3. _Clavate_ (Fig. 91, d): Rods swollen at one pole and cylindrical
(unaltered) at the other; keyhole-shaped; e. g., B. chauvei.
4. _Capitate_ (Fig. 91, e): Rods with a spherical enlargement at one
pole; drumstick-shaped; e. g., B. tetani.
The endo-spores remain within the parent cell for a variable time (in
one case it is stated that germination of the spore occurs within the
interior of the parent cell--"endo-germination"), but are eventually set
free, as a result of the swelling up and solution of the cell membrane
of the parent bacillus in the surrounding liquid, or of the rupture of
that membrane. They then present the following characteristics:
1. Well-formed, dense cell membranes, which renders them extremely
difficult to stain, but when once stained equally difficult to
decolourise.
2. High refractility, which distinguished them from vacuoles.
3. Higher resistance than the parent organism to such lethal agents as
heat, desiccation, starvation, time, etc., this resistance being due to
(a) Low water contents of plasma of the spore.
(b) Low heat-conducting power } of the spore
(c) Low permeability } membrane.
This resistance varies somewhat with the particular species--e. g.,
some spores may resist boiling for a few minutes--but practically all
are killed if the boiling is continued for ten minutes.
~Germination.~--When transplanted to suitable media and placed under
favourable conditions, the spores germinate, usually within twenty-four
to thirty-six hours, and successively undergo the following changes
which may be followed in hanging-drop cultures on a warm stage:
1. Swell up slowly and enlarge, through the absorption of water.
2. Lose their refrangibility.
3. At this stage one of three processes (but the particular process is
always constant for the same species) may be observed:
(a) The spore grows out into the new bacillus without discarding the
spore membrane (which in this case now becomes the cell membrane); _e.
g._, B. leptosporus.
(b) It loses its spore membrane by solution; e. g., B. anthracis.
(c) It loses its spore membrane by rupture.
In this process the rupture may be either polar (at one pole only _e.
g._, B. butyricus), or bipolar (e. g., B. sessile), or equatorial;
(e. g., B. subtilis).
In those cases where the spore membrane is discarded the cell membrane
of the new bacillus may either be formed from--
(a) The inner layer of the spore membrane, which has undergone a
preliminary splitting into parietal and visceral layers; e. g., B.
butyricus.
(b) The outer layers of the cell protoplasm, which become
differentiated for that purpose; e. g., B. megatherium.
The new bacillus now increases in size, elongates, and takes on a
vegetative growth--i. e., undergoes fission--the bacilli resulting
from which may in their turn give rise to spores.
[Illustration: FIG. 92. Simple.]
[Illustration: FIG. 93. Solution.]
[Illustration: FIG. 94. Polar.]
[Illustration: FIG. 95. Bipolar.]
[Illustration: FIG. 96. Equatorial.]
~Food Stuffs.~--1. _Organic Foods._--
(a) The pure parasites (e. g., B. leprae) will not live outside the
living body.
(b) Both saprophytic and facultative parasitic bacteria agree in
requiring non-concentrated food.
(c) The facultative parasites need highly organised foods; e. g.,
proteids or other sources of nitrogen and carbon, and salts.
(d) The saprophytic bacteria are more easily cultivated; e. g.,
1. Some bacteria will grow in almost pure distilled water.
2. Some bacteria will grow in pure solutions of the carbohydrates.
3. _Water_ is absolutely essential to the _growth_ of bacteria.
Food of a definite reaction is needed for the growth of bacteria. As a
general rule growth is most active in media which react slightly acid to
phenolphthalein--that is, neutral or faintly alkaline to litmus. Mould
growth, on the other hand, is most vigourous in media that are strongly
acid to phenolphthalein.
~Environment.~--The influence of physical agents upon bacterial life and
growth is strongly marked.
1. _Atmosphere._--The presence of _oxygen_ is necessary for the growth
of some bacteria, and death follows when the supply is cut off. Such
organisms are termed _obligate aerobes_.
Some bacteria appear to thrive equally well whether supplied with or
deprived of oxygen. These are termed _facultative anaerobes_.
A third class will only live and multiply when the access of free oxygen
is completely excluded. These are termed _obligate anaerobes_.
2. _Temperature._--Practically no bacterial growth occurs below 5 deg. C,
and very little above 40 deg. C. 30 deg. C. to 37 deg. C is the most
favorable for the large majority of micro-organisms.
The maximum and minimum temperatures at which growth takes place, as
well as the optimum, are fairly constant for each bacterium.
Bacteria have been classified, according to their optimum temperature,
into--
MIN. OPT. MAX.
1. Psychrophilic bacteria
(chiefly water organisms) 0 deg. C. 15 deg. C. 30 deg. C.
2. Mesophilic bacteria
(includes pathogenic bacteria) 15 deg. C. 37 deg. C. 45 deg. C.
3. Thermophilic bacteria 45 deg. C. 55 deg. C. 70 deg. C.
The thermal death-point of an organism is another biological constant;
and is that temperature which causes the death of the vegetative forms
when the exposure is continued for a period of ten minutes (see pages
298-301).
3. _Light._--Many organisms are indifferent to the presence of light. On
the other hand, light frequently impedes growth, and alters to a greater
or lesser extent the biochemical characters of the organisms--e. g.,
chromogenicity or power of liquefaction. Pathogenic bacteria undergo a
progressive loss of virulence when cultivated in the presence of light.
4. _Movements._--Movements, if slight and simply of a flowing character,
do not appear to injuriously affect the growth of bacteria; but violent
agitation, such as shaking, absolutely kills them.
A condition of perfect rest would seem to be that most conducive to
bacterial growth.
~The Metabolic Products of Bacteria.~--_Pigment Production._--Many
micro-organisms produce one or more vivid pigments--yellow, orange, red,
violet, fluorescent, etc.--during the course of their life and growth.
The colouring matter usually exists as an intercellular excrementitious
substance. Occasionally, however, it appears to be stored actually
within the bodies of the bacteria. The chromogenic bacteria are
therefore classified, in accordance with the final destination of the
colouring matter they elaborate, into--
_Chromoparous_ Bacteria: in which the pigment is diffused out upon and
into the surrounding medium.
_Chromophorous_ Bacteria: in which the pigment is stored in the cell
protoplasm of the organism.
_Parachromophorous_ Bacteria: in which the pigment is stored in the cell
wall of the organism.
Different species of chromogenic bacteria differ in their requirements
as to environment, for the production of their characteristic pigments;
e. g., some need oxygen, light, or high temperature; others again
favor the converse of these conditions.
_Light Production._--Some bacteria, and usually those originally derived
from water, whether fresh or salt, exhibit marked phosphorescence when
cultivated under suitable conditions. These are classed as "photogenic."
_Enzyme Production._--Many bacteria produce soluble ferments or enzymes
during the course of their growth, as evidenced by the liquefaction of
gelatine, the clotting of milk, etc. These ferments may belong to either
of the following well-recognised classes: proteolytic, diastatic,
invertin, rennet.
_Toxin Production._--A large number, especially of the pathogenic
bacteria, elaborate or secrete poisonous substances concerning which but
little exact knowledge is available, although many would appear to be
enzymic in their action.
These toxins are usually differentiated into--
_Extracellular_ (or Soluble) Toxins: those which are diffused into, and
held in solution by, the surrounding medium.
_Intracellular_ (or Inseparate) Toxins: those which are so closely bound
up with the cell protoplasm of the bacteria elaborating them that up to
the present time no means has been devised for their separation or
extraction.
_End-products of Metabolism._--Under this heading are included--
Organic Acids (e. g., lactic, butyric, etc.).
Alkalies (e. g., ammonia).
Aromatic Compounds (e. g., indol, phenol).
Reducing Substances (e. g., those reducing nitrates to nitrites).
Gases (e. g., sulphuretted hydrogen, carbon dioxide, etc.).
And while the discussion of their formation, etc., is beyond the scope
of a laboratory handbook, the methods in use for their detection and
separation come into the ordinary routine work and will therefore be
described (_vide_ page 276 _et seq._).
X. NUTRIENT MEDIA.
In order that the life and growth of bacteria may be accurately observed
in the laboratory, it is necessary--
1. To _isolate_ individual members of the different varieties of
micro-organisms.
2. To _cultivate_ organisms, thus isolated, apart from other associated
or contaminating bacteria--i. e., in _pure culture_.
For the successful achievement of these objects it is necessary to
provide nutriment in a form suited to the needs of the particular
bacterium or bacteria under observation, and in a general way it may be
said that the nutrient materials should approximate as closely as
possible, in composition and character, to the natural pabulum of the
organism.
The general requirements of bacteria as to their food-supply have
already been indicated (page 142) and many combinations of proteid and
of carbohydrate have been devised, from time to time, on those lines.
These, together with various vegetable tissues, physiological or
pathological fluid secretions, etc., are collectively spoken of as
_nutrient media_ or _culture media_.
The greater number of these media are primarily _fluid_, but, on account
of the rapidity with which bacterial growth diffuses itself through a
liquid, it is impossible to study therein the characteristics of
individual organisms. Many such media are, therefore, subsequently
rendered solid by the addition of substances like gelatine or agar, in
varying proportions, the proportions of such added material being
generally mentioned when referring to the media; e. g., 10 per cent.
gelatine, 2 per cent. agar. Gelatine is employed for the solidification
of those media it is intended to use in the cultivation of bacteria at
the room temperature or in the "cold" incubator. In the percentages
usually employed, gelatine media become fluid at 25 deg. C.; higher
percentages remain solid at somewhat higher temperatures, but the
difficulty of filtering strong solutions of gelatine militates against
their general use.
Media, on the other hand which have been solidified by the addition of
agar, only become liquid when exposed to 90 deg. C. for about ten
minutes, and again solidify when the temperature falls to 40 deg. C.
When it becomes necessary to render these media fluid, heat is applied,
upon the withdrawal of which they again assume their solid condition.
Such media should be referred to as _liquefiable media_; in point of
fact, however, they are usually grouped together with the solid media.
NOTE.--It must here be stated that the designation 10 per
cent. gelatine or 2 per cent. agar refers only to the
quantity of those substances actually added in the process
of manufacture, and _not_ to the percentage of gelatine or
agar, as the case may be, present in the finished medium;
the explanation being that the commercial products employed
contain a large proportion of insoluble material which is
separated off by filtration during the preparation of the
liquefiable media.
Other media, again--e. g., potato, coagulated blood-serum,
etc.--cannot be again liquefied by physical means, and these are spoken
of as _solid_ media.
The following pages detail the method of preparing the various nutrient
media, in ordinary use (see also Chapter XI), those which are only
occasionally required for more highly specialised work are grouped
together in Chapter XII. It must be premised that scrupulous cleanliness
is to be observed with regard to all apparatus, vessels, funnels, etc.,
employed in the preparation of media; although in the preliminary stages
of the preparation of most media absolute sterility of the apparatus
used is not essential.
MEAT EXTRACT.
A watery solution of the extractives, etc., of lean meat (usually beef)
forms the basis of several nutrient media. This solution is termed "meat
extract" and it has been determined empirically that its preparation
shall be carried out by extracting half a kilo of moist meat with one
litre of water. For many purposes, however, it is more convenient to
have a more concentrated extract; one kilo of meat should therefore be
extracted with one litre of water, to form "Double Strength" meat
extract.
It was customary at one time, and is even now in some laboratories to
use either "shin of beef" or "beef-steak"--both contain muscle sugar
which often needs to be removed before the nutrient medium can be
completed. Heart muscle (bullock's heart or sheep's heart) is much to be
preferred and from the point of economy, ease and cleanliness of
manipulation, and extractive value, the imported frozen bullock's hearts
provide the best extract.
Meat extract (Fleischwasser) is prepared as follows:
1. Measure 1000 c.c. of distilled water into a large flask (or glass
beaker, or enamelled iron pot) and add 1000 grammes (roughly, 2-1/2
pounds) of fresh lean meat--e. g., bullock's heart--finely minced in a
mincing machine.
2. Heat the mixture gently in a water-bath, taking care that the
temperature of the contents of the flask does not exceed 40 deg. C. for
the first twenty minutes. (This dissolves out the soluble proteids,
extractives, salts, etc.)
3. Now raise the temperature of the mixture to the boiling-point, and
maintain at this temperature for ten minutes. (This precipitates some
of the albumins, the haemoglobin, etc., from the solution.)
4. Strain the mixture through sterile butter muslin or a perforated
porcelain funnel, then filter the liquid through Swedish filter paper
into a sterile "normal" litre flask, and when cold make up to 1000 c.c.
by the addition of distilled water--to replace the loss from
evaporation.
5. If not needed at once, sterilise the meat extract in bulk in the
steam steriliser for twenty minutes on each of three consecutive days.
Calf, sheep, or chicken flesh is occasionally substituted for the beef;
or the meat extract may be prepared from animal viscera, such as brain,
spleen, liver, or kidneys.
NOTE.--As an alternative method, 5 c.c. of Brand's meat
juice or 3 grammes of Wyeth's beef juice, or 10 grammes
Liebig's extract of meat (Lemco) may be dissolved in 1000
c.c. distilled water, and heated and filtered as above to
form ordinary or single strength meat extract.
Media, prepared from such meat extracts are, however,
eminently unsatisfactory when used for the cultivation of
the more highly parasitic bacteria; although when working in
tropical and subtropical regions their use is well-nigh
compulsory.
~Reaction of Meat Extract.~--Meat extract thus prepared is acid in its
reaction, owing to the presence of acid phosphates of potassium and
sodium, weak acids of the glycolic series, and organic compounds in
which the acid character predominates. Owing to the nature of the
substances from which it derives its reaction, the total acidity of meat
extract can only be estimated accurately when the solution is at the
boiling-point.
Moreover, it has been observed that prolonged boiling (such as is
involved in the preparation of nutrient media) causes it to undergo
hydrolytic changes which increase its acidity, and ~the meat extract only
becomes stable in this respect after it has been maintained at the
boiling-point for forty-five minutes~.
Although meat extract always reacts acid to phenolphthalein, it
occasionally reacts neutral or even alkaline to litmus; and again, meat
extract that has been rendered exactly neutral to litmus still reacts
acid to phenolphthalein. This peculiar behaviour depends upon two
factors:
1. Litmus is insensitive to many weak organic acids the presence of
which is readily indicated by phenolphthalein.
2. Dibasic sodium phosphate which is formed during the process of
neutralisation is a salt which reacts alkaline to litmus, but neutral to
phenolphthalein. In order, therefore, to obtain an accurate estimation
of the reaction of any given sample of meat extract, it is essential
that--
1. The meat extract be previously exposed to a temperature of 100 deg. C.
for forty-five minutes.
2. The estimation be performed at the boiling-point.
3. Phenolphthalein be used as the indicator.
The estimation is carried out by means of titration experiments against
standard solutions of caustic soda, in the following manner:
_Method of Estimating the Reaction._--
_Apparatus Required_: _Solutions Required_:
1. 25 c.c. burette graduated 1. 10N NaOH, accurately
in tenths of a centimetre. standardised.
2. 1 c.c. pipette graduated in 2. n/1 NaOH, accurately
hundredths, and provided standardised
with rubber tube, pinch-cock,
and delivery nozzle.
3. 25 c.c. measure (cylinder or 3. n/10 NaOH, accurately
pipette, calibrated for standardised.
98 deg. C.--_not_ 15 deg. C).
4. Several 60 c.c. conical 4. 0.5 per cent. solution of
beakers or Erlenmeyer phenolphthalein in 50 per
flasks. cent. alcohol.
5. White porcelain evaporating basin, filled with boiling water and
arranged over a gas flame as a water-bath.
6. Bohemian glass flask, fitted as a wash-bottle, and filled
with distilled water, which is kept boiling on a tripod stand.
METHOD.--Arrange the apparatus as indicated in figure 97.
(A) 1. Fill the burette with n/10 NaOH.
2. Fill the pipette with n/1 NaOH.
[Illustration: FIG. 97.--Arrangement of apparatus for titrating media.]
3. Measure 25 c.c. of the meat extract (previously heated in the steamer
at 100 deg. C. for forty-five minutes) into one of the beakers by means of
the measure; rinse out the measure with a very small quantity of boiling
distilled water from the wash-bottle, and then add this rinse water to
the meat extract already in the beaker.
4. Run in about 0.5 c.c. of the phenolphthalein solution and immerse the
beaker in the water-bath, and raise to the boil.
5. To the medium in the beaker run in n/10 NaOH cautiously from the
burette until the end-point is reached, as indicated by the development
of a pinkish tinge, shown in figure 98 (b). Note the amount of
decinormal soda solution used in the process.
NOTE.--Just before the end-point is reached, a very slight
opalescence may be noted in the fluid, due to the
precipitation of dibasic phosphates. After the true
end-point is reached, the further addition of about 0.5 c.c.
of the decinormal soda solution will produce a deep magenta
colour (Fig. 98, c), which is the so-called "end-point" of
the American Committee of Bacteriologists.
[Illustration: FIG. 98.--a, Sample of filtered meat extract or
nutrient gelatine to which phenolphthalein has been added. The medium is
acid, as evidenced by the unaltered colour of the sample. b, The same
neutralised by the addition of n/10 NaOH. The production of this faint
rose-pink colour indicates that the "end-point," or neutral point to
phenolphthalein, has been reached. If such a sample is cooled down to
say 30 deg. or 20 deg. C., the colour will be found to become more distinct
and decidedly deeper and brighter, resembling that shown in c. c, Also
if, after the end-point is reached, a further 0.5 c.c. or 1.0 c.c. n/10
NaOH be added to the sample, the marked alkalinity is evidenced by the
deep colour here shown.]
(B) Perform a "control" titration (occasionally two controls may be
necessary), as follows:
1. Measure 25 c.c. of the meat extract into one of the beakers, wash out
the measure with boiling water, and add the phenolphthalein as in the
first estimation.
2. Run in n/1 NaOH from the pipette, just short of the equivalent of the
amount of _deci_-normal soda solution required to neutralise the 25 c.c.
of medium. (For example, if in the first estimation 5 c.c. of n/10 NaOH
were required to render 25 c.c. of medium neutral to phenolphthalein,
only add 0.48 c.c. of n/1 NaOH.) Immerse the beaker in the water-bath.
3. Complete the titration by the aid of the n/10 NaOH.
4. Note the amount of n/10 NaOH solution required to complete the
titration, and add it to the equivalent of the n/1 NaOH solution
previously run in. Take the total as the correct estimation.
_Method of Expressing the Reaction._--
The reaction or _titre_ of meat extract, medium, or any solution
estimated in the foregoing manner, is most conveniently expressed by
indicating the number of cubic centimetres of normal alkali (or normal
acid) that would be required to render _one litre_ of the solution
exactly neutral to phenolphthalein.
[Illustration: FIG. 99.--Stock bottle for dekanormal soda solution.]
The sign + (plus) is prefixed to this number if the original solution
reacts acid, and the sign - (minus) if it reacts alkaline.
For example, "meat extract + 10," indicates a sample of meat extract
which reacts acid to phenolphthalein, and would require the addition of
10 c.c. of _normal_ NaOH per litre, to neutralise it.
NOTE.--Such a solution would probably react alkaline to
litmus.
Conversely, if as the result of our titration experiments we find that
25 c.c. of meat extract require the addition of 5 c.c. n/10 NaOH to
neutralise, then 1000 c.c. of meat extract will require the addition of
200 c.c. n/10 NaOH = 20 c.c. n/1 NaOH.
And this last figure, 20, preceded by the sign + (i. e., +20), to
signify that it is acid, indicates the reaction of the meat extract.
NOTE.--The standard soda solutions should be prepared by
accurate measuring operations, controlled by titrations,
from a stock solution of 10N NaOH, which should be very
carefully standardised. If a large supply is made or the
consumption is small this stock solution must be kept in an
aspirator bottle to which air can only gain access after it
has been dried and rendered free from CO_{2}. This may be
done by first leading it over H_{2}SO_{4} and soda lime, or
soda lime alone, by some such arrangement as is shown in
figure 99, which also shows a constant burette arrangement
for the delivery of small measured quantities of the
dekanormal soda solution.
STANDARDISATION OF MEDIA.
Differences in the reaction of the medium in which it is grown will
provoke not only differences in the rate of growth of any given
bacterium, but also well-marked differences in its cultural and
morphological characters; and nearly every organism will be found to
affect a definite "optimum reaction"--a point to be carefully determined
for each. For most bacteria, however, the "optimum" usually approximates
fairly closely to +10; and as experiment has shown that this reaction is
the most generally useful for routine laboratory work, it is the one
which may be adopted as the standard for all nutrient media derived from
meat extract.
Briefly, the method of standardising a litre of media to +10 consists in
subtracting 10 from the initial _titre_ of the medium mass; the
remainder indicates the number of cubic centimetres of normal soda
solution that must be added to the medium, per litre, to render the
reaction +10.
~Standardising Nutrient Bouillon.~--For example, 1000 c.c. bouillon are
prepared; at the first titration it is found
1. 25 c.c. require the addition of 5.50 c.c. n/10 NaOH to neutralise.
Two controls give the following results:
2. 25 c.c. require the addition of 5.70 c.c. n/10 NaOH to neutralise.
3. 25 c.c. require the addition of 5.60 c.c. n/10 NaOH to neutralise.