Page 811 - My FlipBook
P. 811
BIOLOGICAL STUDIES ON FUNGI OF HUMAN MOUTH. 821 ; :
We will start with a solution densely impregnated with micro-organ-
isms and a number of tubes of culture gelatin perfectly sterilized. The
gelatin being melted, we add to the first tube one bead (on a loop of
sterilized platinum wire) of the solution ; this is called the//-s^ dilution.
From this tube we add two or three beads to a second tube (second dilu-
tion), and from the second five or six beads to a third tube (tJdrd dilu-
tion). The gelatin 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 exam-
ined after twenty-four to thirty-six hours under a magnification of 100
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 gelatin, 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 appearance. With a steril-
ized platinum wire bent at right angles at the end we now pick up a
number of the colonies of each kind under the microscope (100 diam-
eters), and transfer them directly to tubes of culture gelatin, only one
colony to each tube. We have then (except in case of a possible acci-
dental air-infection) pure cultures. Some experience is necessary to
enable one to pick up the colonies under the microscope. Beginners
should not attempt it with plates where more than one colony is in the
field at once.
The method described on page 813 may also sometimes be used to
great advantage. For fungi which do not grow on gelatin, agar-agar or
congealed blood-serum should be used. The former, 1 to 1^ per cent.,
has a higher melting-point than gelatin, 10 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 tem-
perature 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 gelatin. 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 movable ? Does it produce spores ?
3. What are its growth-characteristics on various media, microscopi-
cally 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 J)athogenic character?
We will start with a solution densely impregnated with micro-organ-
isms and a number of tubes of culture gelatin perfectly sterilized. The
gelatin being melted, we add to the first tube one bead (on a loop of
sterilized platinum wire) of the solution ; this is called the//-s^ dilution.
From this tube we add two or three beads to a second tube (second dilu-
tion), and from the second five or six beads to a third tube (tJdrd dilu-
tion). The gelatin 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 exam-
ined after twenty-four to thirty-six hours under a magnification of 100
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 gelatin, 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 appearance. With a steril-
ized platinum wire bent at right angles at the end we now pick up a
number of the colonies of each kind under the microscope (100 diam-
eters), and transfer them directly to tubes of culture gelatin, only one
colony to each tube. We have then (except in case of a possible acci-
dental air-infection) pure cultures. Some experience is necessary to
enable one to pick up the colonies under the microscope. Beginners
should not attempt it with plates where more than one colony is in the
field at once.
The method described on page 813 may also sometimes be used to
great advantage. For fungi which do not grow on gelatin, agar-agar or
congealed blood-serum should be used. The former, 1 to 1^ per cent.,
has a higher melting-point than gelatin, 10 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 tem-
perature 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 gelatin. 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 movable ? Does it produce spores ?
3. What are its growth-characteristics on various media, microscopi-
cally 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 J)athogenic character?
