cover-glass, break it, destroy our preparation, and, if
examining parasitic bacteria, infect the lens. This may
be avoided by first finding the hanging drop with a low
power lens and thus exactly centre it. The lens of
higher magnification is now very gradually lowered,
while at the same time gently moving the slide back
and forth to the slightest extent possible with the left
hand. If any resistance is felt raise the lens, for it has
gone beyond the point of focus and is touching the
cover-glass.



CHAPTER XIII.

BACTERIOLOGICAL TECHNIQUE Continued.

THE CULTIVATION OF BACTERIA.

IN order to determine the number of living bacteria
in any substance and their nature we have to cultivate
and isolate them.

The Most Common of the Nutrient Media Used for the
Growth of Bacteria.

All of these must have, as noted earlier, food con-
taining the necessary carbon, nitrogen, and mineral
substances in a form easily assimilated and in the
proper concentration. The pathogenic bacteria nearly
all require for good growth peptone, albumins, and
sugar. For each kind the proper food must be found
through experimentation, as slight alterations may
make a great difference.

Physicians will find it, as a rule, convenient to
purchase their media already prepared from some of
the reliable firms that deal in bacteriological products.
Special media, such as those employed for isolation and
identification of the typhoid bacillus and gonococcus,
will be found described along with those bacteria. For
those who may wish to make their own, we will de-
scribe here those in common use:

Nutrient Bouillon or Broth. One part of finely chopped
fresh, lean meat is macerated in two parts of water and



BACTERIOLOGICAL TECHNIQUE.



213



put in an ice-chest for from eighteen to twenty-four
hours. The infusion is strained, when cold, through a
fine cheese-cloth, and to the clear filtrate 1 per cent, of
peptone and 0.5 per cent, of sodium chloride are added.
The medium is then warmed for some minutes until the
peptone is dissolved, and then exposed to live steam
either without pressure in the Arnold steam sterilizer
(Fig. 20) for thirty minutes, or in the autoclave (Fig.



FTG. 20.



STERILIZING CHAMBER

U A




Arnold steam sterilizer.

21) at one atmosphere of pressure for fifteen minutes,
or boiled over a free flame for ten minutes. While
still hot it is filtered through filter-paper or through
absorbent cotton, and the reaction is tested and suf-
ficient hydrochloric acid or sodium hydroxide added



214



BACTERIOLOGY.



to give it the desired reaction, which is for most bac-
teria slightly alkaline to litmus. If the fluid is clear
it is put into flasks and tubes and sterilized; if not
clear, the white of one or two eggs is added to the
fluid after cooling it down to about 55 C. After



FIG. 21.




Autoclave for sterilization with live steam under pressure.

thoroughly mixing the eggs the bouillon is boiled
briskly for a few minutes and then again filtered and
distributed in flasks and tubes and put in the Arnold
sterilizer for one hour on each of two consecutive days,
or in the autoclave for twenty minutes for sterilization.
Instead of meat 2 to 4 grammes of Liebig's or some
other meat extract are added to each litre of water.
For most purposes the extract is as good as the fresh
meat, but for toxin production it is inferior.



BACTERIOLOGICAL TECHNIQUE. 215

Fermentation broth is made usually by adding 1 per
cent, of glucose to the above. For accurate work the
meat sugars are first extracted by allowing the colon
bacillus to grow in the broth over night. The bouillon
is then sterilized and the peptone and salt added, and
the process already given gone through with.

Fermentation bouillon is usually placed in a tube of
special construction, known as a fermentation tube (see
Fig. 14, p. 82). This is essentially a tube 1.5 cm. in
diameter, bent at an acute angle, closed at one end, and
provided with a bulb at the other end, which latter
should be large enough to receive all the fluid in the
closed branch should gas in any considerable quantity
collect there. The tube also serves a most important end
in giving information as to the aerobic and anaerobic
growth of the species under consideration, for the con-
necting tube being constricted serves to prevent, to a
great degree, the entrance of oxygen of the air into the
closed branch, and the free oxygen in the medium is
driven out by the heat during sterilization; from which
it may be seen that growth in the bulb is aerobic and
growth in the closed branch is anaerobic. For the study
of fermentation alone small tubes may be inverted into
larger ones or tubes may be bent on themselves.

Nutrient Gelatin. To the bouillon already prepared
as described add 10 per cent, of sheet gelatin and neu-
tralize. Add the whites of two eggs for each litre and
boil for a few minutes. Filter, place in tubes or flasks,
and sterilize. Instead of adding gelatin to bouillon
already prepared, it may be added to the meat infusion
at the same time the peptone and salt were added in
preparing nutrient bouillon as just described.

Nutrient Agar. This is prepared by adding to stock



216 BACTERIOLOGY.

bouillon 1 or 2 per cent., as desired, of thread agar,
melting it by placing over a free flame or in the auto-
clave or steam sterilizer. When the agar is brought
into solution over a free flame there may be consider-
able loss of fluid by evaporation. This should be com-
pensated for by adding additional water before boiling.
Agar may be added directly to the meat infusion along
with the peptone and salt. Indeed, this is an advan-
tage, as agar-agar is very difficult to bring into solution,
and is not injured in the least by prolonged boiling.
Glycerin agar is simply nutrient agar plus 3 to 6 per
cent, of glycerin. It is added to the hot nutrient agar
just previous to putting it in the flasks. Nutrient agar
begins to thicken at a fairly high temperature, and
should be filtered as hot as possible. When small
amounts are made it is well to place the filter and re-
ceiving-flask in the sterilizer while filtering.

Milk. This fluid is a good culture medium for most
pathogenic bacteria. It should be obtained as fresh as
possible, so that but little bacterial change has occurred.
It is first put in a steam sterilizer for fifteen minutes
and then put in the ice-chest for twelve hours, to allow
the cream to rise. The milk is then siphoned off from
below the cream into a flask and its reaction tested. A f ter
correction it is put in tubes or flasks and sterilized.

Potatoes. Potatoes are used for some special pur-
poses. The potatoes may after thorough scrubbing and
removal of "eyes" be soaked in bichloride of mer-
cury (1 : 1000) for twenty minutes, and then sterilized
on three consecutive days for one-half hour in the
steam sterilizer. To use they are cut in thick slices and
put in deep Petri dishes. For more careful work the
potatoes are first cut into proper sizes for tubes or



BACTERIOLOGICAL TECHNIQUE. 217

dishes, and then soaked for from twelve to eighteen
hours in running water; this removes excessive acidity;
they are then placed in test-tubes and sterilized by steam
on two consecutive days.

Blood-serum and Ascitic Fluid with and without the
Addition of Bouillon. Blood-serum is used in the fluid
state, semi-solid, and firmly coagulated. It is used
alone, with 66 per cent, of bouillon and with 25 per
cent, of bouillon plus 1 per cent, of glucose. Ascitic,
pleuritic, and hydrocele fluids are also used alone, with
bouillon, or with nutrient agar.

The Correction of the Reaction in Media.

Formerly it was customary to use litmus-paper as
the indicator in neutralizing media, adding soda solu-
tion until the mixture turned the red litmus slightly
blue, and the blue litmus just a tinge less blue. This
is still the best method for those who are only going
to cultivate the common pathogenic bacteria for diag-
nostic purposes or for the development of toxin. Most
parasitic bacteria which grow at all on artificial culture
media develop best in them when they have a slightly
alkaline reaction to litmus. If a greater alkalinity is
desired a certain number of c.c. of normal soda solu-
tion can be added for each litre; if an acidity is desired,
normal hydrochloric acid solution is added.

Many bacteriologists consider that litmus is not deli-
cate enough to be entirely satisfactory, especially when
experiments are to be reported or exactly repeated.
For these purposes phenolphthalein has been generally
selected. A little experience will show that different
indicators not only differ in delicacy, but that they
react differently to different substances.



218 BACTERIOLOGY.

A litre of bouillon becomes on the addition of 1 per
cent, of peptone more alkaline to litmus, but decidedly
more acid to phenolphthalein. We have, therefore,
especially with the latter substance, to find by growing
the bacteria just what reaction we want, and then test
the fluid with phenolphthalein as the indicator. With
exactly similar materials we can exactly reproduce at
any time in the future the same reaction, but with dif-
ferent materials this would be impossible. A bou-
illon which contains 1 per cent, of peptone and reacts
neutral to litmus is about 15 points acid to phenolph-
thalein that is, 15 c.c. of normal soda solution must
be added per litre to make the bouillon neutral.

When phenolphthalein is used we must have accu-
rately standardized solutions of caustic soda and hydro-
chloric acid. The test is carried out as follows : To
10 c.c. of the hot nutrient bouillon add one drop of a
1 : 300 solution in alcohol of phenolphthalein; into this
is dropped slowly a 4 per cent, solution of caustic soda
until a faint rose-tint appears. This indicates the be-
ginning of an alkaline reaction. To make a litre neu-
tral we would add 100 times as much of the decinormal
solution of caustic soda as was required to make 10 c.c.
neutral. As a rule, we use 1 per cent, peptone bou-
illon of such an acidity that 15 c.c. of normal soda
solution must be added to each litre to make it neutral.

The Sterilization of Different Media.

Flasks and tubes of nutrient broth and agar are
easily sterilized by placing them in an Arnold steam
sterilizer (Fig. 20) for from fifteen minutes to one hour,
according to the bulk of the fluid, upon two or three
consecutive days. They can also be even more cer-



BACTERIOLOGICAL TECHNIQUE. 219

tainly sterilized by putting them in an autoclave (Fig.
21) at 110 C. for from fifteen to thirty minutes on
two consecutive days.

Gelatin is sterilized in the same manner except, as
already stated, the shorter times are used. Pro-
longed heating destroys the congealing properties of the
gelatin.

Blood-serum may be sterilized by fractional sterili-
zation and remain fluid, or may be rendered solid by
the degree of heat used in sterilizing.

For the sterilization of fluid serum it is requisite that
it be exposed to a temperature of from 62 to 66 C.
for one hour on each of six consecutive days. The
best apparatus for obtaining and maintaining this tem-
perature (about 65 C.) is a small and well-regulated
incubator or chamber surrounded by a water space,
into which the tubes and flasks containing serum are
to be put each day and in which they are to be left for
the prescribed time after having been warmed to the
desired temperature.

Serum may be solidified and still remain translucent
at a temperature of 76 C., but when heated to a higher
degree a more definite coagulation takes place, and the
medium becomes opaque. Care must be taken in coagu-
lating blood-serum at the higher temperatures to run
the temperature up slowly and not to heat above 90 C.
until the serum has firmly coagulated; for unless these
precautions are taken ebullition is likely to occur, which
will lead to the formation of bubbles and an uneven-
ness of the surface upon which growth is to be obtained
and studied. Serum may be solidified at the tempera-
tures mentioned in an incubator, water-oven, or even
in an Arnold steam sterilizer, with the top covered by



220 BACTERIOLOGY.

a cloth instead of the usual lid, and when coagulated
firmly (90 C.) the tubes and their contents may, on
the following day, be sterilized in streaming steam at
100 C. without danger of the subsequent formation
of bubbles. Koch's serum coagulator (Fig. 22) is,
however, the most convenient apparatus.

Serum may be preserved by placing it in flasks
which, after the addition of 5 per cent, of chloroform,
are sealed. When it is to be used it is filled into

FIG. 22.




Blood-serum coagulator.

sterilized culture (test) tubes and sterilized by exactly
the same methods as are employed in sterilizing fresh
serum. The chloroform, being volatile, tends to dis-
appear at ordinary temperatures, but is quickly and
surely driven off at the temperatures used in steril-
izing.

Serum may be efficiently sterilized, when great care
is used, by passing it through a Pasteur or Berkefeld
filter, under pressure. When so treated the fluid is
very clear and light-colored.

Flasks, Dishes, Tubes, etc., Used for the Preservation of
Media and for other Bacteriological Purposes. The nutri-



BACTERIOLOGICAL TECHNIQUE.



221



ent media are stored in large quantities in round or flat-
bottomed Erlenmeyer flasks (Fig. 23). From these, as
needed, glass tubes (Fig. 24) are filled. Glass dishes
with covers (Petri dishes, Fig. 27) and flat flasks are
used for growing bacteria in or upon thin layers of
media. When small amounts of media are taken fre-
quently from flasks, Pasteur's flasks (Fig. 25) are of

FIG. 24.



FIG. 23.




FIG. 25.




Erlenmeyer flask.



Pasteur flask.



Glass test-tubes.

great convenience. They consist of a flask with a
ground-glass neck, over which fits a cap. This cap
may or may not terminate, as desired, in a narrow
tube, which is plugged with cotton. The cap keeps
the edges of the flask free from bacteria and prevents
the cotton from sticking.



222 BACTERIOLOGY.

The Methods of Obtaining and Studying Pure Cultures of
Single Species of Bacteria.

In order to study bacteria, both in culture media
and in the living body, we must separate those devel-
oped from one organism from all others and study
them by themselves in pure cultures. In order to do
this we have to take the greatest precautions to insure
that the materials that we make use of for the growth of
bacteria, the flasks and tubes that hold these materials,
and the instruments with which we transfer the bac-
teria are sterile. We also carefully try to prevent any
bacteria entering from the air or elsewhere.

The Cleansing and Sterilization of Apparatus.

In bacteriological work sterilization is practically
always done by means of dry and moist heat, for no
antiseptic substances can be allowed to remain in any
of the media used for the growth of bacteria or on any
of the apparatus which would come in contact with
them, as such substances would inhibit the growth of
the bacteria which we desired to study.

The platinum wires and loops used in transferring
bacteria are sterilized by holding them for a moment
until red-hot in a gas or alcohol flame. They should
not be used until time enough has elapsed for them to
cool sufficienly not to injure the bacteria touched by
them. Knives, instruments, etc., are, after thorough
cleansing, placed in boiling 1 per cent, soda solution
for three to five minutes. Hypodermic needles are
sterilized by boiling in soda solution, or, when this is
impossible, they are first frequently rinsed with boil-
ing or with very hot water and then filled with a 5 per



BACTERIOLOGICAL TECHNIQUE.



223



cent, carbolic acid solution for at least thirty minutes
and then rinsed again with sterile water. New tubes
and flasks sometimes require to be washed in a 2 per
cent, solution of nitric acid, so as to remove any free
alkali which may be present. They are finally thor-
oughly rinsed in pure water. Old tubes, flasks, and
other glassware are boiled for about thirty minutes in a
5 per cent, solution of washing soda and then thoroughly
rinsed off with water until perfectly clean. If neces-



FIG. 26.




Dry heat sterilizer.



sary, any dirt clinging to the insides of the flasks
and tubes can be removed by bristle brushes or suit-
able swabs. After the tubes and flasks have been
thoroughly cleaned they are plugged loosely with ordi-
nary cotton batting, or, if that is not at hand, the more
expensive absorbent cotton. The tubes and flasks with
their cotton plugs and all other glassware are sterilized
by dry heat at 150 C. for one hour (Fig. 26).

The sterile tubes and flasks are filled with the media,
when small quantities are used, by means of a glass
funnel. The main precaution to be observed is not to
let the media soil the neck of the tubes and flasks, as



224 BACTERIOLOGY.

this would cause the fibres of the cotton plugs to adhere
to the sides of the tubes when the media dried and
make it difficult to remove the plugs wholly when we
wished to inoculate the contents of the tubes.

The tubes and flasks, plugged with sterile cotton
and full of media, are put in the steam sterilizer for
one half hour on three consecutive days, or in the
autoclave for twenty minutes for two consecutive days.
A portion of the tubes containing nutrient agar are
laid in a slanted position before cooling, after the final
sterilization, so that a larger surface may be obtained.

Technique of Making Plate Cultures.

When we make cultures from any material, we are
very apt to find that instead of one variety of bacteria
only there are a number present. If such material
is placed in fluid media contained in test-tubes, we find
that the different varieties all grow together and be-
come hopelessly mixed. When, on the other hand, the
bacteria are placed on solid media they develop about the
spot where they were inoculated. If different varieties,
however, are placed too near together, they overgrow
one another ; it is thus advisable to have a greater sur-
face of nutrient material than is given on the slanted
surface of nutrient agar or blood-serum contained in
test-tubes. This need is met by pouring the media
while warm on flat, cool, glass plates or into shallow
dishes. In making plate cultures two methods are
carried out. In the first the material with its contained
bacteria is scattered throughout the fluid before it
hardens; in the second it is streaked over the surface of
the medium after it has solidified. Nutrient agar and
nutrient gelatin, the two substances used for plate



BACTERIOLOGICAL TECHNIQUE. 225

cultures, differ in two essential points, which cause
some difference in their uses. Nutrient 1 per cent,
agar melts at a high temperature and begins to thicken
at about 36 C. It is not liquefied by bacterial fer-
ments. Nutrient 10 per cent, gelatin melts at the low
temperature of about 23 C. and solidifies at a point
slightly below that. It is liquefied by many bacterial
ferments. When we wish to inoculate fluid nutrient
agar for plate cultures we have to take great care that
in cooling it to a point which will not injure the bac-
teria, about 41 C., we do not allow it to cool too much
and thus solidify and prevent our pouring it into the
plates. To prevent this, when a number of tubes are
to be inoculated they are placed while still hot in a
basin of water which has been heated to about 45 C.
When the temperature of the agar in one of the tubes,

FIG. 27.




Petri dish.

as tested by a thermometer, has fallen to 40 the water,
milk, feces, bacterial culture, or other substances to be
tested are added to the other tubes in whatever quantity
is thought to be proper. After inoculation the con-
tents of the tubes are thoroughly shaken and poured
out quickly into round, flat-bottomed glass dishes (Fig.
27), the covers of which are removed for the required
time only. The bacteria are now scattered throughout
the fluid, and as it quickly solidifies they are fixed
wherever they happen to be, and thus as each individual

15



226 BACTERIOLOGY.

multiplies clusters are formed about it at the spot where
it was fixed at the moment of solidification. The num-
ber of colonies of bacteria (Fig. 28) thus indicate to
us roughly the number of living bacteria in the quan-
tity of fluid added to the liquid agar. Nutrient gelatin
is used exactly as agar, except that, as it does not
congeal until cooled below 22 C., we have no fear of

FIG. 28.




Photograph of a large number of colonies developing in a layer of gelatin
contained in a Petri dish. Some colonies are only pin point in size ; some as
large as a pencil. The colonies here appear in their actual size.

its cooling too rapidly. In order not only to count
the number of colonies which develop, but also to
obtain a characteristic growth, it is desirable not to
have them too near together. As it is impossible to
determine accurately the number in any suspected fluid,
it is usual to make a set of four different plates, to each
of which a different amount of material is added, so that



BACTERIOLOGICAL TECHNIQUE. 227

some one of the four will have the required number of
colonies. In the first tube we place an amount which we
believe will surely contain sufficient and probably too
many bacteria. To the second tube we add 10 per cent,
of the amount added to the first, and to the third 10
per cent, of the second, and to the fourth 10 per cent,
of the third. Thus if the first contained 60,000 colo-
nies, the second would have 6000 (Fig. 28), the third
600, and the fourth 60. If, on the other hand, the first
contained but 60, the second would have about 6, and
the remaining two would probably contain none at all.
When there are many colonies present the dishes are
covered by a glass plate (Fig. 29), ruled in larger and

FIG. 29.




WolffhiigePs apparatus for counting colonies.

smaller squares. With a hand lens the colonies in a
certain number of squares are counted and then the
number for the whole contents estimated.

When the material to be tested is crowded with bac-
teria it is often best to make an emulsion of a portion
of it, and use this rather than the original substance
for making the cultures.

Measured quantities of the diluted material can be
transferred most accurately through a sterilized long
glass pipette graduated in one-hundredth cubic centi-
metres, or, more roughly, by a platinum loop of known
size.



228 BACTERIOLOGY.

The nutrient agar-agar is frequently used in a dif-
ferent manner. A small quantity is poured into the
Petri dish and allowed to harden. The substance to
be tested bacteriologically, or a dilution of it, is then
streaked by means of a platinum loop lightly over its
surface. While in the former method most of the bac-
teria developed under the surface, here all develop upon
it. This is an advantage, as many forms of bacteria
develop more characteristically on the surface than in
the midst of the media, and it is easier to remove them
free from other bacteria with the platinum needle. The
method of using glass plates upon a cooling stage has
now been practically given up for the more convenient
one of Petri dishes. In warm weather the dishes should
be cooled before using, so as to harden quickly the agar
or gelatin that is poured into them.

An old method, which is still sometimes used to find
the number of living bacteria, is, instead of pouring
out the media which has been inoculated, to congeal it
on the sides of the test-tube. This is best done by
laying the tube flat on its side on a cake of ice and
rotating it. Tubes come especially formed for this by
having a slight neck, which prevents the media run-
ning up to the plugged end of the tube. This method,
Esmarch's, is used only when the Petri dishes are not
obtainable or cannot easily be transported.

The Study of Colonies in Plate Cultures in Nutrient Agar.

The plates should be removed after twelve to twenty-
four hours' growth at blood temperature and after one
to three days at 70. The special time allowed varies
according to the rapidity of the growth of the varieties
developing, thus bacteria, such as the streptococci and



BACTERIOLOGICAL TECHNIQUE. 229

influenza bacilli, reach the maximum development of
their colonies in from ten to sixteen hours, while others
continue to spread for several days. If we wait too long
where numerous varieties of bacteria are growing the
colonies of heavier growth may cover up the finer and