that it has been 
 found difficult to secure a rich growth upon the surface of a cheese 
 which has been drained for two or three days before inoculation. Con- 
 trary to directions commonly given for ripening these cheeses, which 
 call for a particular degree of humidity, cheeses have been ripened 
 successfully in our cellars at the saturation point, as well as at various 
 degrees of humidity below that. A good illustration of a mold which 
 has adapted itself to changes of moisture is found in mold No. 198 
 Upon a fresh cheese in a moist room this mold forms a circular, 
 ringlike colony of floccose hyphae standing often 8 mm. high upon 
 the surface of the cheese. In a drier situation, or when the cheese 
 is nearly ripe and the rind becomes harder and dried, the same mold
 
 CAMEMBERT AND ROQUEFORT. 25 
 
 produces conidiophores which barely rise above the substratum, so 
 that the surface of the cheese is covered by a white, powdery layer 
 which is practically pure spores. The Mucors are so sensitive to mois- 
 ture that they scarcely develop upon the cheese, except sometimes 
 during the first few days, when the surfaces are very wet. They 
 appear to be unable to withstand the rate at which surface evapora- 
 tion proceeds in the ripening cellars. 
 
 INOCULATING MATERIAL. 
 
 The problem of propagation of the Camembert Penicillium for inocu- 
 lation purposes presents some difficulties. This species bears spores 
 only upon the surface of the culture medium used, in contrast to the 
 Roquefort species, which, when grown upon bread, develops spores in 
 every air space, as well as on the surface. To produce spores in quan- 
 tity, therefore, material must be capable of sterilization and must pre- 
 sent the largest possible amount of free surface in proportion to the 
 space occupied. For the preparation of such material, quart fruit jars 
 have been used. Various styles of crackers have been tried. Most of 
 these were not successful. The most suitable appears to be the hard, 
 dry " water cracker." The jar is filled with crackers and dry sterilized 
 at 140 to 160 C. for an hour or more, better twice on successive days. 
 The spores may be added directly, or first inoculated into about 100 
 c. c. of sterile water (acidified with 1.5 per cent of lactic acid usually) 
 and this poured into the jar and shaken until all the crackers are wet. 
 Various types of "milk cracker" soften to a pasty mass in this mois- 
 tening process. The best water crackers are not very satisfactory, 
 because the mycelium tends to transform bread or cracker into a soft, 
 gummy mass. The crackers become matted together until they pre- 
 sent much less actual surface than might be expected. The substi- 
 tutes tried have been excelsior, hay, and sheets of cardboard wetted 
 with milk or whey. Although some of these have advantages, they 
 were on the whole less satisfactory than the water crackers. So far, 
 therefore, on account of the very different habit of our mold, no mate- 
 rial has been found so easily prepared and so satisfactory as the 
 " Schimmelbrot " of the Roquefort cheese makers. 
 
 From the point of view of the use of pure cultures the Oidium lactis 
 is even more troublesome. This mold produces a large proportion, 
 and in some strains all of its spores as chains below the surface of the 
 substratum. For pure-culture work Petri-dish cultures have been the 
 only satisfactory vessels used. Its exceedingly rapid development, 
 however, makes possible the propagation of a culture from day to day 
 from the draining boards upon which the cheese is made. These 
 become heavily coated with a slimy mass of mycelium and spores upon 
 standing overnight. Direct transfers from them have been used with 
 apparently no serious trouble from contamination. In fact, so capa-
 
 26 FUNGI IN CHEESE RIPENING. 
 
 ble is the Oidium of self-propagation in dairy work that Epstein 
 declares it to be present in all dairy work. Although Roger in his 
 published statement does not mention it at all, it was found abun- 
 dantly upon the cheese forwarded by him to this station. We have 
 succeeded by careful work in making many cheeses entirely free from 
 Oidium, but with the ordinary treatment of dairy utensils it appears 
 constantly in factory practice. It is practically possible to rely to a 
 considerable extent upon the ability of the Oidium to propagate itself, 
 as has hitherto been done in the factories. 
 
 INOCULATION WITH PENICILLIUM. 
 
 With the Penicillium, however, numerous experiments indicate that 
 there is much advantage in early and effective inoculation from cul- 
 tures of known purity. Whether such inoculation must be always 
 made from specially grown laboratory cultures is questionable. In 
 factory practice, the making room and the ripening cellar are usually 
 adjacent. If precautions are taken always to have on hand some 
 cheeses bearing pure cultures (and the cheese maker must know his 
 mold so well that there will be no question about it), one or two such 
 cheeses will furnish enough inoculation material for much newly made 
 product. This would be indicated by the rough calculation that from 
 the abundance of the chains of fruit and the size of the spores (0.005 
 mm. in diameter) probably about enough spores are produced to 
 cover evenly the surface upon which they grow perhaps 25,000,000 
 to the square inch. Very successful inoculation in 75 pounds of milk 
 has commonly been secured by tapping a Petri-dish culture over the 
 vat, or by braking a piece of cracker about an inch square or less and 
 stirring it into the milk. 
 
 The most economical and successful method of inoculation so far 
 devised has been the use of a sprinkling jar or can. For this purpose 
 holes 1 mm. or less in diameter in the jar lid are demanded. A small 
 amount of water is put into the jar, a piece of cracker or cheese covered 
 with mold is broken into the water, the top is then screwed on, and 
 the jar thoroughly shaken. The water is then sprinkled upon the 
 newly made cheese at the time of first turning, so that both sides of 
 each cheese receive a few drops of water. Excellent results have 
 been obtained in this way with the smallest amount of inoculating 
 material and the least requirement of labor and skill. Such a jar 
 should be emptied and washed immediately after using. The mix- 
 ture is made fresh each time. Milk may be used instead of water, as 
 was first suggested and tried by Doctor Conn; but the water has been 
 found the more easily managed. The practical method for factory 
 use will probably vary with the conditions and skill of the maker.
 
 CAMEMBERT AND ROQUEFORT. 27 
 
 VITALITY OF SPORES. 
 
 Studies have been made upon the vitality of the spores of the spe- 
 cies used. This varies greatly in different species. In some of the 
 most common forms spores have been reputed to remain viable for 
 several years. Decent studies by Wehmer showed that five species 
 of Penicillium used in his experiment were entirely dead in labora- 
 tory cultures at the end of two and one-half years. Cultures of the 
 Camembert Penicillium grown upon potato in test tubes plugged with 
 cotton have refused entirely to germinate at the age of one year. 
 Other -cultures have seemed entirely dead inside of six months. In 
 fact, the spores of this mold are very thin walled and die very rapidly 
 when stored. Under such conditions they lose turbidity and become 
 crenulated or indented. Spores of Monilia Candida and several 
 others have grown after more than a year in laboratory cultures, but 
 their germination was much retarded. Oidium lactis seems to be 
 very easily killed by drying, as would be expected from a species with 
 such thin-walled spores. The Roquefort Penicillium under some con- 
 ditions is more resistant, but loses vitality quite rapidly. It is cer- 
 tain, therefore, that to give the best results material for inoculation 
 should be fresh and vigorous. Under ordinary circumstances it 
 would not be desirable to use material more than a few weeks old. 
 
 CONTAMINATIONS. 
 
 The number of molds found upon market Camembert cheese shows 
 the need of care in guarding against contamination of cultures. Ex- 
 traneous molds may come from the milk or from the utensils used or 
 from the clothes and hands of the w r orkmen. Although the milk^s 
 the primary source of most infections, practical experiments havW 
 shown that if the proper molds are put upon the cheese at the time of 
 making the troubles arising in this way may be minimized. In fact, 
 sufficient contamination -from this source directly to ruin a cheese is 
 very uncommon. 
 
 The very habit in some countries of washing or rinsing cheese- 
 making utensils in whey will account readily for the universal presence 
 of Oidium lactis and perhaps for many of the bacterial infections that 
 result in loss. But the source of the most trouble in a cheese cellar 
 is found to be the cheese maker himself. The cheeses are commonly 
 exposed upon curing boards and turned and examined in the hands. 
 In this way spores from molds or bacteria occurring accidentally as 
 single colonies upon single cheeses are distributed by thousands to 
 hundreds of cheeses. The product of a factory may almost be identi- 
 fied in the markets by the contaminations upon the surface of its 
 cheeses. Certain brands of the cheese always bear Monilia Candida 
 and commonly one or two other Monilias. A species of Fusarium is 
 distinctive of another brand, with Acrostalagmus cinnabarinus occa-
 
 28 FUNGI IN CHEESE KIPENING. 
 
 sionally present. After numerous experiences with all sorts of con- 
 tamination this trouble has been practically eliminated from our 
 experimental work by putting the fresh cheeses, as soon as they are 
 drained, salted, and comparatively dry upon the surface, into boxes 
 which are slightly larger than the cheeses, leaving air space and room 
 for mold to develop normally. In this way fingering is done away 
 with, the cheese is turned by turning the box, and examined by 
 removing the lid without touching the surface, so that a colony of 
 mold appearing upon one cheese is no longer distributed throughout 
 the cellar. 
 
 It is therefore possible to produce cheeses practically free from 
 molds other thaji those inoculated upon their surface. Although 
 such boxing upon a large scale may be practically undesirable on 
 account of expense, it remains certain that it may be useful in elimi- 
 nating certain troubles without so large a loss as would come from dis- 
 carding all infected cheeses, many of which would ripen satisfactorily 
 but for the danger of spreading obnoxious fungi over great numbers of 
 cheeses. 
 
 ROQUEFORT CHEESE. 
 
 The well-known Roquefort cheese is another highly flavored cheese 
 in which mold has long been known to play a part. In manufacture 
 this cheese approaches the hard type, but the ripened cheese bears a 
 closer relation to the soft v cheeses. Many complete descriptions give 
 the details of its making and curing. These need not be repeated 
 here. Roquefort is by description a goat's or sheep's milk cheese, 
 made in France principally, though cheese of nearly the same quality 
 is said to be made in other parts of Europe from mixed cow's and 
 sheep's milk or from cow's milk alone. 
 
 The great popularity of Roquefort cheese makes information as to 
 the biology of its ripening processes very desirable. To this end nu- 
 merous specimens of Roquefort have been purchased and analyzed. 
 The results of this work have been very much simpler than the stud- 
 ies of Camembert. The ordinary Roquefort cheese before it is sent to 
 the market is carefully cleaned and covered with tin foil. Its surface 
 would, therefore, tell very little. When cut it is seen to be traversed 
 by channels or ho es made by the prickle machine ( Stechmaschine) 
 and by cracks. Every air space is lined with green Penicillium, so 
 that the cut surface is said to be marbled with green. The texture of 
 the cheese is reasonably uniform, with every indication that ripening 
 is simultaneous throughout the cheese or at least approximately so. 
 Its texture is rather crumbling than waxy, with a tendency to dissolve 
 readily in the mouth. The taste is a characteristic sharp flavor, in 
 which a rather high salt content is noticeable. Its odor is strong, 
 cheesy rather than offensive in any way, except as pronounced
 
 CAMEMBERT AND ROQUEFORT. 29 
 
 putrefactive odors are sometimes developed in the rind. Cultures from 
 the surface often show various species of fungi. There is no regu- 
 larity about the surface, however, while uniformity of texture and ap- 
 pearance is universal on the inside. Cultures from the interior show 
 a remarkable uniformity. In many cheeses examined a pure culture 
 of a single species of Penicillium has been found. The extremely rare 
 appearance of any other mold in the cultures has been remarkab e. 
 Similarly the bacterial content is usually limited to typical lactic forms. 
 Sufficient analyses have been made to establish clearly that a first- 
 class Roquefort cheese should conta n only lact'c bacteria and the 
 Roquefort Penicillium. This Penicillium is often referred to by writ- 
 ers as P. glaucum and regarded as the common green species, but as it 
 has very characteristic morphological and physiological characters it 
 seems best to designate it as the Roquefort Penicillium, even though 
 it quite often occurs upon other substrata. 
 
 The cultures which have been conducted in connection with the 
 study of Camembert cheese have shown that the Roquefort Peni- 
 cillium is capable of digesting curd very completely. Here, as in 
 Camembert cheese, chemical analyses have shown that the derivatives 
 of casein become almost completely water soluble. Further pure- 
 culture experiments upon sterile curd have shown that this mold dur- 
 ing the process of digestion produces bitter flavors during the first few 
 weeks, but that its continued action changes these to typical flavors 
 of the Roquefort cheese. Here, then, we have a definite, positive re- 
 sult. It is thus shown that the Roquefort Penicillium, acting with the 
 lactic bacteria, is capable of ripening Roquefort cheese without the in- 
 troduction of other enzyme-producing or flavor-producing organisms. 
 The investigations of the chemical nature of these changes have barely 
 been touched upon at this time. In a recent experiment a cheese of 
 the Roquefort type was made of cow's milk inoculated with the 
 Roquefort Penicillium and kept in a room at a temperature of about 
 60 F. At the end of five weeks this cheese was found to have ac- 
 quired both the texture and the flavor of genuine Roquefort. There 
 seems to be no doubt that it will be possible to develop methods of 
 making and ripening that will produce the Roquefort type of cheese 
 successfully in the United States. Details of making and handling 
 will then be offered. 
 
 CHEESES BELATED TO ROQUEFORT. 
 
 Single studies have been made from the Italian Gorgonzola, Eng- 
 lish Stilton, and Hungarian Brinse (Brindze or Briinse). Gorgonzola 
 and Stilton are made from cow's milk. Brinse is described as made 
 from sheep's milk, mixed sometimes with goat's milk. These three 
 varieties of cheese are found marbled with green Penicillia in pure 
 cultures, which are unquestionably one or more strains of the Roque-
 
 30 FUNGI IN CHEESE RIPENING. 
 
 fort Penicillium. In the Gorgonzola and Stilton cheeses examined 
 lactic species were the only bacteria found. Comparison of the flavors 
 in these cheeses shows that the differences lie in the qualities of the 
 materials used in the making and the handling of the cheeses rather 
 than in the qualities attributable to ripening organisms. It is pecul- 
 iarly interesting to find the same species of mold in the interior of 
 ripened cheese in four countries so widely separated, where no efforts 
 at the use of pure cultures are known to be made. Experiments show 
 that in every locality so far studied there are many green species of 
 Penicillium. It is evident, then, that the food material or the condi- 
 tions, or both, presented by these types of cheese must exert a selective 
 influence upon the molds, which results in the dominance of the one 
 species so universally found. This species has been introduced into 
 experimental cheeses at this station. 
 
 AMERICAN BRIE AND ISIGNY. 
 
 Cheeses of the type referred to in our previous bulletin as the Ameri- 
 can Brie have been studied for comparison. This was a collective 
 term suggested to cover cheese sold under various labels as Brie, 
 Isigny, Wiener, Miniature, and others, designated commonly by the 
 retailer simply as Brie. The name "Brie" seems to be applied in the 
 French dairy literature to a cheese which differs from the Camembert 
 in the process of making, but ripened by the same fungi and approxi- 
 mately in the same way as Camembert. The domestic product so far 
 as examined is quite different, with the exception of the output of one 
 factory, which is conducted by imported cheese makers. The cheese 
 met in the eastern markets under these names show^s no trace of the 
 Camembert Penicillium. Numerous brands have been examined in 
 the market and many hundreds of cheeses have been seen in the cel- 
 lars of two of the largest cheese companies. Oidium lactis is univer- 
 sally present upon these cheeses, but its presence goes practically un- 
 noticed by the makers, since it produces neither color nor aerial 
 mycelium. All noticeable molds are washed or scraped from the sur- 
 face of the cheese. The washing produces exactly the best conditions 
 for the growth of bacteria and Oidium. This treatment results in a 
 cheese without a very definite fungous rind and with a strong flavor 
 and smell. 
 
 Cultures from this type of cheese indicate that there is an asso- 
 ciative action between the Oidium lactis and various species of bacte- 
 ria. Several species of Penicillium occur as contaminations in these 
 cellars and sometimes are found upon the cheeses in the market. 
 Every effort is made to eliminate mold action other than that of 
 Oidium lactis, which usually passes unrecognized. Cheeses of this 
 type usually bear rich growths of yeasts, giving a characteristic greasy 
 feeling to the surface. Exactly what parts these various organisms 
 play in the production of Brie is as yet undetermined.
 
 CAMEMBERT AND ROQUEFORT. 31 
 
 Single studies have shown that Oidiwn lactis is the dominant mold 
 upon the surface of some brands of Limburger, brick, and Port du 
 Salut. There is, then, good reason to believe that this fungus is asso- 
 ciated with nearly every type of highly flavored, ripened soft cheese 
 met in the American market. 
 
 MOLDS REFERRED TO IN THIS PAPER. 
 
 The Camembert and Roquefort molds belong to the hyphomycete 
 genus Penicillium, which has been characterized by one author 
 
 Hyphse broadly effused, creeping; conidiophore branched at the apex in an irregularly 
 verticillate manner, producing brush or broom-like forms; conidia in chains, hyaline or 
 bright colored, spherical or elliptical. 
 
 This genus of fungi contains a large number of very poorly de- 
 scribed forms which are everywhere abundant as the "green" or 
 "blue" mold of the household, the dairy, and the granary. They 
 form patches upon and just under the surface of the materials upon 
 which they grow. The patches are composed of delicate threads of 
 mold, which are matted together, forming more or less cottony sur- 
 faces, never rising more than a small fraction of an inch above the sub- 
 stratum. At first these areas are always white, but in most species 
 the ripening of a crop of spores is indicated by the change to a color 
 which is usually some shade of green, though this may later give 
 place to a brown. In a few species other colors appear. These spores 
 (conidia), or propagating bodies, are minute thin-walled cells averag- 
 ing possibly one five-thousandth of an inch in diameter, and so light 
 that they float freely in the air. A breath upon the surface of such a 
 colony carries away thousands of them, when if held in a proper posi- 
 tion they may commonly be seen to rise in a cloud. If the colony be 
 held to the nose and inhaled they give the sensation commonly called 
 the "smell of mold." They are, then, exceedingly light; they are pro- 
 duced in immense numbers; they are capable of growing in almost 
 every conceivable situation, upon anything which is not definitely and 
 strongly poisonous. Some of these spores are short lived, others cling 
 tenaciously to their power to germinate. Of the species, probably a 
 dozen common ones may be expected in any locality, perhaps more. 
 Our studies have shown that they affect very differently the sub- 
 stances upon which they grow. It is, then, clearly necessary that by 
 thorough study of their characters and habits we know the forms we 
 are to use, and just as important that we know how to get rid and stay 
 rid, if it be possible, of those we do not want. The discussion of the whole 
 group will be reserved for another paper. Here we may describe in 
 simple terms the two cheese fungi we find important, but it may as 
 well be acknowledged at the outset that, with the possible exception 
 of the Camembert species, safe recognition of species without technical 
 knowledge and cultural study is out of the question.
 
 32 
 
 FUNGI IN CHEESE RIPENING. 
 
 o. 
 
 THE CAMEMBERT MOLD (PENICILLIUM CAMEMBERTl). 
 
 The spores of the Camembert mold grow rather slowly in compari- 
 son with the other molds of the group. They first swell to nearly 
 double size, and then produce fine threads or hyphse at from one to 
 three points on their surface. Upon a cheese or in laboratory culture 
 the subsequent growth of these threads forms a colony large enough to 
 be visible to the naked eye, in ordinary room temperature, in about two 
 days. Usually in four or five days the colony will have become loosely 
 white, cottony, about one-half inch or less in diameter, and perhaps 
 
 standing one - twentieth of an inch 
 above the surrounding surface. At 
 or about this stage the center of this 
 colony begins to turn a shade of green- 
 ish gray, which is characteristic of 
 this species, though one or two other 
 forms produce colors closely resem- 
 bling this shade, and difficult to dis- 
 tinguish from it except to one very 
 familiar with the colors in question. 
 This is due to the presence of ripe 
 spores. Upon the cheese in the cellar 
 this color often does not appear in less 
 than a week or even ten days. Micro- 
 scopic examination shows that the 
 submerged threads of mycelium of 
 such a colony do not go deeper into the 
 solid media than one-sixteenth of an 
 inch, and that the superficial portion 
 of the mycelium spreads as fast, or 
 nearly so, as the part beneath the sur- 
 face of the substratum. This fungus 
 
 of large fructifications ( x 
 germinating conidia. 
 
 FIG. 1. Camembert Penicillium (P. ca- 
 memberti). a, conidiophore showing a 
 common type of branching and the pro- 
 duction of basidia and conidia, highly 
 magnified; 6, a common form showing 
 much less branching; c, rf, /, diagrams gTOWS and fruits for about two Weeks 
 
 7. *. j- m some cases this may be prolonged 
 to three weeks and at the end of 
 that period no further growth is to be expected from the primary 
 colonies, nor, if the medium is undisturbed, is there a secondary 
 growth from the germination of the spores produced by the first 
 colony. In case the rind of the cheese is broken so that a fresh 
 surface is presented, the spores will develop new colonies upon such 
 areas. A colony, then, produces a single crop of spores and dies, 
 under ordinary circumstances, and in undisturbed cultures there is 
 usually no second growth from the spores or from the old mycelium, 
 although the contrary has been claimed for this fungus by a recent 
 writer (Maze 9 ). A cheese inoculated with this mold will become
 
 CAMEMBERT AND ROQUEFORT. 33 
 
 covered with pure white cottony mycelium in about a week. The 
 color will then begin to show the gray -green shade characteristic 
 of the species, which spreads, until at the end of the second week the 
 entire surface, if left undisturbed, will be colored. 
 
 Persistent search has failed to find a single colony in America whose 
 presence can be attributed to anything but Camembert cheese im- 
 ported from Europe. The mold may then be regarded as a typical 
 dairy form which is not well adapted to cosmopolitan conditions and 
 to the struggle for existence on all sorts of media. In fact, in the 
 course of laboratory practice involving thousands of cultures, even in 
 the laboratories of this station, this mold rarely appears as a contam- 
 ination, although it has been cultivated in quantity and used in the 
 inoculation of large numbers of cheeses in the same building with the 
 bacteriological laboratory. Moreover, the spores are easily killed by 
 heat and retain their vitality for only a few weeks in ordinary cultures 
 allowed to dry in the air at room temperature. 
 
 TECHNICAL CHARACTERIZATION OF THE CAMEMBERT MOLD." 
 
 The following technical characterization of Penicillium camemberti 
 (fig. 1) may be offered, based upon studies made upon the sugar 
 gelatin and potato agar described in this paper: 
 
 Colonies effused, white, slowly changing to gray-green (glaucous); surface of colony floe- 
 cose, of loosely felted hyphae about 5 ft in diameter; reverse of colony yellowish white; 
 conidiophores 300 to 800 /< in length, 3 to 4 f.i in diameter, septate, cells thin-walled, often 
 collapsing in age, arising as branches of aerial hyphen; fructification sometimes 175 n in 
 length, but usually much less, consisting commonly of one main branch and one lateral 
 sparingly branched to produce rather few basidia, which l>ear long, loosely divergent chains 
 of conidia. Basidia 8 to 11 by 2.4 to 3 //; conidia at first cylindrical, then elliptical, and 
 finally globose when ripe, smooth, bluish-green by transmitted light, thin-walled and com- 
 monly guttulate, 4.5 to 5.5 /< in diameter, swelling in germination to 8 to 10 //. Germ- 
 tubes one to several. Cells of mycelium about 5 by 20 to 40 f.i ; liquefies sugar gelatin only 
 under the center of the colony. Changes blue litmus to red strongly at first, then after four 
 to six days begins to turn the red back to blue at the center and continues outward concen- 
 trically until all has become blue. Growing and fruiting period about two weeks. Fruits 
 only upon exposed surfaces of the substrata never produces spores in cavities not very 
 broadly open. Habitat, cheese. 
 
 a Penicillium camemberti (nomen novum). This species is unquestionably the one 
 referred to by Maze" in his recent papers as P. album Epstein. Professor Maze" was kind 
 enough to show me the cultures. But the name P. album was already used by Preuss some 
 fifty years earlier for a sjx'cies of Penicillium, hence by the rules of nomenclature should not 
 be used again for a species whose identity with /'. album Preuss is not claimed by Epstein. 
 Upon this ground Lindau, in Ralx>nhorst's Kryptogamcnflora, has changed the name of 
 Epstein's fungus to /'. fpxteini Lindau. and extracted from the article written by Epstein a 
 brief and totally insufficient diagnosis. A careful study of the physiological data given by 
 Epstein shows that they differ from the data so far found for this species so materially as to 
 lead to the probability that he was studying another form entirely. I therefore give /'. 
 album Epstein in the list of possible synonymy only, localise the name is accepted by 
 for what I know to be this species.
 
 84 FUNGI IN CHEESE RIPENING. 
 
 THE ROQUEFORT MOLD (PENICILLIUM ROQUEFORTl). 
 
 The spores of the Roquefort mold grow very rapidly, often produc- 
 ing new mycelium and ripe spores within thirty-six hours. The colo- 
 nies are white at the very first, but begin to become green at the cen- 
 ter within two days in a rapidly growing colony. Such a colony may 
 become a half inch in diameter in the first two days. The mycelium 
 is mostly submerged, but very close to the surface, and grows rapidly 
 outward from the starting point in a radial manner, w^hich is rendered 
 prominent by certain of the threads lying just under the surface for the 
 most part, but making loops into the air by rising just above the sub- 
 stratum for a little way, then reentering the medium again. This 
 gives a grayish, almost cobwebby (arachnoid) , appearance to the mar- 
 gin of the young colony. The rate of growth is not uniform in the cir- 
 cumference of such a colony, which makes the border of a colony 
 uneven instead of regularly circular, as most species appear. The 
 superficial portion of the Roquefort mold is almost entirely composed 
 of the fruiting hyphae or conidiophores, the vast majority of which 
 arise as branches of submerged hyphae and consequently stand sepa- 
 rately as short, unbranched threads of approximately equal length, 
 which gives the surface a velvety appearance. They are usually 0.2 
 or 0.3 mm. or less in length, say one seventy-fifth of an inch. Such a 
 colony spreads indefinitely in the substratum, so that the center will 
 be composed of ripe fruit, while the margin is still actively growing. 
 In laboratory culture, however, the development is so rapid that the 
 entire surface is covered within the first few days; then growth ceases. 
 The mycelium here, as in the Camembert mold, produces but a single 
 crop of spores, then dies. These spores are a bright green at first, but 
 in a short time become a dirty-brown color in dry culture. The spores 
 of this fungus are much more resistant than those of the Camembert 
 mold both to heat and to natural exposures. They will retain their 
 viability for months in old cultures under the ordinary conditions of 
 exposure in the laboratory. Upon a cheese this mold produces a 
 bright green area which extends rapidly. Its action can be detected 
 in a few days by the bitter taste of the curd near to the mycelium. A 
 similar taste is, however, produced at least in some measure by other 
 green forms, so that it is not diagnostic except as between this and the 
 Camembert species. A colony upon the surface of a cheese becomes 
 brown in two or three weeks, but colonies growing in the cavities 
 which are so characteristic of the center of this type of cheese retain 
 their bright green color for long periods. 
 
 This mold is not limited to dairy products, but is widely distributed. 
 It has been sent to the laboratory from the most distant correspond- 
 ents. It has been found in silage, and in laboratory cultures from 
 many substances. It has been found to be the green mold of Stilton, 
 Gorgonzola, and Brinse, as well as in certain tvpes of prepared cheese
 
 CAMEMBERT AND KOQUEFORT. 
 
 35 
 
 purchased in the market. Once in a laboratory it stays and seems 
 to get into everything. In other words, this is one of the cosmopoli- 
 tan and omnivorous species of the genus. One character seems to 
 differentiate this mold from most of the others that is, its power of 
 growing into and fruiting normally within narrow cavities, such as 
 appear in cheese. It appears that this character exerts a sort of 
 automatic (perhaps we may call it a truly "natural") selection which 
 eliminates all other species from the ripening processes of Roquefort 
 and related types of cheese. 
 
 n 
 
 PIG. 2. Roquefort Penicillium (P. roqueforti). a, part of conidiophore and of has of fructification, 
 highly magnified, showing the production of basidia on the sides as well as at the apex of the 
 basidiophore; b, c, other types of branching; d, young conidiophore just branching; e, f, basidia 
 and the formation of conidia, highly magnified; g, h,j, diagrams of types of fructification as seen 
 under low power (x 80); k, I, m, n, germination of c-onidia and new conidia produced directly on 
 the first hyphse. 
 
 TECHNICAL CHARACTERIZATION OF THE ROQUEFORT MOI.D.O 
 
 A technical characterization is offered of Penicillium roqueforti 
 (fig. 2), as follows: 
 
 Colonies quickly turning green, becoming a dirty brown in age, velvety strict, indetermi- 
 nately spreading by large main radiating, branching hyphse, giving a somewhat uneven or 
 
 a Penicillium roqueforti (nomen novum). In offering a new specific name for this well- 
 known fungus, the author is perfectly aware that the mold is often referred to in the litera- 
 ture as P . glaucum. A careful study of the literature fails to disclose a single description 
 which indicates that this is identical with the plant descril>ed as P. glaucum. As a prelimi- 
 nary step, therefore, to the proper determination of the green species of Penicillium which 
 have hitherto l>een collectively referred to as P. glaucum, this very distinct and easily rec- 
 ognized form is named from its universal (x-currence P. roqueforti.
 
 36 FUNGI IN CHEESE RIPENING. 
 
 indefinite margin, which gets a white, fibrous, almost spider-web appearance from its alter- 
 nation of submerged parts of hyphir with short prostrate aerial loops; reverse of colony yel- 
 lowish white. Conidiophores arising separately and in acropetal succession from the grow- 
 ing parts of submerged hypha* (comparatively few from aerial parts, but some), 200 to 300 /.i 
 septate. Fructification 90 to 120 ;< or at times 160 /< by 30 to 60 // at broadest place, 
 usually appearing double by the divergence of the lowest branch ; branchlets (basidiophores) 
 irregularly verticillate, bearing crowded verticils of appressed basidia 9 to 11 // by 2.5 // 
 with long divergent chains of conidia. Conidia bluish green, cylindrical to globose, smooth, 
 rather firm-walled, 4 to 5 // in diameter, germinating by a straight tube. Colonies do not 
 liquefy sugar gelatin, though they soften it somewhat. The fungus changes litmus from 
 red to blue very rapidly and strongly, almost from the beginning of growth. Fruiting period 
 short, but one crop of spores upon the mycelium. Cosmopolitan and omnivorous, or nearly 
 so. Characteristic of Roquefort and related types of cheese. 
 
 OIDIUM LACTIS. 
 
 The mold (fig. 3) variously known as Oidium, or Oospera, lactis 
 is another cosmopolitan organism. This fungus differs widely from 
 the species previously described. Inoculated into any suitable 
 medium it grows with enormous rapidity. A single spore (or oidium) 
 may give rise to several centimeters of mycelium and hundreds of 
 spores in twenty-four hours. It prefers very moist situations, since 
 almost the entire mycelium is developed below the surface of the sub- 
 stratum. It is therefore passed unnoticed many times or produces 
 changes which are attributed by the observer to bacteria. Descrip- 
 tion, therefore, must depend upon microscopic characters. The study 
 of the border of the young colony shows numerous vegetative hyphse 
 radiating outward. Each of these is found to divide dichotomously 
 (fig. 3, a, &), so that the border is a crowded series of forking branches. 
 In the older parts of the mycelium a branch may be produced at each 
 end of eA r ery cell, or several at each end, and these branch indefinitely. 
 The fruiting branches are mostly produced as outgrowths from the dis- 
 tal ends of the cells. These extend upward into the air or remain en- 
 tirely submerged in many cases. From the ends of these outgrowths 
 one to several rows of oblong or cylindrical cells begin to be pinched off. 
 If extending above the surface this gives rise to chains of delicate shim- 
 mering cells appearing as a powdery covering upon the surface, which 
 can be seen with a good lens to be arranged in chains. In some strains 
 of Oidium all of these chains (and some of the chains in all strains) of 
 spores remain submerged and germinate at once, so that they give 
 rise to unintelligible mats of hyphae. Oidium produces a very slight 
 acid reaction to litmus at first, then a strong and continued alkaline 
 reaction. It liquefies sugar gelatin under the colonies, but does not 
 extend the area of liquefaction beyond the edge of the colony. 
 Oidium always and everywhere tested has produced a strong and very 
 characteristic odor. Once familiar with this odor the worker may 
 recognize its presence by its spores or oidia, which are hyaline,
 
 CAMEMBERT AND ROQUEFORT. 
 
 37 
 
 smooth, cylindrical, 3.5 to 5 /* by 6 to 30 /*, varying with the condi- 
 tions and the substratum and perhaps at times exceeding these limits. 
 These swell variously and germinate in many ways, so that no germi- 
 nation characters are definite. Upon some media this mold may be 
 induced to produce a large growth of aerial mycelium, but the limits 
 here denned will include the variations to be found upon the usual 
 culture media. 
 
 Oidium lactis is described as universally present on milk and its 
 products. Epstein even suggests that experiments upon milk and 
 cheese can not be freed from its presence without sterilizing. The 
 
 FIG. 3. Oidium lactis. a, b, dichotomous branching of growing hyphac; c. d, (j, simple chains of oidia 
 breaking through substratum at dotted line y-ij, dotted portions submerged; e, f, chains of oidia 
 from a branching outgrowth of a submerged cell; h, branching chain of oidia; k, I, m, n, o, p, s, 
 types of germination of oidia under varying conditions; t, diagram of a portion of a colony show- 
 ing habit of Oidium lactis as seen in culture media. 
 
 same or almost indistinguishable forms are found upon decaying vege- 
 tables and fruits, which may give reason for the statement that the 
 odor produced by Oidium is that of rotten cabbage. There seems to 
 be good reason for saying that all these forms are but varieties or 
 strains of the same species. Comparison of several of them shows 
 that under uniform conditions the morphology of all these forms is 
 very nearly the same. This is largely true also of their physiological 
 effects. This mold has been much studied and numerous papers dis- 
 cuss its nature and physiological effects as well as its relationships.
 
 38 FUNGI IN CHEESE RIPENING. 
 
 It will be sufficient to describe here the fungus and to give figures to 
 assist in its recognition. Its relations to the problems of cheese ripen- 
 ing have already been indicated. 
 
 SUMMARY. 
 CAMEMBERT CHEESE. 
 
 The acidity of the curd resulting from the action of lactic organ- 
 isms reduces where it does not entirely eliminate the growth of objec- 
 tionable bacteria. 
 
 Many species of dairy fungi exert in the course of their development 
 the power of changing this reaction to alkaline. The Camembert 
 Penicillium and Oidium lactis possess this power, but not in greater 
 degree than many other species. 
 
 Many species of fungi possess the ability to change curd to a greater 
 or less extent. 
 
 The breaking down of curd by fungi is due in the cases studied to 
 the production of enzymes. 
 
 The texture, appearance, and flavor of curd acted upon by such 
 fungi are different for different species. 
 
 The Camembert Penicillium (P. camemberti) is the only species so 
 far studied with, which the particular appearance and texture sought 
 in the ripened Camembert can be produced from curd soured by 
 lactic bacteria without producing any objectionable flavor. 
 
 Oidium lactis is always found upon Camembert cheese and so closely 
 associated with the presence of the flavor as to indicate its agency in 
 flavor production, though only circumstantial proof of such function 
 has been possible thus far. The participation of bacteria in flavor 
 production is not excluded by these results. 
 
 Other species of fungi have been shown to produce variations in 
 this flavor such as have been often found in certain market cheeses. 
 In this way it is possible to look for the cause of differences in flavor 
 in contamination of the cultures upon the cheeses. This points 
 toward the use of pure cultures for inoculation, with the addition of 
 special organisms if certain variations from what we have regarded as 
 typical flavor are found to be of value in the market rather than 
 dependence upon accidental occurrence of the desired species in the 
 factory. 
 
 ROQUEFORT CHEESE. 
 
 In the ripening of Roquefort cheese the only organisms found neces- 
 sary are lactic bacteria and the Roquefort species of Penicillium. 
 
 The Roquefort Penicillium has been shown to possess the power to 
 reduce the acidity, to digest the curd, and to produce the typical 
 flavor.
 
 CAMEMBERT AND ROQUEFORT. 39 
 
 OTHER VARIETIES OF CHEESE. 
 
 The Roquefort species of Penicillium is found in the imported Stil- 
 ton, Gorgonzola, and Brinse, as well as in Roquefort cheese. 
 
 Oidium lactis alone of the forms studied has been found upon the 
 various brands of Limburger, Brie (American type), Isigny, and 
 related cheeses found in the market. Other species incidentally 
 occur, but not uniformly, and such occurrence is avoided as far as 
 possible by the makers. 
 
 BIBLIOGRAPHY. 
 
 (1) CONN, HERBERT WILLIAM; THOM, CHARLES; BOSWORTH, A. W.; STOCKING, W. A., Jr., 
 
 and ISSAJEFF, T. W. The Camembert type of soft cheese in the United States. 
 Bull. No. 71, U. S. Department of Agriculture, Bureau of Animal Industry. 
 Washington, 1905. Also published as Bull. No. 35 of the Storrs Agricultural 
 Experiment Station, Storrs, Conn., Apr., 1905. 
 
 (2) CONN, HERBERT WILLIAM. Bacteria in milk and its products. Illus. 306. pp. Phila- 
 
 delphia, Blakiston's Sons & Co., 1903. See p. 268. 
 
 (3) EPSTEIN, STANISLAUS. Untersuchungen iiber die Reifung von Weichkasen. Arch. f. 
 
 Hyg., Bd. 43, Hft. 1, pp. 1-20; Bd. 45, Hft. 4, pp. 354-376. Munich and Leipzig, 
 1902. 
 
 (4) JOHAN-OLSEN, OLAV. Die bei der Kasereifung wirksamen Pilze. Cent. f. Bakt., Abt. 
 
 2. Bd. 4, No. 5, pp. 162-169. Jena, March 5, 1898. 
 
 (5) CONSTANTIN, J., and RAY, J. Sur les champignons du fromage de Brie. Compt. rend. 
 
 Soc. de biol., Paris, ser. 10, t. 5, No. 16, pp. 504-507. Paris, May 13, 1898. 
 
 (6) ROGER, GEORGES. [Article in] Revue hebdomadaire, v. 7, p. 334. Paris. 
 
 (7) MARGARET, pseudonym. The practice of cheesemaking at home and abroad. The 
 
 Creamery Journal, v. 1, No. 11, pp. 313-315. London, July 20, 1905. 
 <8) EPSTEIN, STANISLAUS. See Citation 3, above, p. 373. 
 
 (9) MAZE, P. Les microbes dans 1'industrie fromagere. Ann. de 1'Inst. Past., ann. 19, 
 
 No. 6, pp. 378-403, June 25; No. 8, pp. 481-493, August 25. Paris, 1905. 
 
 (10) SMITH, ERWIN F., and SWINGLE, DEAN B. The dry rot of potatoes, due to Fusarium 
 
 oxysporum. Bull. No. 55, U. S. Department of Agriculture, Bureau of Plant 
 
 Industry. Washington, February 16, 1904. 
 LANG, M.. and FREUDENREICH, EDUARD VON. Uber Oidium lactis. Landwirthschaftl. 
 
 Jahrbuch der Schweiz, Bd. 7, pp. 229-237. Bern, 1893. 
 MARPMANN, G. Beitrage zur Kaseflora. Ztschr. f. angewandte Mikroskopie, Bd. 2, Hft. 
 
 3, pp. 68-79. Berlin, June, 1896. 
 
 TEICHERT, KURT. Beitrage zur Biologic einiger in Molkereiproduction vorkommenden 
 Schimmelpilzen. Milch-Xeitung, v. 32, No. 50, pp. 786-787. Bremen, December 
 12, 1903. 
 
 THOM, CHARLES. Some suggestions from the study of dairy fungi. Jrn. of Mycology, v. 2, 
 No. 77, pp. 117-124. Columbus, Ohio, May, 1905. 
 
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