IBIOUOGIGAL STODIEiS 
OF THE 
Fungi of the Humag Hjoutlj 
PROF. DR. W. D. MILLER, 
BERLIN, GERMANY. 
RtTOUOtO VRQW jwt PBACTVYORER 01 Mm MVO Iwi, \SSb 
NEWYORK DENTAL JOURNAL ASSOCIATION, 
No. 35 West 46th St., New York, and No. 11 West Chippewa St., Buffalo, N. Y. BIOLOGICAL STUDIES ON THE FUNGI OF THE 
HUMAN MOUTH.* 
PE OF. DR. W. D. MILLER, BERLIN. 
In order to be able to determine upon the proper course to be 
taken in the attempt to remove or check the progress of any dis- 
ease, it is necessary that our ideas of the cause and course of that 
affection be established upon the most certain, exact and scientific 
data which we are capable of attaining. Unfortunately for the den- 
tal profession, the attempt to furnish a scientific solution of the 
problems of dental caries has, until recently, been confined to a 
very few, and even now a majority of the investigators in dental 
pathology are content to restrict their observations to the clinical 
aspect of the question, a course which could never produce a satis- 
factory solution; while others even openly advocate a speculative 
course, and do not hesitate to ascribe to every new factor discov- 
ered in nature, a role in the production of caries of the teeth. 
Consequently we have had presented to us, in turn, worms, acids, 
inflammation, electricity, infusoria, bacteria, putrefaction, toxic 
agents, etc., etc., as causes or conditions of caries dentium, some of 
* German mycologists use the term ‘ Pilz ’ indiscriminately to designate either Schizomy- 
cetes, Blastomycetes, Hyphomycetes or Myxomycetes. When it is desirable to refer to any 
one of these groups in particular, they use the prefixes Spalt, Spross, Schimmel or Faden, 
and Schleim, giving Spaltpilz, Sprosspilz, Schimmel- or Fadenpilz and Schleimpilz. Follow- 
ing their example, I have in previous papers used the term fungus for all of the four groups 
of mycetes mentioned above, and shall also use the term in this paper, in which only Schizomy- 
cetes are treated of. 2 
these theories containing some truth and some a surprising amount 
of absurdity. In the last two or three years, however, a great ad- 
vance has been made in the methods of study, and a number of 
important points have been firmly established. 
1. The observation of Leber and Eottenstein that micro-organ- 
isms are constantly present in decaying dentine, has been confirmed. 
(Weil, Milles, Underwood, Miller.) 
2. The softening of dentine in caries has been shown to be 
chemically identical with that produced by certain weak organic 
acids. (Miller, Jeserich, Bennefeld.) 
3. It has been established that various organisms found in the 
human mouth, produce the decalcifying acid by first converting non- 
ferraentable sugars into fermentable varieties, and secondly, by 
splitting fermentable sugars into lactic acid. (Miller, Hueppe.) 
4. The same organisms have been found capable of dissolving 
decalcified dentine, while they have no apparent effect, even after 
two or three years, on sound dentine. (Miller.) 
5. Caries of dentine, chemically and morphologically identical 
with natural caries, has been produced outside of the mouth. 
(Miller.) 
6. It has been furthermore shown that certain of the organisms 
of the human mouth are capable of developing under exclusion of 
air, thus making it possible for them to propagate within the sub- 
stance of the dentine. (Miller, Hueppe.) 
I propose to describe, in this and the following article, a series of 
experiments made for the purpose of obtaining more definite infor- 
mation respecting the number and morphology of the fungi of the 
human mouth, and their physiology, as far as is necessary to an 
understanding of the part which they may perform in the produc- 
tion of caries of the human teeth. 
At the meeting of the American Dental Association, at Saratoga, 
a number of tubes containing pure cultures of fungi were passed 
around; with regard to these a reporter remarked that “they were 
evidently beyond the information of the majority.” It is not very 
flattering to American dentistry if its representative Association 
allows a question of so great importance to remain beyond its com- 
prehension, nor is there any excuse for such a condition of things 
now, so widespread have the methods of pure culture become. I 3 
rather incline to the opinion that the reporter misinterpreted the 
apathy of the members of the Society. I shall, at any rate, here de- 
scribe in a few words the methods now universally employed in 
isolating any given fungus, and then, more in detail, give the means 
which I have used to ascertain the physiological characteristics of 
the different fungi when obtained in pure culture. 
We will start with a solution densely impregnated with micro- 
organisms, and a number of tubes of culture gelatine, perfectly 
sterilized. The gelatine being melted, we add to the first tube one 
bead (on a loop of sterilized platinum wire) of the solution. This 
is called the first dilution. From this tube we add two or three 
beads to a second tube {second dilution), and from the second five or 
six beads to a third tube {third dilution.) The gelatine is then 
poured upon horizontally placed, sterilized, cold glass plates. It 
congeals in a few seconds, and the three plates are placed in a pile 
(on glass benches) in a moist cell. The plates are examined after 
twenty-four to thirty-six hours, under a magnification of one hun- 
dred diameters. 
By this means the fungi are so separated that on the third plate 
there will generally not be more than two to ten; (on the second there 
may be one hundred or two hundred, while on the first, of course, 
there are very many more.) As each micro-organism develops, being 
fixed in the gelatine, we will have at that point a pure culture of 
that particular kind. At another point we obtain a colony of a 
second kind, and so on. In general, colonies of different fungi may 
be distinguished with the greatest ease, by their microscopic appear- 
ance. With a sterilized platinum wire, bent at right angles at the 
end, we now pick up a number of the colonies of each kind, under 
the microscope (one hundred diameters), and transfer them directly 
to tubes of culture gelatine, only one colony to each tube. We 
have then (except in case of a possible accidental air-infection) 
pure cultures. Some experience is necessary to enable one to pick 
up the colonies under the microscope. Beginners should not at- 
tempt it with plates where more than one colony is in the field at 
once. 
The method described in this journal (page 340, 1884), may also 
sometimes be used to great advantage. For fungi which do not 
grow on gelatine, Agar-Agar, or congealed blood serum, should be 
used. The former, one to one and a half per cent, has a higher 4 
melting point than gelatine, ten per cent, and remains solid at the 
temperature of the human blood. When it is used for plate-cul- 
tures, it must be melted in hot water, and the infection made at a 
temperature of 40° to 42° C. Below this temperature it becomes 
solid, and cannot be poured; above it the germs would be liable to 
suffer. In other respects the Agar-Agar media are treated as the 
gelatine. Congealed blood serum cannot, of course, be poured 
upon plates. It is prepared in test tubes so inclined as to give the 
greatest possible surface, and a minimum quantity of the substance 
containing the fungus or fungi spread over the surface. Having 
obtained a pure culture of any fungus, the points to be determined 
regarding it are the following : 
1. Its morphology; (bacillus, spirillum, micrococcus.) 
2. Is it moveable ? does it produce spores? 
3. What are its growth-characteristics on various media, micros- 
copically and to the naked eye ? 
4. What are its relations to oxygen ? 
5. Does it produce fermentation ? If so, what fermentation, 
under what conditions, and with or without development of 
gas? 
6. Does it cause putrefaction ? 
7. Does it have a diastatic, inverting, or peptonizing action ? 
8. Has it a pathogenic character ? 
9. Does it produce coloring matter ? 
10. What is its susceptibility to the action of the various anti- 
septics ? 
The first and second of these questions are, of course, determined 
by the microscope alone; the third, by the microscope and the 
naked eye combined ; the fourth by the methods described in this 
journal, page 62, 1884, or by placing a thin strip of mica upon one 
half of the culture-plate before the gelatine solidifies; the mica then 
adapts itself closely to the surface of the gelatine, excluding the 
air, and if the fungus requires oxygen for its development the col- 
onies beneath the mica either will not develop at all, or they 
will be very small compared with those on the other half of 
the plate, their growth ceasing as soon as the oxygen in the gela- 
tine has been consumed. (Koch.) The fifth point is answered by 5 
infecting fermentable solutions with the fungus in question, placing 
it under various conditions of temperature, etc., and determining 
the products of fermentation (if any); the sixth by analogous 
methods; the seventh question is determined by the action of the 
fungi upon starch, cane sugar, and albumen, (boiled white of egg); 
the eighth by experiments on animals ; the ninth by the appearance 
or non-appearance of color in the vegetation itself, or in the sur- 
rounding medium; the tenth by experiments that will readily sug- 
gest themselves. 
Other points to be investigated will be mentioned further on. 
Boiled potato is a medium of great value in the determination of Schi- 
zomycetes. No medium, however, requires greater care in prepara- 
tion and after treatment than this, in order to obtain satisfactory re- 
sults. Any sound potato which does not become mealy or crack open 
on boiling, will do for the purpose ; it is first thoroughly washed 
and brushed, and all defective spots and deep eyes being removed, 
it is placed for one hour in a corrosive sublimate solution, five to 
one thousand, then in the steam sterilizer for one-half to one hour. 
In the mean time the moist cell is sterilized, and the bottom lined 
with filter paper wet with sublimate solution, five to one thousand. 
The potatoes are, while hot, removed from the sterilizer with steril- 
ized forceps, cut into halves with a cold sterilized knife, and placed 
directly upon the sublimate paper (the cut surface up), and the cell 
closed. Potato sections prepared in this way should remain un- 
changed indefinitely. When the potato has become cool, the cover 
of the cell is carefully removed, and the fungus which is to be cul- 
tivated is spread upon a space about as large as a dime, in the centre 
of the section. Fungi which, morphologically as well as in their 
reaction upon gelatine, Agar-Agar and blood serum, show no ap- 
preciable differences, may sometimes be easily distinguished by aid 
of the potato culture. The potato can seldom be used to separate 
fungi, (i. e. to prepare pure culture). It is chiefly used as a reagent 
in distinguishing between fungi already in pure culture. For ex- 
ample, all comma bacilli yet discovered grow on potato, except the 
one found by Dencke in old cheese, which does not develop at all 
on potato, and is thereby at once distinguished as an entirely dif- 
ferent fungus. 
Eggs may often be used to great advantage. They are pre- 
pared as follows. The fresh egg is placed in sublimate, five to one thousand, for ten minutes, then in the steam sterilizer for 
one hour. The cell for eggs is prepared as for potatoes, except that 
a sterilized glass plate, resting on a glass bench, is placed in the 
bottom to support the egg sections. As the eggs must be handled 
with the fingers, the hands must be thoroughly washed, then soaked 
in sublimate, five to one thousand, and then washed again in alco- 
hol absolutus, to remove the sublimate. The eggs are shelled while 
still hot, and cut into two, three, or four sections. They are vac- 
cinated in points upon the white; the yellow is not so well adapted 
to culture experiments, since it cannot be cut with a smooth sur- 
face. 
I always keep on hand sections of potato and egg, also tubes of 
gelatine, Agar-Agar, and blood serum, and when in my practice 
particularly good material, or anything uncommon presents itself, 
a portion of it is at once transferred to these different culture 
media, so that it is pretty sure to develop in one of them, at least. 
For example, I have several times met with a fungus in the human 
mouth which produces a yellowish coloring matter, and which 
absolutely refuses to grow on anything which I have tried, except 
potato. 
By use of the methods described I have isolated twenty-two dif- 
ferent fungi from the secretions or deposits of the human mouth, 
and have endeavored to determine, as far as possible, their separate 
peculiarities of growth, physiological action, etc. It will, however, 
at once suggest itself to every one, that a thorough study of twenty- 
two different fungi involves an enormous amount of labor, and 
might constitute almost a life task for one experimenter. The task 
is, moreover, rendered still more difficult by reason of the fact that 
many of these fungi show differences of action when cultivated in 
different media, rendering the number of experiments necessary to 
come to a definite conclusion doubly great. I shall, therefore, not 
attempt to present an exhaustive treatment of the subject, but rather 
an introduction, hoping, at the same time, to establish some points 
which may be of use in bringing about a clearer understanding of 
the factors involved in the production of dental caries. 
Regarding the first point to be considered—the morphology of 
the fungi—it is not at all necessary to enter into a minute descrip- 
tion of all the different forms here presented. The figures will 
give a sufficiently clear idea of their diversity, and the appearance of their colonies under a low power. For the rest, suffice it to say 
that ten of them are micro- or diplococci, five are bacteria and six 
bacilli. Some show more than one form of development. It would, 
however, lead us too far from our subject to discuss this fact here. 
In liquid media, three grow out into long leptothrix, forming 
bundles or meshes of intertwining uni- or multicelluar threads, 
while one develops into spirilli; eight are motile, fourteen are non- 
motile, while three only have been seen to form spores. The others 
multiply by division alone. 
With reference to the latter point, however, I have not made ex- 
aminations sufficiently careful or extensive to be able to speak 
decidedly. Eight liquify nutritive gelatine, one converts it into a 
paste, thirteen leave it unchanged. On agar-agar, 
the differences of growth are not sufficiently pro- 
nounced to deserve particular mention. In gelatine, 
the microscopic appearance of the colonies of a suf- 
ficient number of these fungi, is shown in the figures 
Fig. 1 
(b). It will be seen that the appearance of the colonies forms a 
much safer means of differentiation than the morphological charac- 
teristics of the fungi, it being very seldom that, in growing, two fungi 
present exactly the same appearance. An exception is, however, 
presented by 6 and 7, which to the naked eye and under the micro- 
scope grow on gelatine exactly alike; moreover, on potato, white of 
egg, blood-serum, agar-agar and milk, their effect is identical. One, 
however, produces a yellow coloring matter, the other not, and 
thereby they are easily distinguished. The others may all be readily 
distinguished by their growth on potato. 
In relation to oxygen, they show great differences. Ten art 
strictly aerobian ; i. e. they grow only where the air has free access, 
Four are not strictly aerobian ; i. e. they propagate also when the 
atmospheric air is excluded, though not so rapidly. Eight grow 
equally well with or without access of air. Sixteen produce an acid 
reaction in a solution of beef extract, peptone and sugar. Four 
produce an alkaline reaction without the appearance of 
bad smelling products, and appear to leave the solution 
neutral. With regard to the six, however, the results 
were not satisfactory, sometimes the reaction being acid, 
at other times neutral or alkaline, depending somewhat upon the 
material used for the cultures. 
Fig. 2 8 
Some which produce an acid reaction in fermentable solutions, 
give rise to an alkaline reaction in non-fermentable solutions. The 
acid produced is probably, in all, or in nearly all these cases, lactic 
acid. This fact I established for No. Iby chemical analysis, for 
No. 2 by forming the zinc salt and crystallizing, for No. 5 by the 
color test.* In the other cases the acid was not determined. 
Thirteen were repeatedly cultivated on potato. Of these, five grew 
rapidly, one in particular covering the whole surface of the section 
in forly-eight hours, and completely liquifying it to a depth of one 
to two mm., the liquified mass flowing off at the sides; the others 
develop very slowly, and attain only a limited growth. lam not 
able to say whether any of them possesses a diastatic action. It is, 
however, highly probable. Fifteen were cultivated on boiled white of 
egg. Four grew very rapidly, No. 19 (see fig. 13) 
in particular, in from two to four days, converting 
the egg into a semi-transparent, pasty mass, which 
gradually disappeared. In the first two days large 
quantities of sulphuretted hydrogen are developed; 
later, ammonia. Seven grew slowly on the white of egg, and four 
scarcely at all. The nourishment of the fungi naturally takes place 
at the expense of the albumen of the egg, which is converted into a 
soluble variety by the peptonizing action of the fungus. In two 
cases the presence of peptone could be detected in the dissolved mass 
after separation from the albumen, by the biuret reaction, the or- 
ganisms producing more peptone than they needed for their own 
Fig. s 
consumption. 
Some of them produce in fermentable solutions considerable 
quantities of gas. If a glass bulb, with a fine stem 
drawn out to a point, be filled with milk inoculated 
with No. 3, (see fig. 2), otherwise sterile, and kept at 
blood temperature, in twenty-four hours so much gas 
will be generated that on breaking off the point the whole contents 
of the bulb will be ejected with considerable force. The same effect 
Fig. 4 
*Two drops carbolic acid, one drop chloride of iron, twenty ccm. water, produce a violet color 
which becomes yellow on the addition of lactic acid, even in very dilute form. lam not pre- 
pared to say that this is an absolutely sure test for lactic acid. It is the test used by Prof. 
Ewald and others, for detecting lactic acid in the stomach, and is considered by them to be 
decisive. Of course the culture material itself must not give this reaction. Beef extract, for 
example, cannot be used, as it already contains lactic acid. A few other substances also give 
this reaction, but none, I believe, which are likely to be produced in these cultures. 9 
may sometimes be produced, though not so markedly, when non- 
fermentable solutions are used. We may expect a similar action 
to take place when we seal up a dead pulp in a tooth, not only the 
gas itself escaping through the apical foramen, but, 
if its exit is hindered, ultimately forcing particles of 
the decomposing pulp through with it. The ques- 
tion suggests itself, whether certain configurations 
seen in carious dentine may not owe their origin 
in part to the pressure of gas. 
Fig. 5 
Four produce coloring matter, Nos. 5 and 7 (figures 4 and 5), in 
gelatine cultures some days’ old, forming brick-yellow masses, such 
as may be seen occasionally on the buccal surface of teeth which are 
not kept well cleaned. 
On potato they appear bright yellow. Nos. 10 and 13 give the 
gelatine for a space one cm. in diameter around the colony, a grass- 
green tinge. I doubt very much whether either of these organisms 
has anything to do with the production of green stain, 
all my attempts to isolate a chromogenic fungus directly 
from green stain having thus far failed. Cultures of 
some of these fungi were made on dentine and enamel. 
Sections of dentine, when decalcified neutralized and 
soaked in saliva and sugar, formed, when kept in a per- 
fect damp cell, a medium on which a considerable development 
took place, microtome sections of the dentine after two weeks 
showing a destruction of substance at the point of inoculation. 
Fig. 6 
On sections of normal dentine, the fungi in some cases appeared 
to maintain an existence until the organic matter exposed upon 
the surface of the section was consumed, after which the develop- 
ment ceased, while normal enamel, as might have been expected, 
formed about as good a culture substratum as glass or porcelain. 
A description of the cultures in milk, blood-serum, etc., is not 
necessary for our present purpose. Also, experiments on animals 
have been made in too limited a number to lead to 
accurate results. It is very plain, however, that a 
study of the pathogenic character of twenty-two fungi 
is out of the question. No. 19, which possesses 
peculiar interest on account of its similarity to the 
cholera-bacillus, was tested on mice, guinea-pigs 
and rabbits. A small quantity from a pure culture injected into 
Fig. 7 10 
the abdominal cavity of mice, almost invariably caused death in a 
few hours. Guinea-pigs and rabbits have thus far shown them- 
selves proof against it, even when large quantities were injected 
into the duodenum (the ductus choledochus not being 
ligated.) Experiments were made with a number of 
antiseptics, in adition to those reported in this journal 
(Vol. V. page 283.) Arsenious acid, contrary to the 
repeated statements of one of our journals, possesses 
an antiseptic power at least half as great as that of carbolic acid, 
and about twenty-five times greater than absolute alcohol. Chlorate 
of potassium, on the other hand, possesses scarcely any available 
power whatever. Peroxide of hydrogen proved to be particularly 
active. These experiments are not yet completed, and will therefore 
be given in a separate paper. 
Fig. 8 
The following practical conclusions appear to follow from the 
experiments above recorded: 
1. A great majority of the fungi found in the human mouth 
are capable of producing acid from cane or grape sugar, and it is 
probable that, with very feiv exceptions, all can, when the proper 
conditions are presented to them. In nearly all cases which have 
been examined with special reference to this question, the acid has 
appeared to be lactic. The acetic acid fermentation, which cannot 
go on at temperatures above 35° 0. (Fluegge), is out of the ques- 
tion in the human mouth, nor is there, as yet, any proof of the 
presence of more than minute traces of butyric acid. 
2. In non-fermentable substances, the reaction will be found 
either neutral or alkaline, in some cases considerable quantities of 
ammonia and sulphuretted hydrogen being produced. If, there- 
fore, a decomposing pulp is sealed up in a tooth, its reaction can- 
not be acid, and caries cannot take place either in the pulp cham- 
ber or root canals. 
3. Of considerable interest is the fact that the 
same fungus may produce an acid reaction in one sub- 
stratum, and an alkaline in another. If, for example, 
No. 19 (Fig. 13) be cultivated in certain neutral, non- 
fermentable substances, an alkaline reaction will 
appear. If, then, sugar be added, the reaction will in 
a few hours change to acid. In such a case we undoubtedly have 
two distinct processes going on; first, the nutrition of the organ- 
Fig. 9 11 
ism accompanied by the appearance of alkaline products, and 
secondly, its fermentive action accompanied by acid products. 
Ordinarily, the latter so outweigh the former that the resultant 
reaction will be acid. This is, however, by no means neces- 
sarily the case. On the other hand, conditions may readily 
be produced under which the resultant reaction will be neutral or 
alkaline, especially in the human mouth, 
where so many different fungi and so various 
conditions are present. In such a case, the 
result would be to put a temporary check upon 
the advance of the decalcifying process; in 
other words, upon the caries itself. In the 
case of particularly foul-mouthed persons, the 
foulness itself may become a preventive of caries. 
Fig. 10 
4. The possession of a peptonizing action by a large number of 
these fungi, readily accounts for the solution of the decalcified den- 
tine.* 
5, Any one of these fungi which can produce acid by fer- 
mentation of carbohydrates, or can dissolve 
the decalcified dentine, may aid in the produc- 
tion of caries, while any one which combines 
both these properties, as many of them do, may 
alone bring about the phenomenon of dental 
carries. A solution of the dentine or enamel, 
withotit previous decalcification, cannot take place. The fact 
which I have so often affirmed, and which was denied by Milles 
and Underwood, that one continually meets with large tracts of 
softened, non-infected dentine, has been completely confirmed by 
Arkovy and Matrai. They say, “the invasion extends, however, 
only to a certain depth, and only isolated tubules show a deeper in- 
Fig. 11 
*Not a little confusion has been introduced by attempted artificial definitions of putre- 
flcation and fermentation. The idea that every change in nitrogenous organic substances 
must be of the nature of putrefaction, is particularly misleading. A ferment of the nature of 
pepsine, which dissolves coagulated albumen, is widely distributed among the fungi of fer- 
mentation, as well as putrefaction, and the schizomycetes in general require nitrogenous 
substances in some shape, for their nutrition. The dissolution of the organic portion of den- 
tine is by no means dependent upon the presence of putrefactive organism, but may be accom- 
plished equally well by fermentation. As stated in previous papers, I never found a putre- 
factive organism in the deeper portions of carious dentine. Moreover, the acid reaction of 
carious dentine is highly unfavorable to the development of such organisms. I intend to 
repeat and extend my experiments on this point. The presence of putrefactive organisms, 
while it would accelerate the second stage of caries, could only retard the first vasion, sometimes to twice the depth, and reach the border of the 
normal dentine,” the whole territory between the isolated tubules 
being free from invasion. 
12 
6. The comparative or complete independ- 
ence of many of these organisms of the free 
access of air, renders their propagation within 
the dentine, or under fillings where softened, 
non-sterilized dentine has been left, an easy 
matter. 
7. The fact that dentine and enamel form 
so exceedingly poor culture substrata forschizo- 
mycetes, is an additional proof of the position 
that their attack upon the teeth is only second- 
ary ; i. e*, they owe their rapid development to 
the secretions, deposits, etc., of the oral cavity, and not until the 
tissue of the tooth has undergone a certain change—first decalcifica- 
tion, second peptonization—can they adapt it to their nourishment. 
The decalcification is produced chiefly by acid, resulting from the 
action of the organisms upon certain carbohydrates in the human 
mouth, while the peptonization is produced, either by the direct 
action of the protoplasm of the organisms upon the decalcified den- 
fino Ktr tVia anfir\n nf a. fpvmenf wliiaTi tVitiv nvnrlnrio 
Fig. 12 
A knowledge of the properties of the fungi of the human mouth, 
as given above, combined with a microscopic and chemical exam- 
ination of carious tissue, and comparative studies of 
caries of living and dead teeth, appear to me to furnish 
a fair solution of the phenomena of dental caries. 
That other agents than those of a parasitic nature 
are also often concerned, there can be no doubt. To 
saj nothing of predisposing causes, an acid reaction 
of the oral secretions, acid medicines, acid foods, etc., 
may give rise to caries at points which otherwise probably would 
have escaped. 
Fig. 13