METHODS OF STUDYING BACTERIA.
=Necessity of artificial cultivation.= The bacteria are so extremely
small, that it is impossible to study individual germs separately
without the aid of powerful microscopes. Little advance was made in
the knowledge of these lower forms of plant life until the
introduction of culture methods, whereby a single organism could be
cultivated, and the progeny of this cell increased to such an extent
in a short course of time that the resulting mass of cells would be
visible to the unaided eye. This is done by growing the bacteria on
various kinds of nutrient media that are prepared for the purpose,
but inasmuch as bacteria are so universally distributed, it becomes
an impossibility to cultivate any special form alone, unless the
medium in which they are grown is first freed from all pre-existing
forms of germ life.
=Food materials.= Many kinds of food substances are used for the
cultivation of bacteria in the laboratory. In fact, bacteria will
grow on almost any organic substance, whether it is solid or liquid,
provided the other essential conditions of growth are furnished. The
food substances that are used for culture purposes are divided into
two classes,--solids and liquids.
Solid culture media may be either permanently solid, like potatoes
and coagulated egg, or they may retain their solid properties only
at certain temperatures, like gelatin or agar. The latter two, which
were devised by Robert Koch, are of utmost importance in
bacteriological research, for their use permits the separation of
the different forms of bacteria that may happen to be in any
mixture. Gelatin is advantageously used, because the majority of
bacteria present wider differences, due to growth upon this medium,
than upon any other. It remains solid at ordinary temperatures,
becoming liquid at about 80 deg. F. Agar, a gelatinous product derived
from a Japanese seaweed, has a much higher melting point, and is
used especially with those organisms whose optimum temperature for
growth is above the melting point of gelatin.
Besides these solid culture media, different liquid substances are
extensively used, such as beef broth, milk and infusions of various
vegetable and animal tissues. Skim milk is of especial value in
studying the milk bacteria, and may be used in its natural
condition, or a few drops of litmus solution may be added, in order
to detect any change in its chemical reaction due to the bacteria.
=Sterilization.= The various ingredients that are used in the
preparation of culture media are not free from micro-organisms,
hence the media would soon spoil if they were not destroyed, and the
media subsequently protected from contamination from the air, etc.
The process of rendering the media free from living micro-organisms
is known as _sterilization_. It may be accomplished in a number of
ways, but most often is done by the use of heat. For culture
material, which is always organic in character, moist heat is
employed. The various culture media, in appropriate containers, are
subjected to a thorough steaming in a steam cooker. This destroys
all of the vegetating cells but not the resistant spores that may be
present. The media are then stored, for twenty-four hours, at
temperatures favorable for the germination of the spores and are
then again heated. Three such applications on successive days are
usually sufficient to free the media from all living germs, since
between the heating periods the spores germinate and the resulting
vegetative cells are more easily destroyed. The sterile media will
keep for an indefinite period in a moist place.
The media are usually placed in glass containers which may be
sterilized before use by heating them in an oven, it being possible
to thus secure a much higher temperature than with streaming steam.
All glass or metal articles may be sterilized by the use of dry heat
but for organic media, to avoid burning, moist heat must be used.
All kinds of materials may be sterilized by treatment with steam
under pressure. An exposure for a few moments at 250 deg. F., a
temperature attained with 15 pounds steam pressure, will destroy all
kinds of bacteria and their spores. This method of sterilization is
used in the canning of meats and vegetables and in the preparation
of evaporated milk. To avoid contamination of the media after
sterilization, the flasks and tubes are, after being filled,
stoppered with plugs of cotton-wool, which effectually filter out
all bacteria and mold spores from the air, and yet allow the air to
pass freely in and out of the containers.
=Methods of determining the number of bacteria.= The method of
determining the number and kinds of bacteria in any substance can be
illustrated by the process as applied to milk. For this purpose the
method of procedure is as follows: Sterile gelatin in glass tubes is
melted and then cooled until it is barely warm. To this melted
gelatin a definite quantity of milk is added. The medium is gently
shaken, so as to thoroughly mix the milk and gelatine, and the
mixture then poured into a sterile, flat, glass dish, and quickly
covered, where it is allowed to cool until the gelatin hardens.
After the culture plate has been left for twenty-four to thirty-six
hours at the proper temperature, tiny spots will begin to appear on
the surface, or in the depth of the culture-medium. These spots are
called _colonies_, and are composed of an almost infinite number of
individual cells, the result of the continued growth of a single
organism that was in the drop of milk and which was firmly held in
place when the gelatin solidified. The number of these colonies
represents approximately the number of living bacteria that were
present in the amount of milk added to the tube of gelatin. If the
plate is not too thickly sown with the bacteria, the colonies will
continue to grow and increase in size, and as they do, minute
differences will begin to appear. These differences may be in the
color, the contour, and the texture of the colony, or the manner in
which it acts toward gelatin.
[Illustration: Fig. 4.--Plate Culture.
Each of the dots is a colony that has been formed by the growth of
an organism embedded in the solid culture-medium. By counting the
colonies, the number of living bacteria in the amount of milk added
to the culture is determined.]
In order to make sure that the number of colonies is not so numerous
as to prevent counting and further study of their characteristics, a
series of plate cultures is usually made in which varying amounts of
milk are added to the tubes of gelatine. This is attained by adding
a definite amount of the milk or other substance to be examined to a
measured amount of sterile water, e.g., one cubic centimeter of milk
to ninety-nine cubic centimeters of water. One cubic centimeter of
this mixture may be used for the inoculation of the plate culture.
This dilution may be carried on to any desired extent; in the
examination of many dairy products, it is necessary to use very
minute quantities of material, often only one one-millionth of a
cubic centimeter.
To study further the peculiarities of the different bacteria, small
portions of the individual colonies are transferred to tubes of
sterile culture-media. In order to do this the colony is touched
with a piece of platinum wire; the minute amount of growth that
adheres to the wire is sufficient to seed the tube of fresh
culture-medium. The inoculating needle must always be sterilized
before use by passing it through a gas flame.
A culture thus obtained is called a _pure culture_ since it contains
but a single kind of an organism, as the colony is the result of the
growth of a single cell. These cultures then serve as a basis for
continued study, and must be planted and grown upon the different
kinds of media that are obtainable. In this way the slightest
variations in the growth of different forms are detected, and the
peculiar characteristics are determined, so that the student is able
to recognize this form when he meets it again.
[Illustration: Fig. 5.--Different Kinds of Bacteria Growing in
Gelatin.
A, meager growth, no liquefaction or surface growth; B, profuse
surface growth, radiating filaments from the growth below the
surface; C, a rapid liquefying form; D, a gas producer that grows
equally well in the presence or absence of air; E, form that grows
only in the absence of air, an anaerob.]
These culture methods are of essential importance in bacteriology,
as it is the only way in which it is possible to secure a quantity
of germs in a pure state.
=The microscope in bacterial investigations.= In order to verify the
purity of the cultures, the microscope is in constant demand
throughout all the different stages of the isolating process. For
this purpose it is essential that the instrument used shall be one
of high magnifying powers (600 to 800 diameters), combined with
sharp definition.
The microscopical examination of any germ is quite as essential as
the determination of culture characteristics, in fact, the two must
go hand in hand. The examination reveals not only the form and size
of the individual germs but the manner in which they are united with
each other, as well as any peculiarities of movement that they may
possess.
In carrying out the microscopical part of the work, not only is the
organism examined in a living condition, but colored preparations
are made by using solutions of anilin dyes as staining agents. These
are of great service in bringing out almost imperceptible
differences. The art of staining has been carried to the highest
degree of perfection in bacteriology, especially in the detection of
germs that are found in diseased tissues in the animal or human
body.
In studying the peculiarities of any special organism, not only is
it necessary that these cultural and microscopical characters should
be closely observed, but special experiments must be made in
different ways, in order to determine any special properties that
the germ may possess. Thus, the ability of any form to act as a
fermentative organism can be tested by fermentation experiments; the
property of causing disease, studied by the inoculation of pure
cultures into experimental animals, like rabbits, guinea pigs and
white mice.
The methods of the bacteriologist in his laboratory are in their
effect not dissimilar to those which the farmer employs in securing
his crop of pure-bred grain. The laboratory farmer kills the weed
seeds in his culture field by the application of heat. His field,
which is embraced in his culture dish, has been fertilized and
prepared by the addition of certain favorable ingredients. When he
has garnered his crop, he maintains its purity by keeping his
selected seed, the pure culture, free from all contamination. The
dairyman, even though he may not expect to carry on the detailed
operations of the laboratory, will understand the reason for the
directions which he is often required to follow much better if he
knows how the simple operations of the laboratory are carried out.
For a fuller knowledge of these matters, the reader is referred to
the special texts on bacteriology.