6. Incubate at 37 deg. C. for forty-eight hours and eliminate any
contaminated tubes; store the remainder for future use.
~Egg-albumen (Tarchanoff and Kolesnikoff).~--
1. Place unbroken hens' eggs in dekanormal caustic soda solution for ten
days. (After this time the white becomes firm like gelatine.)
2. Carefully remove the shell and cut the egg into fine slices.
3. Wash for two hours in running water.
4. Place the egg slices in a large beaker and sterilise in the steamer
at 100 deg. C. for one hour.
5. Transfer each slice of egg by means of a pair of sterilised forceps
to a Petri dish or large capsule.
6. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of
three consecutive days.
~Egg Albumin Broth (Lipschuetz).~--
1. Weigh out
Egg albumin (extra fine powder, Merck). 4 grammes
and place in a 2-litre flask with a number of sterile glass beads.
2. Measure out distilled water 200 c.c. into a half-litre flask and warm
to 37 deg. C. in the incubator.
3. Add the water to the flask containing the albumin and beads and
dissolve by shaking.
4. Add n/10-NaOH, 40 c.c. Allow the mixture to stand for thirty minutes
with frequent shaking.
5. Filter through Swedish filter paper.
6. Sterilise by boiling two or three times at intervals of two hours.
7. Add ordinary nutrient bouillon 600 c.c.
8. Fill into small Erlenmeyer flasks in quantities of 50 c.c.
9. Incubate for forty-eight hours at 37 deg. C.--discard any contaminated
flasks and store the remainder for future use.
~Egg Albumin Agar.~--
1. Prepare egg albumin solution as above 1-6.
2. Liquefy and measure out ordinary nutrient agar 600 c.c. and add to
the egg albumin solution (in place of the nutrient broth).
3. Complete as above 8-9.
_Diplococcus Meningitidis Intracellularis._
~Ascitic Fluid Agar (Wassermann)~ _Synonym_ ~N-as-gar (Mervyn Gordon).~
1. Liquefy and measure out into a sterile flask:
Nutrient agar 600 c.c.
2. Measure out into a half litre flask
Distilled water 210 c.c.
and add to it
Ascitic fluid 90 c.c.
Nutrose 6 grammes
3. Heat over a bunsen flame, shaking constantly until the fluid boils,
and the nutrose is dissolved.
4. Add the nutrose ascitic solution to the fluid agar.
5. Heat in the steamer for thirty minutes, then filter.
6. Tube and sterilise as for nutrient agar.
NOTE.--The finished medium in this case measures 900 c.c.
only since inconvenient fractions would be introduced in
making up to one litre exactly.
_Diplococcus Pneumoniae._
~Blood Agar (Washbourn).~--
1. Melt up several tubes of nutrient agar (_vide_ page 167) and allow
them to solidify in the oblique position.
2. Place the tubes, in the horizontal position, in the "hot" incubator
for forty-eight hours, to evaporate off some of the condensation water.
3. Kill a small rabbit with chloroform and nail it out on a board (as
for a necropsy). Moisten the hair thoroughly with 2 per cent. solution
of lysol.
4. Sterilise several pairs of forceps, scissors, etc., by boiling.
5. Reflect the skin over the thorax with sterile instruments.
6. Open the thoracic cavity by the aid of a fresh set of sterile
instruments.
7. Open the pericardium with another set of sterile instruments.
8. Sear the surface of the left ventricle with a red-hot iron and remove
fluid blood from the heart by means of sterile pipettes (e. g., those
shown in Fig. 13, c).
9. Deliver a small quantity of the blood on the slanted surface of the
agar in each of the tubes, and allow it to run over the entire surface
of the medium.
10. Place the tubes in the slanting position and allow the blood to
coagulate.
11. Return the "blood agar" to the hot incubator for forty-eight hours
and eliminate any contaminated tubes. Store the remainder for future
use.
_Media for the Study of Mouth Bacteria Generally._
~Potato Gelatine (Goadby).~--
1. Prepare glycerine potato broth (see page 203, sections 1 to 5).
2. Add 10 per cent. gelatine to the potato decoction and bubble live
steam through the mixture for ten minutes.
3. Estimate the reaction; adjust the reaction of the medium to +5.
4. Cool the medium to below 60 deg. C., clarify with egg as for nutrient
gelatine.
5. Filter through papier Chardin.
6. Tube, and sterilise as for nutrient gelatine.
_Media for the Study of Protozoa._
~Tissue Medium (Noguchi).~--_For spirochaetes (cultivations must be grown
anaerobically)._
1. Plug and sterilise test-tubes 20 x 2 cm.
2. Kill a small rabbit with chloroform vapour. Open the abdomen with
all aseptic precautions, remove kidneys and testicles and transfer to a
sterile glass dish. Cut up the organs with sterile scissors into small
pieces--say 4 millimetre cubes. The four organs should yield from 25 to
30 pieces of tissue.
3. Drop a small piece of sterile tissue into the bottom of each
sterilised tube.
4. Take a flask containing about 400 c.c. nutrient agar (+10 reaction),
liquefy the medium by heat and cool in a water bath to 50 deg. C.
5. Add 200 c.c. ascitic or hydrocele fluid (horse or sheep serum may be
employed, but is not so good) to the liquid agar and mix carefully to
avoid formation of air bubbles.
6. Fill about 20 c.c. of the ascitic agar into each of the sterilised
tubes which already contains a piece of sterile rabbit's tissue, stand
all the tubes upright in racks or a jar, and allow agar to set.
7. After solidification pour sterile paraffin oil on the surface of the
medium in each tube to the depth of 3 centimetres.
8. Incubate tubes at 37 deg. C. for several days and discard any which
prove to be contaminated.
9. Store such tubes as are sterile for future use.
XIII. INCUBATORS.
[Illustration: FIG. 113.--Incubator.]
An incubator (Fig. 113) consists essentially of a chamber for the
reception of cultivations, etc., surrounded by a water jacket, the walls
of which are of metal, usually copper, and outside all an asbestos or
felt jacket, or wooden casing. The water in the jacket is heated by gas
or electricity and maintained at some constant temperature by a
thermo-regulator. The cellular incubator (Fig. 114) which was made for
me[7] some years ago is of the greatest practical utility. Here the
central cavity is subdivided by five double-walled partitions (in which
water circulates in connection with the water tanks at the top and base
of the incubator) and again by iron shelves to form twenty-four pigeon
holes. Into each of these slides an iron drawer 35 cm. long x 12 cm.
wide x 22 cm. high forming a self-contained incubator. The drawer is
fitted with a wooden form to which is fixed a handle and a numbered
label. The thermo-regulating apparatus is the well-known Hearson
capsule.
[Illustration: FIG. 114.--Cellular incubator.]
Two incubators at least are required in the laboratory, for the
cultivation of bacteria the one regulated to maintain a temperature of
37 deg. C., and known as the "hot" incubator; the other, 20 deg. C. to
22 deg. C., and known as the "cool" or "cold" incubator.
Two other incubators, regulated to 42 deg. C. and 60 deg. C. respectively,
whilst not absolutely, necessary very soon justify their purchase.
~Thermo-regulators.~--The thermo-regulator is the most essential portion
of the incubator, as upon its efficient working depends the maintenance
of a constant temperature in the cultivation chamber. It is also used in
the fitting up of water and paraffin baths, and for many other purposes.
[Illustration: FIG. 115.--Reichert's thermo-regulator.]
Of the many forms and varieties of thermo-regulator (other than
electrical), two only are of sufficiently general use to need mention.
In one of these the flow of gas to the gas-jet is controlled by the
expansion or contraction of mercury within a glass bulb; in the other,
by alterations in the position of the walls of a metallic capsule
containing a fluid, the boiling-point of which corresponds to the
temperature at which the incubator is intended to act. They are:
(a) _Reichert's_ (Fig. 115), consists of a bulb containing mercury
which is to be suspended in the medium, whether air or water, the
temperature of which it is desired to regulate. Gas enters at A, and
passes out to the jet by B. As the temperature rises the mercury expands
and cuts off the main gas supply. As the temperature falls the mercury
contracts and reopens the narrow tube C. By means of a thumbscrew D
(which mechanically raises or lowers the column of mercury irrespective
of the temperature) and the aid of a thermometer the apparatus can be
set to keep the incubator at any desired temperature. With this form a
special gas burner is required, with separate supply of gas to a pilot
jet at the side.
(b) _Hearson's capsule regulator_ consists of a metal capsule
hermetically sealed and filled with a liquid which boils at the required
temperature, this is adjusted in the interior of the incubator. Soldered
to the upper side of the capsule is a thick piece of metal having a
central cup to receive the lower end of a rigid rod, through which the
movements of the walls of the capsule are transmitted to the gas valve
fixed outside the incubator.
The gas valve or governor is shown in figure 116. A is the inlet for
gas, C the outlet to burner heating the water jacket, B D a lever
pivoted to standards at G, and acted upon by the capsule, through the
rigid rod which enters the socket below the screw P.
[Illustration: FIG. 116.--Capsule thermo-regulator.]
The construction of the valve is such that, whenever the short arm of
the lever B D presses on the disc below the end B, the main supply of
gas is entirely cut off. At such times, however, a very small portion of
gas passes from A to C, through an aperture inside the valve, the size
of which aperture can be adjusted by the screw needle S, hence the gas
flame below the incubator is never extinguished.
The expansion of the metal walls of the capsule, which takes place upon
the boiling of its contents, provides the motive force, transmitted
through the rigid rod to raise the long arm of the lever B D, and as
this expansion only takes place at a predetermined temperature, the
lever will only be acted upon when the critical temperature is reached,
no sensible effect being produced at even 1 deg. C. below that at which
the capsule is destined to act.
W is a weight sliding on the lever rod D; by increasing the distance
between the weight and the fulcrum of the lower increased pressure is
brought to bear upon the walls of the capsule with the result that the
boiling-point of the liquid in the capsule is slightly raised, and a
range of about two degrees can thus be obtained with any particular
capsule.
FOOTNOTES:
[7] Made by the firm of Chas. Hearson & Co., 235 Regent St., London, W.
XIV. METHODS OF CULTIVATION.
Cultivations of micro-organisms are usually prepared in the laboratory
in one of three ways:
~Tube cultures.~
~Plate cultures.~
~Hanging-drop cultures.~
These may be incubated either ~aerobically~ (i. e., in the presence of
oxygen) or ~anaerobically~ (i. e., in the absence of oxygen, or in the
presence of an indifferent gas, such as hydrogen, nitrogen, or carbon
dioxide).
With regard to the temperature at which the cultivations are grown, it
may be stated as a general rule that all media rendered solid by the
addition of gelatine are incubated at 20 deg. C., or at any rate at a
temperature not exceeding 22 deg. C. (that is, in the "cold" incubator);
whilst fluid media and all other solid media are incubated at 37 deg. C.
(that is, in the "hot" incubator). Exceptions to this rule are numerous.
For instance, in studying the growth of the psychrophylic bacteria, the
yeasts and the moulds, the cold incubator is employed for all media.
Tube cultivations are usually packed in the incubator in small tin
cylinders, such as those in which American cigarettes are sold, or in
square tin boxes. Beakers or tumblers may be used for the same purpose,
but being fragile are not so convenient. Metal test-tube racks, long
enough to just fit into the interior of the incubator and each
accommodating two rows of tubes, are also exceedingly useful.
~AEROBIC.~
~The Preparation of Tube Cultivations.~
The preparation of a tube cultivation consists in:
(a) Inoculating a tube of sterile nutrient medium with a portion of
the material to be examined.
(b) Incubating the inoculated tube at a suitable temperature.
The details of the first of these processes must be varied somewhat
according to whether the tubes of nutrient media are inoculated or
"planted" from--
1. Pre-existing cultivations.
2. Morbid material previously collected (_vide_ page 373).
3. Fluids, tissues, etc., or from the animal body direct.
The method of preparing tube cultivations from pre-existing cultivations
is as follows:
[Illustration: FIG. 117.--Inoculating tubes, seen from the front.]
~1. Fluid Media~ (e. g., Nutrient Bouillon).--
1. Flame the cotton-wool plug of the tube containing the cultivation and
also that of the tube of sterile bouillon.
2. Hold the two tubes, side by side, between the left thumb and the
first and third fingers, allowing the sealed ends to rest on the dorsum
of the hand, and separating the mouths of the tubes (which are pointed
to the right) by the tip of the second finger. Keep the tubes as nearly
horizontal as is possible without allowing the fluid in the bouillon
tube to reach the cotton-wool plug (Fig. 117).
3. Sterilise the platinum loop and allow it to cool.[8]
4. Grasp the plug of the tube containing the cultivation between the
little finger and palm of the hand and remove it from the tube.
5. Grasp the plug of the bouillon tube between the fourth finger and the
ball of the thumb and remove it from the tube.
6. Pass the platinum loop into the tube containing the culture--do not
allow the loop to touch the sides of the tube, or the handle to touch
the medium--and remove a small portion of the growth; withdraw the loop
from the tube, keeping the infected side of the loop downward.
7. Pass the loop into the bouillon tube almost down to the level of the
fluid, reverse the loop so that the infected side faces upward, emulsify
the portion of the growth in the moisture adhering to the side of the
tube which is uppermost. Withdraw the loop.
8. Replug both tubes.
9. Sterilise the platinum loop.
10. Label the bouillon tube with (a) the name of the organism and
(b) the date of inoculation.
11. Incubate.
~2. Solid Media.~--Solid media are stored in tubes in one of two ways:
1. Oblique tube or slanted tube (Fig. 118), in which the medium has been
allowed to solidify whilst the tube was retained in an inclined
position, so forming an extensive surface of medium extending from the
bottom of the tube almost to its mouth.
This is employed for "streak" or "smear" cultivations (_Strichcultur_).
2. Straight tube (Fig. 119), in which the medium forms a cylindrical
mass in the lower portion of the tube and presents an upper surface
which is at right angles to the long axis of the tube.
This is employed for "stab" or "stick" cultivations (_Stichcultur_), or
by inoculating the medium whilst fluid, and allowing to solidify in this
position, for "shake" cultivations.
_Streak Culture._--
1. Flame the plugs, sterilise the platinum loop (or spatula). Open the
tubes and charge the loop as in previous inoculation.
2. Pass the infected loop to the bottom of the tube to be inoculated and
draw it, as lightly as possible, along the centre of the surface of the
medium, terminating the "streak" over the thin layer of medium near the
mouth of the tube.
3. Replug the tubes, sterilise the platinum loop.
4. Label the newly inoculated tube and incubate.
_Smear Culture._--Proceed generally as in streak culture, but rub the
infected loop all over the surface of the medium, instead of restricting
the inoculation to a narrow line.
NOTE.--Gelatine and agar oblique tubes should be freshly
"slanted" before use.
_Stab Culture._--
1. Flame the plugs, open the tubes, sterilise the platinum needle and
charge it with the inoculum as in the previous cultivations.
2. Pass the platinum needle into the tube to be inoculated until it
touches the centre of the surface of the medium. Now thrust it deeply
into the substance of the medium, keeping the needle as nearly as
possible in the axis of the cylinder of medium. Then withdraw the
needle.
3. Replug the tubes. Sterilise the platinum needle.
4. Label the newly planted tube and incubate.
NOTE.--When gelatine is stored for some time the upper
surface of the cylinder becomes concave owing to
evaporation. Tubes showing this appearance should be
liquefied and again allowed to set before use for stab
culture, otherwise when the needle enters the medium, the
surface tension will cause the gelatine cylinder to split.
[Illustration: FIG. 118.--Sloped or slanted medium for streak or smear
culture.]
[Illustration: FIG. 119.--Straight tube.]
_Shake Culture._--
1. Liquefy a tube of nutrient gelatine (or agar, or other similar
medium), by heating in a water-bath (Fig. 121).
2. Inoculate the liquefied medium and label it, etc., precisely as if
dealing with a tube of bouillon.
3. Place the newly planted tube in the upright position (e. g., in a
test-tube rack) and allow it to solidify.
4. Label the tube; when solid, incubate.
_Esmarch's Roll Cultivation._--
1. Liquefy three tubes of gelatine by heat.
2. Prepare three dilutions of the inoculum (as described for
plate cultivations, page 228, steps 4 to 7).
3. Roll the tubes, held almost horizontally, in a groove
made in a block of ice, until the gelatine has set in a thin
film on the inner surface of tube (Fig. 120); or under the
cold-water tap.
[Illustration: FIG. 120. Esmarch's roll culture on block of
ice.]
In order that the medium may adhere firmly to the glass, the
agar used for roll cultivation should have 1 per cent.
gelatine or 1 per cent. gum arabic added to it before
sterilisation.
Roll cultivations, which served a most important purpose in
the days before the introduction of Petri dishes for plate
cultivations, are now obsolete in modern laboratories and
are merely mentioned for the benefit of students, since
examiners who are interested in the academic and historical
aspects of bacteriology sometimes expect candidates to be
acquainted with the method of preparing them.
The Preparation of Plate Cultures.
If a small number of bacteria are suspended in liquefied gelatine, agar,
or other similar medium, and the infected medium spread out in an even
layer over a flat surface and allowed to solidify, each individual
micro-organism becomes fixed to a certain spot and its further
development is restricted to the vicinity of this spot. After a variable
interval the growth of this organism becomes visible to the naked eye
as a "colony." This is the principle upon which the method of plate
cultivation is based and its practice enables the bacteriologist to
study the particular manner of development affected by each species of
microbe when growing (a) unrestricted upon the surface of the medium,
(b) in the depths of the medium. The method itself is as follows:
~Apparatus Required.~--
1. Tripod levelling stand.
2. Large shallow glass dish, with a square sheet of plate
glass to cover it.
3. Spirit level.
4. Case of sterile Petri dishes.
5. Tubes of sterile nutrient media, gelatine (or agar)
previously liquefied by heating in the water-bath and cooled
to 42 deg. C., otherwise the heat of the medium would destroy
many, if not all, of the bacteria introduced.
6. Tube of cultivation to be planted from.
7. Platinum loop.
8. Bunsen burner.
9. Grease pencil.
[Illustration: FIG. 121.--Handy form of water-bath for melting tubes of
agar and gelatine previous to slanting them; or to making shake cultures
or pouring plates.]
Method of "Pouring" Plates.--
1. Place the glass dish on the levelling tripod (Figs. 122, 123); if
gelatine plates are to be poured fill the dish with ice water--gelatine
solidifies so slowly that it is necessary to hasten the process; if agar
is to be used fill with water at 50 deg. C.--agar sets almost immediately
at the room temperature and by slightly retarding the process lumpiness
is avoided; cover the dish with the square sheet of glass.
2. Place the spirit level on the sheet of glass and by means of the
levelling screws adjust the surface of the glass to the horizontal.
This leveling is an important matter since the development of a colony
is to some extent proportionate to the supply of medium available for
its nutrition. Thus in a "smear" on sloped tube culture, the colonies at
the upper part of the medium are stunted and small but increase in size
and luxuriance of growth the nearer they approach to the bottom of the
tube, where there is the greatest depth of medium.
[Illustration: FIG. 122.--Plate-levelling stand.]
3. Place three sterile Petri dishes in a row on the surface of the glass
plate and number them 1, 2, and 3, from left to right.
[Illustration: FIG. 123.--Plate-levelling stand, side view.]
4. Number the previously liquefied tubes of nutrient media 1, 2, and 3.
Flame the plugs and see that each plug can be readily removed from the
mouth of its tube.
5. Add one loopful of the inoculum to tube No. 1, treating the
liquefied medium as bouillon. After replugging, grasp the tube near its
mouth by the thumb and first finger of the right hand, and with an even
circular movement of the whole arm, diffuse the inoculum throughout the
medium; avoid jerky movements, as these cause bubbles of air to form in
the medium.
[Illustration: FIG. 124.--Mixing emulsion for plates.]
The knack of mixing evenly without producing air bubbles, is not always
easily acquired, by this method. An alternative plan is to hold the
inoculated tube vertically upright between the opposed palms and to
rotate it between them by rapid backward and forward movements of the
two hands (Fig. 124).
[Illustration: FIG. 125.--Pouring plates.]
6. Sterilise the platinum loop, and add two loopfuls of diluted inoculum
to tube No. 2, and mix as before.
7. In a similar manner transfer three loopfuls of liquefied medium from
tube No. 2 to tube No. 3, and mix thoroughly.
8. Flame the plug of tube No. 1, remove it, then flame the lips of the
tube; slightly raise the cover of Petri dish No. 1, introduce the mouth
of the tube; then, elevating the bottom of the tube, pour the liquefied
medium into the Petri dish, to form a thin layer. Remove the mouth of
the tube and close the "plate." If the medium has failed to flow evenly
over the bottom of the plate, raise the plate from the levelling
platform and by tilting in different directions rectify the fault.
9. Pour plates No. 2 and No. 3, in a similar manner, from tubes Nos. 2
and 3.
10. Label the plates with the distinctive name or number of the
inoculum, also the date; the number of the dilution having been
previously indicated (step 3).
11. Place in the cool incubator for three or more days, as may be
necessary.
In this way colonies may be obtained quite pure and separate from each
other.
In plate No. 1, probably, the colonies will be so numerous and crowded,
and therefore so small, as to render it useless. In plate No. 2 they
will be more widely separated, but usually No. 3 is the plate reserved
for careful examination, as in this the colonies are usually widely
separated, few in number, and large in size.
_Agar plates_ are poured in a similar manner, but the agar must be
melted in boiling water and then allowed to cool to 45 deg. C. or 42 deg.
C. in a carefully regulated water-bath before being inoculated, and the
entire process must be carried out very rapidly, otherwise the agar will
have solidified before the operation is completed.
NOTE.--In pouring plates, since tube No. 1 (for the first
dilution) rarely gives a plate that is of any practical
value it is frequently replaced by a tube of bouillon or
sterile salt solution, and in such case plate No. 1 is not
poured.
~Surface Plates.~--