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A. D. MELVIN, Chief of Bureau. 





Mycologist in Cheese Investigations, Dairy Division, Bureau of 
Animal Industry. 



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. 

A. D. Melvin, 

Chief of Bureau. 

Hon. James Wilson, 

Secretary of Agriculture. 




Introduction 5 

Camembert cheese 5 

Resume 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 ? 17 

Camembert Penicillium upon cheese 18 

Comparative studies of fungous digestion 18 

Flavors 21 

Temperature 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 Camembert 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 lactis 36 

Summary 38 

Camembert cheese 38 

Roquefort cheese 38 

Other varieties of cheese 39 

Bibliography 40 


Fig. 1. Camembert Penicillium (P. camemberti) 32 

2. Roquefort Penicillium (P. roqueforti) 35 

3. Oidium lactis 37 





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, 
Koquefort, 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 

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. 



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 1 "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 The figure references are to bibliography at end of bulletin. 




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, in 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. 


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 



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 

Petri-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 hyphse 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 



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. 


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. 


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 



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. 


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 Limburger, 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. 82—06 2 



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 


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 



action, he must depend entirely upon the culture for evidence of its 

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 

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. 


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 



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. 


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 Soger, 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 Eoquefort 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. 



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 not 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 tface of the phenolphthalein 
reaction was gone. The other species used did not give this reaction. 
There are forms including some species of Penicillium, Aspergillus 
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 



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 hyphae, 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 has thus been shown by many experiments that the Camembert 
Penicilhum 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 
Penicilhum 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 


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 

(1) The chemist describes the general course and extent of these 
processes 1 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. 



(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 
thfe 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 Koquefort 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. 


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 fufigi 
upon gelatin gives, however, some very intefesting 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 



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 almost 

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 Eoquefort 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. 


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.. 




' 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 for several months until it forms 
tough, irregular masses of felted hyphse, 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. 


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 hyphse 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. 82—06 3 



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. 


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 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 



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. 


Camembert P . . . 

Roquefort P 


Mucor 12 

Mueor 191 






Monilia Candida 

P. brevicaule 



A 8pergillus niger 




Red, then blue 







Red, then slowly 

Red, then blue 

Red, then blue 







Red to purple blue 

of gelatin. 

Partial. . . 

Incomplete. . 
Incomplete . . 
Incomplete . . 





Partial . 
Rapid . . 






Partial soft- 

Rate of 
of curd. 

Medium.. . 





to rapid. 

Slow to 







Rate of diges- 
tion of milk. 

Medium . 
Rapid . . . 

Rapid . 
Rapid . 


Medium . 
Rapid . . . 


Rather slow. 

Rapid . 

o° to 10° C. 

Grow, slow fruit- 


Poor growth. 


Slow growth. 
Slow fruiting. 


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. 



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 in 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 



cheeses were entirely worthless. This mold was originally isolated 
from a market Camembert cheese, where it was found mixed with 

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 the best type of Camembert cheese, with no objectionable 
flavors or colors. 


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- 



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 



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 ? 


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- 



lium 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. 


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 hyphse 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 



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. 


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 we'll 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- 



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. 


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 breaking 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. 



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. Recent 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 turgidity 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. 


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 workmen. Although the milk is 
the primary source of most infections, practical experiments have 
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 op two other Monilias. A species of Fusarium is 
distinctive of another brand, with Acrostalagmus cinnabarinus occa- 



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 than 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 


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 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 luxes 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 



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. 


Single studies have been made from the Italian Gorgonzola, Eng- 
lish Stilton, and Hungarian Brinse (Brindze or Brimse). 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 Ro'que- 



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. 


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 shows 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. 



Single studies have shown that Oidium 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. 


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. 




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 6n 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 
grows and fruits for about two weeks — 
in 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 (Maz6 9 ) . A cheese inoculated with this mold will become 

Fig. 1. — Camembert Penicilllum (P. ca- 
memberti). a, conidiophore showing a 
common type of branching and the pro- 
duction of basidia and conldia, highly 
magnified; 6, a common form showing 
much less branching; c, d, /, diagrams 
of large fructifications (X 80); g, i, j, 
germinating conidia. 



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. 


The following technical characterization of Penicillium camemherti 
(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 floc- 
cose, of loosely felted hypha? about 5 fi in diameter; reverse of colony yellowish white; 
conidiophores 300 to 800 /i in length, 3 to 4 //in diameter, septate, cells thin-walled, often 
collapsing in age, arising as branches of aerial hyphen; fructification sometimes 175 fi in 
length, but usually much less, consisting commonly of one main branch and one lateral 
sparingly branched to produce rather few basidia, which bear long, loosely divergent chains 
of conidia. Basidia 8 to 11 by 2.4 to 3 n; 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 f.i. Germ- 
tubes one to several. Cells of mycelium about 5 by 20 to 40 /z; 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 PeniciUium camemherti (nomen novum). This species is unquestionably the one 
referred to by Maz6 in his recent papers as P.. album Epstein. Professor Maz6 was kind 
enough to show me the cultures. But the name P. album was already used by Preuss some 
fifty years earlier for a species of Penicillium, hence by the rules of nomenclature should not 
be used again for a species whose identity with P. album Preuss is not claimed by Epstein. 
Upon this ground Lindau, in Rabenhorst's Kryptogamenflora, has changed the name of 
Epstein's fungus to P. epsteini 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 P. 
album Epstein in the list of possible synonymy only, because the name is accepted by Maz6 
for what I know to be this species. 




The spores of the Koquefort 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, which 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 hyphse or conidiophores, the vast majority of which 
arise as branches of submerged hyphse 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, bfft 
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 types of prepared cheese 



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. 

Fig. 2. — Roquefort Penicillium (P. roqueforti). a, part of conidiophore and of bas of fructification, 
highly magnified, showing the production of basidia on the sides as well as at the apex of the 
basidiophore; 6, 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 conidia and new conidia produced directly on 
the first hyphse. 


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 

<• 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 described as P. glaucum. As a prelimi- 
nary step, therefore, to the proper determination of the green species of Penicillium which 
have hitherto been collectively referred to as P. glaucum, this very distinct and easily rec- 
ognized form is named from its universal occurrence P. roqueforti. 



indefinite margin, which gets a white, fibrous, almost spider-web appearance from its alter- 
nation of submerged parts of hypha? 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 hyphse (comparatively few from aerial parts, but some), 200 to 300 fi 
septate. Fructification 90 to 120 /« or at times 160 n 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 )i by 2.5 /u 
with long divergent chains of conidia. Conidia bluish green, cylindrical to globose, smooth, 
rather firm-walled, 4 to 5 n 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. 


The mold (fig. 3) variously known as Oidium, or Oospera, laetis 
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, I), 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 every 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, 



smooth, cylindrical, 3.5 to 5 }x by 6 to 30 M, 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 defined 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, ft, dichotomous branching of growing hyphre; c, d, g, simple chains of oidia 
breaking through substratum at dotted line x-y, dotted portions submerged; e,f, chains of oidia 
from a branching outgrowth of a submerged cell; A, branching chain of oidia; k, I, m, n, o, p, s, 
types of germination of oidia under varying conditions; (, 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. 



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. 



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 


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 




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. 


(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 fiber die Reifung von Weichkasen. Arch. f. 

Hyg., Bd. 43, Hft. 1, pp. 1-20; Bd. 45, Hft. 4, pp. 354-376. Munich and Leipzig, 

(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, aer. 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 l'industrie fromagere. Ann. de l'Inst. Past., ann. 19, 

No. 6, pp. 378-403, June 25; No. 8, pp. 481-493, August 25. Paris, 1905. 

(10) Smith, Erwtn F.,and Swingle, Dean B. The dry rot of potatoes, due to Fusarium 

oxysporurn. 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 Biologie einiger in Molkereiproduction vorkommenden 
Schimmelpilzen. Milch-Zeitung, 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.