METHODS OF STUDYING BACTERIA.
~Necessity of bacterial masses for study.~ The bacteria are so extremely
small that it is impossible to study individual germs separately without
the aid of first-class microscopes. For this reason, but 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 they would be visible to the unaided eye.
This is done by growing the bacteria in masses on various kinds of food
media that are prepared for the purpose, but inasmuch as bacteria are so
universally distributed, it becomes an impossibility to cultivate any
special form, unless the medium in which they are grown is first freed
from all pre-existing forms of germ life. To accomplish this, it is
necessary to subject the nutrient medium to some method of
sterilization, such as heat or filtration, whereby all life is
completely destroyed or eliminated. Such material after it has been
rendered germ-free is kept in sterilized glass tubes and flasks, and is
protected from infection by cotton stoppers.
~Culture media.~ For culture media, many different substances are
employed. In fact, bacteria will grow on almost any organic substance
whether it is solid or fluid, 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 media may be either permanently solid like potatoes, or they may
retain their solid properties only at certain temperatures like gelatin
or agar. The latter two are of utmost importance in bacteriological
research, for their use, which was introduced by Koch, permits the
separation of the different forms that may happen to be in any mixture.
Gelatin is used advantageously 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 70 deg.
F. Agar, a gelatinous product derived from a Japanese sea-weed, has a much
higher melting point, and can be successfully used, especially with
those organisms whose optimum growth point is above the melting point of
gelatin.
Besides these solid 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.
[Illustration: FIG. 2. A gelatin plate culture showing appearance of
different organisms in a sample of milk. Each mass represents a
bacterial growth (colony) derived from a single cell. Different forms
react differently toward the gelatin, some liquefying the same, others
growing in a restricted mass. _a_, represents a colony of the ordinary
bread mold; _b_, a liquefying bacterium; _c_, and _d_, solid forms.]
~Methods of isolation.~ Suppose for instance one wishes to isolate the
different varieties of bacteria found in milk. The method of procedure
is as follows: Sterile gelatin in glass tubes is melted and cooled down
so as to be barely warm. To this gelatin which is germ-free a drop of
milk is added. The gelatin is then gently shaken so as to thoroughly
distribute the milk particles, and poured out into a sterile flat glass
dish and quickly covered. This is allowed to stand on a cool surface
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 patches are called _colonies_ and are composed of an
almost infinite number of individual germs, the result of the continued
growth of a single organism that was in the drop of milk which was
firmly held in place when the gelatin solidified. The number of these
colonies represents approximately the number of germs that were present
in the milk drop. If the plate is not too thickly sown with these germs,
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. In order to make sure that the seeding in
not too copious so as to interfere with continued study, an
_attenuation_ is usually made. This consists in taking a drop of the
infected gelatin in the first tube, and transferring it to another tube
of sterile media. Usually this operation is repeated again so that these
culture plates are made with different amounts of seed with the
expectation that in at least one plate the seeding will not be so thick
as to prevent further study. For transferring the culture a loop made of
platinum wire is used. By passing this through a gas flame, it can be
sufficiently sterilized.
[Illustration: FIG. 3. Profile view of gelatin plate culture; _b_, a
liquefying form that dissolves the gelatin; _c_ and _d_, surface
colonies that do not liquefy the gelatin.]
To further study the peculiarities of different germs, the separate
colonies are transferred to other sterile tubes of culture material and
thus _pure cultures_ of the various germs are secured. These cultures
then serve as a basis for continued study and must be planted and grown
upon all the different kinds of media that are obtainable. In this way
the slight 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.
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 of
the same kind.
~The microscope in bacterial investigation.~ 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 strong magnifying
powers (600-800 diameters), combined with sharp definition.
[Illustration: FIG. 4. Pure cultures of different kinds of bacteria in
gelatin tubes. _a_, growth slight in this medium; _b_, growth copious at
and near surface. Fine parallel filaments growing out into medium
liquefying at surface; _c_, a rapid liquefying form; _d_, a
gas-producing form that grows equally well in lower part of tube as at
surface (facultative anaerobe); _e_, an obligate anaerobe, that develops
only in absence of air.]
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 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 carried out along
different lines, 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
animals. A great many different methods have been devised for the
purpose of studying special characteristics of different bacteria, but a
full description of these would necessarily be so lengthy that in a work
of this character they must be omitted. For details of this nature
consult standard reference books on bacteriological technique.