Chapter XI: Appendix: 492 (9)

8. Pipette 10 c.c. of the diluted suspension into each of ten sterile test-tubes.

9. Label one of the tubes "Control" and place it in the incubator at 18 deg. C.

10. Add to each of the remaining tubes a sufficient quantity[11] of a concentrated solution of the disinfectant to produce the percentage previously determined upon (_vide_ step 5).

11. Incubate the tubes at 18 deg. C. to 20 deg. C.

12. At hourly intervals remove the control tube and one of the tubes with added disinfectant from the incubator.

13. Make a subcultivation from both the control and the test suspension, upon the surface of nutrient agar; incubate under optimum conditions.

14. Observe these culture tubes from day to day until the completion of seven days, and determine the shortest exposure necessary to cause the death of vegetative forms.

~Superior Lethal Coefficient.~--

1. Prepare surface cultivations of the "test" organisms upon nutrient agar in a culture bottle, and incubate under optimum conditions, for three days, for the formation of their spores.

2. Transfer the emulsion to a sterile test-tube and heat in the differential steriliser for ten minutes at 80 deg. C. to destroy all vegetative forms.

3. Employing that percentage solution of the disinfectant determined in the previous experiment, and complete the investigations as detailed therein, steps 7 to 14, increasing the interval between planting the subcultivations to two, three, or five hours if considered advisable.

NOTE.--Where it is necessary to leave the organisms in
contact with a strong solution of the disinfectant for
lengthy periods, some means must be adopted to remove every
trace of the disinfectant from the bacteria before
transferring them to fresh culture media; otherwise,
although not actually killed, the presence of the
disinfectant may prevent their development, and so give rise
to an erroneous conclusion. Consequently it is essential in
all germicidal experiments to determine first of all the
inhibition coefficient of the germicide employed. Under the
circumstances referred to above it is usually sufficient to
prepare the subcultures in such a volume of fluid nutrient
medium as would suffice to reduce the concentration of the
germicide to about one hundredth of the inhibition
percentage, assuming that the entire bulk of inoculum was
made up of that strength of germicide employed in the test.
In some cases it is a simple matter to neutralise the
germicide and render it inert by washing the organisms in
some non-germicidal solution (such for example as ammonium
sulphide when using mercurial salts as the germicide). When,
however, it is desired to remove the last traces of
germicide proceed as follows:

1. Transfer the suspension of bacteria to sterile
centrifugal tubes; add the required amount of disinfectant,
and allow it to remain in contact with the bacteria for the
necessary period.

2. Centrifugalise thoroughly, pipette off the supernatant
fluid; fill the tube with sterile water and distribute the
deposit evenly throughout the fluid.

3. Centrifugalise again, pipette off the supernatant fluid;
fill the tube with sterile water; distribute the deposit
evenly throughout the fluid, and transfer the suspension to
a litre flask.

4. Make up to a litre by the addition of sterile water;
filter the suspension through a sterile porcelain candle.

5. Emulsify the bacterial residue with 5 c.c. sterile
bouillon.

6. Prepare the necessary subcultivations from this emulsion.

PATHOGENESIS.

_Living Bacteria._--

(a) Psychrophilic Bacteria: When the organism will only grow at or below 18 deg. to 20 deg. C.,

1. Prepare cultivations in nutrient broth and incubate under optimum conditions.

2. After seven days' incubation inject that amount of the culture corresponding to 1 per cent. of the body-weight of a healthy frog, into the reptile's dorsal lymph sac.

3. Observe until death takes place, or, in the event of a negative result, until the completion of twenty-eight days (_vide_ Chapter XVIII).

4. If, and when, death occurs, make a careful post-mortem examination (_vide_ Chapter XIX).

(b) Mesophilic Bacteria: When the organism grows at 35 deg. to 37 deg. C.,

1. Prepare cultivations in nutrient broth and incubate under optimum conditions for forty-eight hours.

2. Select two white mice, as nearly as possible of the same age, size, and weight.

3. Inoculate the first mouse, subcutaneously at the root of the tail, with an amount of cultivation equivalent to 1 per cent. of its body-weight.

4. Inoculate the second mouse intraperitoneally with a similar dose.

5. Observe carefully until death occurs, or until the lapse of twenty-eight days.

6. If the inoculated animals succumb, make complete post-mortem examination.

If death follows shortly after the injection of cultivations of bacteria, the inoculation experiments should be repeated two or three times. Then, if the organism under observation invariably exhibits pathogenic effects, steps should be taken to ascertain, if possible, the minimal lethal dose (_vide infra_) of the growth upon solid media for the frog or white mouse respectively. Other experimental animals--_e. g._, the white rat, guinea-pig, and rabbit--should next be tested in a similar manner.

7. If the inoculated mice are unaffected, test the action of the organism in question upon white rats, guinea-pigs, rabbits, etc.

_Minimal Lethal Dose_ (_m. l. d._); If the purpose of the inoculation is to determine the minimal lethal dose, a slightly different procedure must be followed. For this and other exact experiments a special platinum loop is manufactured, some 2.5 mm. by 0.75 mm., with parallel sides, and calibrated by careful weighing, to determine approximately the amount of moist bacterial growth, the loop will hold when filled.

1. The cultivation must be prepared on a solid medium of the optimum reaction, incubated at the optimum temperature, and injected at the period of greatest activity and vigour, of the particular organism it is desired to test.

2. Arrange four sterile capsules in a row and label them I, II, III, and IV. Into the first deliver 10 c.c. sterile bouillon by means of a sterile graduated pipette; and into each of the remaining three, 9.9 c.c.

3. Remove one loopful of the bacterial growth from the surface of the medium in the culture tube, observing the usual precautions against contamination, and emulsify it evenly with the bouillon in the first capsule. Each cubic centimetre of the emulsion will now contain one-tenth of the organisms contained in the original loopful (written shortly 0.1 loop).

4. Remove 0.1 c.c. of the emulsion in the first capsule by means of a sterile graduated pipette and transfer it to the second capsule and mix thoroughly. Drop the infected pipette into a jar of lysol solution. This makes up the bulk of the fluid in the second capsule to 10 c.c., and therefore every cubic centimetre of bouillon in capsule II contains 0.001 loop.

5. Similarly, 0.1 c.c. of the mixture is transferred from capsule II to capsule III (1 c.c. of bouillon in capsule III contains 0.00001 loop), and then from capsule III to capsule IV (1 c.c. of bouillon in capsule IV contains 0.0000001 loop).

The dilutions thus prepared may be summarised in a table;

Capsule I = 1 loopful + 10 c.c. water [.'.] 1 c.c.=0.1 loop. Capsule II = 0.1 c.c. capsule I + 9.9 c.c. water [.'.] 1 c.c.=0.001 loop. Capsule III = 0.1 c.c. capsule II + 9.9 c.c. water [.'.] 1 c.c.=0.00001 loop. Capsule IV = 0.1 c.c. capsule III + 9.9 c.c. water [.'.] 1 c.c. = 0.0000001 loop.

6. With sterile graduated pipettes remove the necessary quantity of bouillon corresponding to the various divisors of ten of the loop from the respective capsules, and transfer each "dose" to a separate sterile capsule and label; and to such doses as are small in bulk, add the necessary quantity of sterile bouillon to make up to 1 c.c.

7. Multiples of the loop are prepared by emulsifying 1, 2, 5, or 10 loops each with 1 c.c. sterile bouillon in separate sterile capsules.

8. Inoculate a series of animals with these measured doses, filling the syringe first from that capsule containing the smallest dose, then from the capsule containing the next smallest, and so on. If care is taken, it will not be found necessary to sterilise the syringe during the series of inoculations.

9. Plant tubes of gelatine or agar, liquefied by heat, from each of the higher dilutions, say from 0.0000001 loop to 0.01 loop; pour plates and incubate. When growth is visible enumerate the number of organisms present in each, average up and calculate the number of bacteria present in one loopful of the inoculum.

10. The smallest dose which causes the infection and death of the inoculated animal is noted as the minimal lethal dose.

_Toxins._--

Prepare flask cultivations of the organism under observation in glucose formate broth, and incubate for fourteen days under optimum conditions.

(a) Intracellular or Insoluble Toxins:

1. Heat the fluid culture in a water-bath at 60 deg. C. for thirty minutes. (The resulting sterile, turbid fluid is often spoken of as "killed" culture,)

2. Inoculate a tube of sterile bouillon with a similar quantity, and incubate under optimum conditions. This "control" then serves to demonstrate the freedom of the toxin from living bacteria.

3. Inject intraveneously that amount of the cultivation corresponding to 1 per cent. of the body-weight of the selected animal, usually one of the small rodents.

4. Observe during life or until the completion of twenty-eight days, and in the event of death occurring during that period, make a complete post-mortem examination.

5. Repeat the experiment at least once. In the event of a positive result estimate the minimal lethal dose of "killed" culture for each of the species of animals experimented upon.

(b) Extracellular or Soluble Toxins:

1. Filter the cultivation through a porcelain filter candle (Berkefeld) into a sterile filter flask, arranging the apparatus as in the accompanying figure (Fig. 160).

2. Inoculate mice, rats, guinea-pigs, and rabbits subcutaneously with that quantity of toxin corresponding to 1 per cent. of the body-weight of each respectively, and observe, if necessary, until the completion of one month.

3. Inoculate a "control" tube of bouillon with a similar quantity and incubate, to determine the freedom of the filtered toxin from living bacteria.

4. In the event of a fatal termination make complete and careful post-mortem examinations.

5. Repeat the experiments and, if the results are positive, ascertain the minimal lethal dose of toxin for each of the susceptible animals.

The estimation of the _m. l. d._ of a toxin is carried out on lines similar to those laid down for living bacteria (_vide_ page 316) merely substituting 1 c.c. of toxin as the unit in place of the unit "loopful" of living culture.

It frequently happens, during the course of casual investigations that a bouillon-tube culture is available for a toxin test whilst a flask cultivation is not. In such cases, Martin's small filter candle and tube (Fig. 161) specially designed for the filtration of small quantities of fluid, is invaluable. This consists of a narrow filter flask just large enough to accommodate an ordinary 18 x 2 cm. test-tube. The mouth of the tubular Chamberland candle 15 x 1.5 cm. is closed by a perforated rubber cork into which fits the end of the stem of a thistle headed funnel, whilst immediately below the butt of the funnel is situated a rubber cork to close the mouth of the filter flask. When the apparatus is fixed in position and connected to an exhaust pump, the cultivation is poured into the head of the funnel and owing to the relatively large filtering surface the germ free filtrate is rapidly drawn through into the test-tube receiver.

~Raising the Virulence of an Organism.~--If it is desired to raise or "exalt" the virulence of a feebly pathogenic organism, special methods of inoculation are necessary, carefully adjusted to the exigencies of each individual case. Among the most important are the following:

1. _Passage of Virus._--The inoculation of pure cultivations of the organism into highly susceptible animals, and passing it as rapidly as possible from animal to animal, always selecting that method of inoculation-e. g., intraperitoneal--which places the organism under the most favorable conditions for its growth and multiplication.

2. _Virus Plus Virulent Organisms._--The inoculation of pure cultivations of the organism together with pure cultivations of some other microbe which in itself is sufficiently virulent to ensure the death of the experimental animal, either into the same situation or into some other part of the body. By this association the organism of low virulence will frequently acquire a higher degree of virulence, which may be still further raised by means of "passages" (_vide supra_).

3. _Virus Plus Toxins._--The inoculation of pure cultivations of the organism into some selected situation, together with the subcutaneous, intraperitoneal, or intravenous injection of a toxin--e. g., one of those elaborated by the proteus group--either simultaneously with, before, or immediately after, the injection of the feeble virus. By this means the natural resistance of the animal is lowered, and the organism inoculated is enabled to multiply and produce its pathogenic effect, its virulence being subsequently exalted by means of "passages."

~Attenuating the Virulence of an Organism.~--Attenuating or lowering the virulence of a pathogenic microbe is usually attained with much less difficulty than the exaltation of its virulence, and is generally effected by varying the environment of the cultivations, as for example:

1. Cultivating in such media as are unsuitable by reason of their (a) composition or (b) reaction.

2. Cultivating in suitable media, but at an unsuitable temperature.

3. Cultivating in suitable media, but in an unsuitable atmosphere.

4. Cultivation in suitable media, but under unfavorable conditions as to light, motion, etc.

Attenuation of the virus can also be secured by

5. Passage through naturally resistant animals.

6. Exposure to desiccation.

7. Exposure to gaseous disinfectants.

8. By a combination of two or more of the above methods.

IMMUNISATION.

The further study of the pathogenetic powers of any particular bacterium involves the active immunisation of one or more previously normal animals. This end may be attained by various means; but it must be remembered that immunisation is not carried out by any hard and fast rule or by one method alone, but usually by a combination of methods adapted to the exigencies of each particular case. The ordinary methods include:

A. Active Immunisation.

I. By inoculation with dead bacteria (i. e., bacteria
killed by heat; the action of ultra-violet rays, of chemical
germicides, or by autolysis).

II. By the inoculation of attenuated strains of bacteria.

III. By the inoculation of living virulent bacteria (exalted
in virulence if necessary).

B. Combined Active and Passive Immunisation:

IV. By the inoculation of toxin-antitoxin mixtures.

ACTIVE IMMUNISATION.

The immunisation of the rabbit against the Diplococcus pneumoniae may be instanced as an example of the general methods of immunisation of laboratory animals.

1. Take a full grown rabbit weighing not less than 1200 to 1500 grammes (large rabbits of 2000 grammes and over are the most suitable for immunising experiments). Observe weight and temperature carefully during the few days occupied in the following steps.

2. Inoculate a small rabbit intraperitoneally with one or two loopfuls of a twenty-four-hour-old blood agar cultivation of a _virulent_ strain of Diplococcus pneumoniae.

Death should follow within twenty-four hours, and in any case will not be delayed beyond forty-eight hours.

3. Under aseptic precautions, at the post-mortem, transfer a loopful of heart blood to an Erlenmeyer flask containing 50 c.c. sterile nutrient broth. Incubate at 37 deg. C. for twenty-four hours.

4. Prepare also several blood agar cultures from the heart blood of the rabbit, label them all O.C. (original culture). After twenty-four hours incubation at 37 deg. C. place an india-rubber cap over the plugged mouth of the tube of all but one of these cultures and paint the cap with Canada balsam or shellac varnish, dry, and replace in the hot incubator.

This will prevent evaporation, and cultures thus sealed will remain unaltered in virulence for a considerable time.

5. Make a fresh subcultivation on blood agar from the uncapped O.C. cultivation and after twenty-four hours incubation at 37 deg. C. determine the minimal lethal dose of this strain upon a series of mice (see page 316).

6. Suspend the flask containing the twenty-four-hour-old broth culture (step 3) in the water-bath at 60 deg. C. for one hour. Cool the flask rapidly under a stream of cold water.

7. Determine the sterility of this (?) killed cultivation by transferring one cubic centimetre to each of several tubes of nutrient broth, and incubate at 37 deg. C. for twenty-four hours. If growth of Diplococcus pneumoniae occurs, again heat culture in water-bath at 60 deg. C. for one hour and again test for sterility.

8. Inject the selected rabbit intravenously (see page 363) with 2 c.c. of the killed cultivation, and inject a further 10 c.c. into the peritoneal cavity.

During the next few days the animal will lose some weight and perhaps show a certain amount of pyrexia.

9. When the temperature and weight have again returned to normal--generally about seven days after the inoculation--again inject killed cultivation, this time giving a dose of 5 c.c. intravenously and 20 c.c. intraperitoneally. A temperature and weight reaction similar to, but less marked than that following the first injection will probably be observed, but after about a week's interval the animal will be ready for the next injection.

10. When ready to give the third injection prepare a fresh blood agar subculture from another O.C. tube and after twenty-four hours incubation prepare a minimal lethal dose (as determined in 5) and inject it subcutaneously into the rabbit's abdominal wall.

A slight local reaction will probably be observed as well as the weight and temperature reactions.

11. A week to ten days later inject a similar minimal lethal dose into the peritoneal cavity.

12. Observe the weight and temperature of the rabbit very carefully, and regulating the dates of inoculation by the animal's general condition, continue to inject living cultivations of the pneumococcus into the peritoneal cavity, gradually increasing the dose by multiples of ten.

13. At intervals of two months samples of blood may be collected from the posterior auricular vein and the serum tested for specific antibodies.

14. Under favourable conditions it will be found after some six months steady work that the rabbit may be injected intraperitoneally with an entire blood agar cultivation without any ill effects being apparent; and this characteristic--resistance to the lethal effects of large doses of the virus--is the sole criterion of _immunity_. Further, the serum separated from blood withdrawn from the animal about a week after an injection, if used in doses of .01 c.c., will protect a mouse against the lethal effects of at least ten minimal lethal doses of living pneumococci.

In the foregoing illustration it has been assumed that complete acquired active immunity has been conferred upon the experimental rabbit in consequence of the formation of antibody, specific to the diplococcus pneumoniac, sufficient in amount to ensure the destruction of enormous doses of the living cocci--the _antigen_ (that is the substance injected in response to which _antibody_ has been elaborated) in this particular case being the bacterial protoplasm of the pneumococcus with its endo-toxins.

But provided death does not immediately follow the injection of the antigen, specific antibody is always formed in greater or lesser amount; and in experimental work a sufficient amount of any required antibody can often be obtained without carrying the process of immunisation to its logical termination.

For instance, if the immunisation of a rabbit toward Bacillus typhosus is commenced on the lines already set out it will often be found, after a few injections of "killed" cultivation that the blood serum of the animal (even when diluted with several hundred times its volume of normal saline) contains specific agglutinin for B. typhosus--and if the sole object of the experiment has been the preparation of agglutinin the inoculations may well be stopped at this point, although the animal is not yet immune in the strict meaning of the word.

Again, antibodies may be formed in response to antigens other than infective particles--thus the injection into suitable animals of foreign proteins such as egg albumin, heterologous blood sera or red blood discs from a different species of animal, will result in the formation of specific antibodies possessing definite affinities for their respective antigens.

The most important antibody of this latter type is Haemolysin, a substance that makes its appearance in the blood serum of an animal previously injected with washed blood cells from an animal of a different species. The serum from such an animal possesses the power of disintegrating red blood discs of the variety employed as antigen and causing the discharge of their contained haemoglobin, and is specific in its action to the extent of failing to exert any injurious effect upon the red blood cells of any other species of animal.

The action of this serum is due to the presence of two distinct bodies, complement and haemolysin.

_Complement_ (or alexine) is a thermo-labile readily oxidised body present in variable but unalterable amount in the normal serum of every animal. It is a substance which exerts a lytic effect upon all foreign matter introduced into the blood or tissues; but by itself is a comparatively inert body, and is only capable of exerting its maximum lytic effect in the presence of and in combination with a specific antibody, or immune body.

Complement is obtained (unmixed with antibody) by collecting fresh blood serum from any healthy normal (that is uninoculated) animal. Guinea-pigs' serum is that most frequently employed for experimental work.

_Haemolysin_ (immune body, copula, sensitising body, amboceptor) is a _thermostable_ antibody formed in response to the injection of red cells which although in itself inert is capable of linking up complement present in the normal serum to the red cells of the variety used as antigen--a combination resulting in haemolysis.

Haemolysin is obtained by collecting fresh blood serum from a suitably inoculated animal and exposing it to a temperature of 56 deg. C. (to destroy the thermo-labile complement) for 15 to 30 minutes before use. It is then referred to as _inactivated_, and is _reactivated_ by the addition of fresh normal serum--that is serum containing complement.

Haemolysin is of importance academically owing to the fact that many of the problems of immunity have been elucidated by its aid; but its present practical importance lies in the application of the _haemolytic system_ (that is haemolysin, corresponding erythrocyte solution and complement) to certain laboratory methods having for their object either the identification of the infective entity or the diagnosis of the existence of infection.

For use in these laboratory methods of diagnosis it is most convenient to prepare haemolytic serum specific for human blood--whether the laboratory is isolated or attached to a large hospital. Ox blood, sheep blood or goat blood if readily obtainable, may however be used instead, and although the following method is directed to the preparation of human haemolysin the same procedure serves in all cases.

THE PREPARATION OF HAEMOLYTIC SERUM.

_Apparatus Required:_

Small centrifuge, preferably electrically driven, with two
receptacles for tubes, and enclosed in a safety shield (Fig. 162).
Sterile centrifuge tubes (10 c.c. capacity), Fig. 163.
Sterile pipettes (10 c.c. graduated) in case.
Sterile glass capsules (in case).
Sterile test-tubes.
Sterile all glass syringe (5 c.c. or 10 c.c. capacity)
and needle.

_Reagents Required:_

Normal saline solution.
10 per cent. sodium citrate solution in normal saline.
Human blood (_vide infra_).

METHOD.--

1. Select a healthy full-grown rabbit of not less than 2500 grammes weight in accordance with the directions already given (page 322) and prepare it for intraperitoneal inoculation.

2. Measure out 2 c.c. citrated human blood (collected at a surgical operation or a venesection, or withdrawn by venipuncture from the median basilic or median cephalic vein of a normal adult) into a centrifuge tube and centrifugalise thoroughly.

3. Wash with three changes of normal saline (_vide_ also page 388).

4. Transfer the washed cells to a sterile capsule by means of a sterile pipette. Add 5 c.c. of normal saline and mix thoroughly.

5. Take up the mixture of cells and saline in the all-glass syringe and inject into the peritoneal cavity of the rabbit.

6. Seven days later inject intraperitoneally the washed cells from 5 c.c. human blood mixed with 5 c.c. normal saline.

7. Seven days later inject the washed cells from 10 c.c. human blood mixed with 5 c.c. normal saline.

8. After a further interval of seven days repeat the injection of washed cells from 10 c.c. human blood mixed with 5 c.c. normal saline.

NOTE.--Better results are obtained if the second and
subsequent injections are made intravenously, even when
smaller quantities of washed red cells are employed. If,
however, the intravenous route is selected exceeding great
care must be exercised to avoid the introduction of air into
the vein--an accident which is followed, within a few
minutes, by the death of the rabbit from pulmonary embolism.

9. Allow five days to elapse, then collect a preliminary sample of blood, say about 2 c.c., from the rabbit's ear. Allow it to clot, separate off the serum and transfer to a sterile test-tube. Place the test-tube in a water-bath at 56 deg. C. for fifteen minutes (to inactivate) and test the serum quantitatively for haemolytic properties in the following manner:

THE TITRATION OF HAEMOLYTIC SERUM.

_Apparatus Required:_

Electrical centrifuge.
Sterile centrifuge tubes.
Water-bath regulated at 56 deg. C.
Sterilised pipettes 10 c.c. graduated in tenths.
Sterilised pipettes 1 c.c. graduated in tenths.
Sterile test-tubes, 16 x 2 cm.
Small sterile test-tubes, 9 x 1 cm.
Small test-tube rack, or roll of plasticine.
Capillary teat pipettes.
Stout rubber band or length of small rubber tubing.

_Reagents Required and Method of Preparation:_

1. Normal saline solution.

2. Haemolytic serum inactivated by preliminary heating to 56 deg.
C. for 15 minutes (_vide supra_) in test-tube labelled H. S.

3. Complement. Fresh guinea-pig serum in test-tube labelled
C.

Kill a normal guinea-pig with chloroform vapour.

Open the thorax with all aseptic precautions, and collect as
much blood as possible from the heart with a sterile Pasteur
pipette.

Transfer it to a sterile centrifuge tube and place the tube
in the incubator at 37 deg. C. Two hours later separate the clot
from the sides of the tube, and centrifugalise thoroughly.

Pipette off the clear serum to a clean sterilised test-tube.

4. Erythrocyte solution, in test-tube labelled E.

Collect and wash human red blood cells (see page 388, 1-8).
Measure the volume of red cells available and prepare a 2
per cent. suspension in normal saline solution.

METHOD.--

1. Take two test-tubes and number them 1 and 2, and pipette into each 9 c.c. of normal saline solution.

2. Add 1 c.c. of haemolytic rabbit serum to tube No. 1 and mix thoroughly: take up 1 c.c. of the mixture and add it to tube No. 2; mix thoroughly.

3. Set up ten small test-tubes in test-tube rack or in roll of plasticine, and number 1 to 10.

4. Pipette into tube No. 1 0.5 c.c. = 0.5 c.c.}
haemolytic serum } From tube
Pipette into tube No. 2 0.1 c.c. = 0.1 c.c. } H. S.
haemolytic serum }

Pipette into tube No. 3 0.5 c.c. = 0.05 c.c. }
haemolytic serum }
Pipette into tube No. 4 0.3 c.c. = 0.03 c.c. }
haemolytic serum } From
Pipette into tube No. 5 0.2 c.c. = 0.02 c.c. } tube 1.
haemolytic serum }
pipette into tube No. 6 0.1 c.c. = 0.01 c.c. }
haemolytic serum }

Pipette into tube No. 7 0.5 c.c. = 0.005 c.c. }
haemolytic serum }
Pipette into tube No. 8 0.3 c.c. = 0.003 c.c. }
haemolytic serum } From
Pipette into tube No. 9 0.2 c.c. = 0.002 c.c. } tube 2.
haemolytic serum }
Pipette into tube No. 10 0.1 c.c. = 0.001 c.c. }
haemolytic serum }

5. To each tube add 1 c.c. of erythrocyte solution.

6. When necessary (that is to say in tubes 2, 4, 5, 6, 8, 9 and 10) add normal saline solution to the mixture in the test-tubes till the column of fluid in each reaches to the same level.

7. Shake each tube in turn, so as to thoroughly mix its contents. Plug the mouth of each tube with cotton wool, and place entire set in the incubator at 37 deg. C. for one hour.

8. Remove the tubes from the incubator and into each tube pipette 0.1 c.c. complement (guinea-pig's serum) and replace tubes in incubator at 37 deg. C. for further period of one hour.

9. Remove the tubes from the incubator, and if complete haemolysis has not taken place in every tube, stand on one side, preferably in the ice chest, for an hour.

10. Then examine the tubes.

Complete haemolysis is indicated by a clear red solution,
with no deposit of red cells at the bottom of the test-tube.

Absence of haemolysis is indicated by a clear or turbid
colourless fluid, with a deposit of red cells at the bottom
of the test-tubes.

The smallest amount of haemolytic serum that has caused complete haemolysis is known as the minimal haemolytic dose (_M. H. D._) and if haemolysis has occurred in all the tubes down to No. 7--the m. h. d. of this particular serum is .005 c.c. = 200 minimal haemolytic doses per cubic centimetre. Such a serum is strong enough for experimental work; indeed, for many purposes, complete haemolysis down to tube 6 will indicate a serum sufficiently strong(= 100 m. h. d. per cubic centimetre). If, however, only the first one or two tubes are completely haemolysed, this is an indication that the rabbit should receive further injections in order to raise the haemolytic power to a sufficiently high level.

STORAGE OF HAEMOLYSIN.

If, and when the haemolysin content of the rabbit's serum is found to be sufficient, destroy the animal by chloroform vapour.

Remove as much of its blood as possible from the heart under aseptic precautions into sterilized centrifuge tubes.

Transfer the tubes of blood to the incubator at 37 deg. C. for two hours--then centrifugalize thoroughly.

Pipette off the clear serum, and fill in quantities of 1 c.c., into small glass ampoules or pipettes, and hermetically seal in the blowpipe flame, care being taken to avoid scorching the serum.

Place the ampoules when filled with serum and sealed, in a water-bath at 56 deg. C. for 30 minutes. This destroys the complement, i. e., inactivates the serum, and at the same time, provided the various operations have been carried out under aseptic precautions, ensures its sterility. A longer exposure reduces the haemolytic power.

Place the ampoules in a closed metal box and store in the ice chest for future use.

FOOTNOTES:

[10] The quantities here given are not absolutely correct. If exactitude is essential the student must calculate the amount required by the aid of the Percentage Formula, Appendix, page 496.

[11] See Percentage Formula, Appendix, page 496.

XVII. EXPERIMENTAL INOCULATION OF ANIMALS.

The use of living animals for inoculation experiments may become a necessary procedure in the Bacteriological Laboratory for some one or more of the following reasons:

A. ~Determination of Pathogenetic Properties of Bacteria already Isolated in Pure Culture~ (see page 315).

The exact study of the conditions influencing the virulence (including its maintenance, exaltation and attenuation) of an organism, and precise observations upon the pathogenic effects produced by its entrance into, and multiplication within the body tissues can obviously only be carried out by means of experimental inoculation; whilst many points relating to vitality, longevity, etc., can be most readily elucidated by such experiments.

B. ~Isolation of Pathogenetic Bacteria.~

Certain highly parasitic bacteria (which grow with difficulty upon the artificial media of the laboratory) can only be isolated with considerable difficulty from associated saprophytic bacteria when cultural methods alone are employed; but if the mixture of parasite and saprophytes is injected into an animal susceptible to the action of the former, the pathogenic organism can readily be isolated from the tissues of the infected animal. The pneumococcus for example occurs in the sputum of patients suffering from acute lobar pneumonia, but usually in association with various saprophytes derived from the mouth and pharynx. The optimum medium for the growth of the pneumococcus, blood agar, is also an excellent pabulum for the saprophytes of the mouth, and plate cultures are rapidly overgrown by them to the destruction of the more delicate pneumococcus. But inoculate some of the sputum under the skin of a mouse and three or four days later the pneumococcus will have entered the blood stream (leaving the saprophytes at the seat of inoculation) and killed the animal. Cultivations made at the post-mortem (see page 398) from the mouse's heart blood will yield a pure growth of the pneumococcus.

C. ~Identification of Pathogenetic Bacteria.~

The resemblances, morphological and cultural, existing between certain pathogenetic bacteria are in some cases so great as to completely overwhelm the differences; again the same bacterium may under varying conditions assume appearances so different from those regarded as typical or normal as to throw doubt on its identity. In each case a simple inoculation experiment may decide the point at once. As a concrete example may be instanced an autopsy on an animal dead from an unknown infection. Cultivations from the heart blood gave a pure growth of a typical (capsulated) pneumococcus. Cultivations from the liver gave a pure growth of what appeared to be a typical (non-capsulated) Streptococcus pyogenes longus. The latter inoculated into a rabbit caused the death of the animal from pneumococcic septicaemia, and cultures from the rabbit's blood gave a pure growth of a typical (capsulated) pneumococcus.

~D. Study of the Problems of Immunity.~

It is only by a careful and elaborate study of the behaviour of the animal cell and the body fluids vis-a-vis with the infecting bacterium that it becomes possible to throw light upon the complex problem whereby the cell opposes successful resistance to the diffusion of the invading microbe, or succeeds in driving out the microbe subsequently to the occurrence of that diffusion.

At the moment, however, our attention is directed to the first of these broad headings, for it is by the application of the knowledge acquired in its pursuit that we are able to deal with problems arising under any of the remainder.

For whatever purpose the inoculation is performed, it is essential that the experiment should be planned to secure the maximum amount of information and the minimum of discomfort to the animal used. Every care therefore must be taken to ensure that the virus is introduced into the exact tissue or organ selected; and the operation itself must be carried out with skill and expedition, and under strictly aseptic conditions.

In the course of inoculation studies many instances of natural immunity, both racial and individual, will be met with; but it must be recollected that natural immunity is relative only and never absolute, and care be taken not to label an organism as _non-pathogenic_ until many different methods of inoculation have been performed upon different species of animals, combined when necessary with various procedures calculated to overcome any apparent immunity, and have invariably given negative results.

In some countries experiments upon animals are only permitted under direct license from the Government, and then only within premises specially licensed for the purpose. In England this license is in the grant of the Home Secretary, and confers the permission to experiment upon animals under general anaesthesia, provided that after the experiment is completed the animal must be destroyed before regaining consciousness. If it is intended to carry out simple hypodermic inoculations and superficial venesections, Certificate A, granting this specific permission and dispensing with the necessity for general anaesthesia must be obtained _in addition to the license_; whilst if the inoculation entails more extensive operative procedures, and it is necessary to observe the subsequent course of the infection, should such occur, the license must be _coupled with Certificate B_--since this certificate removes the compulsion to destroy the animal whilst under the anaesthetic. Further special certificates and combinations of certificates are required if cats, dogs, horses, asses or cattle are to be the subjects of experiment. Under every certificate it is expressly stipulated that if the animal shows signs of pain it must be destroyed immediately.

The animals generally employed in the study of the pathogenic properties of the various micro-organisms are:

_Cold Blooded._ _Warm Blooded._ _Hot Blooded._
Frog. Mouse. Fowl.
Toad. Rat. Pigeon.
Lizard. Guinea pig.
Rabbit.
Monkey.

~Preparation.~--Before inoculation, the experimental animals should be carefully examined, to avoid the risk of employing such as are already diseased: since it must be remembered that in a state of nature, as well as in captivity, the animals employed for laboratory inoculations are subject to infection by various animal and vegetable parasites, and in some instances such infection presents no symptoms which are obvious to the casual examination; the sex should be noted, the weight recorded, and the rectal temperature taken. The remaining items of importance are the time of the inoculation, the material that is inoculated, and the method of inoculation, and finally under what authority the experiment is performed. In the author's laboratory these data are entered upon a pink card which forms part of a card index system. The card further provides space for notes on the course of the resulting infection, and carries on the reverse the weight and temperature chart (Figs. 164 and 165).

~Preliminary Inspection and Examination.~--The preliminary examination should comprise observation of the animal at rest and in motion; the appearance of the fur, feathers or scales, inspection of the eyes, and of external orifices of the body; tactile examination of the body and limbs, and palpation of the groins and abdomen; and in many cases the microscopical examination of fresh and stained blood-films.

Some of the commoner forms of naturally acquired infection may be briefly mentioned, without however touching upon the various fleas, lice and ticks which at times infect the ordinary laboratory animals.

~The Rabbit~, particularly in captivity, is subject to attacks of Psoric Acari, and the infection is readily transmitted to rabbits in neighbouring cages and also to guinea pigs, but not to rats and mice. One species (_Sarcoptes minor_ var. _cuniculi_) gives rise to the ordinary mange. The infection first shows itself as thick yellowish scales and crusts around the nose, mouth and eyes, spreads to the bases and outer surfaces of the ears (never to the inside of the concha), to the fore and hind legs and into the groins and around the genitals. The acari can be readily demonstrated microscopically in scrapings of the skin, treated with liquor potassae. Another form of scabies (due to Psoroptes _communis cuniculi_) commences at the bottom of the concha, which is filled with whitish-yellow masses consisting of dried crusts, scales, faeces, and dead acari. The base of the ear is hard and swollen, and lifting the animal by the ears--as is usually done--gives rise to considerable pain; indeed this symptom may be the one which first attracts attention to an infection, which causes progressive wasting and terminates in death. A mixed infection--sarcoptic plus psorotic acariasis--is sometimes seen.

If it is decided to try and save animals suffering from infection by these parasites, they must be segregated, the scabs carefully cleaned from the infected areas and the denuded surfaces washed with 5 per cent. solution of Potassium persulphate (a few drops being allowed to run into the concha), or with a preparation containing equal parts of soft paraffin and vaseline with a few drops of lysol. This treatment should be repeated daily until the acarus is destroyed and the animal has regained its normal condition. The cages should be disinfected and all neighbouring animals carefully examined, and any which show signs of infection should be treated in a similar manner. Favus also attacks the rabbit, and the typical spots are first noted around the base of the ear.

Infection by _Coccidium oviforme_ is very common, without however presenting any symptoms by which the infection may be recognised. Usually the condition is only noted post-mortem, when the liver is found to be studded with numerous cascating tubercles, which on examination prove to be cystic areas crowded with coccidia. Sometimes too the liver of a rabbit dead from some intentional or accidental bacterial infection is found at the post-mortem to be marked by fine yellowish streaks and small tubercles due to the embryos of _Taenia serrata_, while the cystic form (_Cysticercus pisiformis_) is often noted free in the peritoneal cavity, or invading the mesentery.

Abscess formation from infection with ordinary pyogenic bacteria occurs naturally in the rabbit, and frequently the animal house of a laboratory is decimated by an infective septicaemia due to _B. cuniculicida_.

The ~Mouse~ and ~Rat~ suffer from septicaemia, and from the cysticercus form of _Taenia murina_; the cystic form (_Cysticercus fasciolaris_) of _T. crassicollis_ has its habitat in their livers. These small rodents are frequently infected with scabies, but if freely provided with clean straw will clean themselves by rubbing through it. The mouse is also attacked by favus, and the rat is often infected with _Trypanosoma Lewisi_.

The ~Guinea pig~, like the rabbit, suffers from scabies and coccidiosis. In addition it is often naturally infected with _B. tuberculosis_, and it is a wise precaution to test animals as soon as they reach the laboratory by injecting Koch's Old Tuberculin--0.5 c.c. causing death in the tuberculous cavy within 48 hours.

The ~Monkey~ is naturally prone to tuberculosis, and should be injected with 1 c.c. Old Tuberculin on arrival in the laboratory. The tissues of the monkey also serve as the habitat for a Nematode worm parasitic in cattle (_Oesophagostoma inflatum_) resembling the Anchylostomum, and this parasite frequently bores through the intestinal wall, and provokes the formation of small cysts in the immediately adjacent mesentery. The presence of these cysts may give rise to considerable speculation at the post-mortem.

The ~Pigeon~ may be infected by _Haemosporidia_, and its blood show the presence of halteridia. This bird may also be the subject of a bacterial infection known as pigeon diphtheria; while the fowl may be subject to scabies and ringworm, or suffer from fowl cholera or fowl septicaemia--infections due to members of the haemorrhagic septicaemia group.

~Weighing.~--The larger animals are most conveniently weighed in a decimal scale provided with a metal cage for their reception instead of the ordinary pan (Fig. 166). Mice and rats are weighed in a modification of the letter balance, weighing to 250 grammes, which has a conical wire cage, (carefully counterpoised) substituted for its original pan (Fig. 167).

~Temperature.~--To take the rectal temperature of any of the laboratory animals, the animal should be carefully and firmly held by an assistant. Introduce the bulb of an ordinary clinical thermometer, well greased with vaseline, just within the sphincter ani. Allow it to remain in this position for a few seconds, and then push it on gently and steadily until the entire bulb and part of the stem, as far as the constriction, have passed into the rectum. Three to five minutes later, the time varying of course with the sensibility of the thermometer used, withdraw the instrument and take the reading. The thermometers employed for recording temperature should be verified from time to time by comparison with a standard Kew certified Thermometer kept in the laboratory for that purpose.

~Cages.~--During the period which elapses between inoculation and death, or complete recovery, the experimental animals must be kept in suitable receptacles which can easily be kept clean and readily disinfected.

The _mouse_ is usually stored in a glass jar (Fig. 168) 11 cm. high and 11 cm. in diameter, closed by a wire gauze cover which is weighted with lead or fastened to the mouth of the jar by a bayonet catch. A small oblong label, 5 cm. by 2.5 cm., sand-blasted on the side of the cylinder, is a very convenient device as notes made upon this with an ordinary lead pencil show up well and only require the use of a damp cloth to remove them (Fig. 168).

The _rat_ is kept under observation in a glass jar similar, but larger, to that used for the mouse.

A layer of sawdust at the bottom of the jar absorbs any moisture and cotton-wool or paper shavings should be provided for bedding. The food should consist of bran and oats with an occasional feed of bread-and-milk sop.

The use of a metal tripod, on the platform of which are soldered two small cups for the reception of the food, inside the cage, prevents waste of food or its contamination with excreta (Fig. 169).

After use the jars and tripods are sterilised either by chemical reagents or by autoclaving.

The _rabbit_ and the _guinea-pig_ are confined in cages of suitable size, made entirely of metal (Fig. 170). The sides and top and bottom are of woven wire work; beneath the cage is a movable metal tray filled with sawdust, for the reception of the excreta. The cage as a whole is raised from the ground on short legs. The sides, etc., are generally hinged so that the cage packs up flat, for convenience of storing and also of sterilising.

The ordinary rat cage, a rectangular wire-work box, 30 cm. from front to back, 20 cm. wide, and 14 cm. high, makes an excellent cage for guinea-pigs if fitted with a shallow zinc tray, 35 cm. by 24 cm., for it to stand upon.

A plentiful supply of straw should be provided for bedding and the food should consist of fresh vegetables, cabbage leaves, carrot and turnip tops and the like for the morning meal and broken animal biscuits for the evening meal. Occasionally a little water may be placed in the cage in an earthenware dish.

The tray which receives the dejecta should be cleaned out and supplied with fresh sawdust each day, and the soiled sawdust, remains of food, etc., should be cremated.

These cages are sterilised after use either by autoclaving or spraying with formalin.

As ~animal inoculation~ is purely a surgical operation, the necessary instruments will be similar to those employed by the surgeon, and, like them, must be sterile. In the performance of the inoculation strict attention must be paid to asepsis, and suitable precautions adopted to guard against accidental contamination of the material to be introduced into the animal. In addition, the hands of the operator should be carefully disinfected.

The list of apparatus used in animal inoculations given below comprises practically everything needed for any inoculation. Needless to remark, all the apparatus will never be required for any one inoculation.

Apparatus Required for Animal Inoculation:

1. Water steriliser (_vide_ page 33). It is also convenient
to have a second water steriliser, similar but smaller (23
by 7 by 5 cm.), for the sterilisation of the syringes.

2. Injection syringe. The best form is one of the ordinary
hypodermic pattern, 1 c.c. capacity graduated in twentieths
of a cubic centimeter (0.05 c.c.), fitted with finger rests,
but with the leather washers and the packing of the piston
replaced by those made of asbestos (Fig. 171). The
instrument must be easily taken to pieces, and spare parts
should be kept on hand to replace accidental breakage or
loss. Other useful syringes are those of 2 c.c., 5 c.c., 10
c.c., and 20 c.c. capacity. A good supply of needles must be
kept on hand, both sharp-pointed and with blunt ends. To
sterilise the syringe, fill it with water, loosen the
packing of the piston and all the screw joints, place it in
the steriliser and boil for at least five minutes. Disinfect
the syringe _after use_, in a similar manner. The needles,
which are exceedingly apt to rust after being boiled, should
be stored in a pot of absolute alcohol when not in use.

3. Operating table.

4. Surgical instruments. Sterilise these before use by
boiling, and disinfect them _after use_ by the same means.
Wipe perfectly dry immediately after the disinfection is
completed.

Scissors, probe and sharp-pointed.

Dissecting forceps of various patterns.

Pressure forceps.

Retractors (small self retaining Fig. 172).

Aneurism needles, sharp and blunt.

Scalpels, } Keratomes, } with metal handles. Trephines, }

Michel's steel clips and special forceps for applying the
same. These small steel clips enable the operator to easily
and rapidly close skin incisions and are most satisfactory
for animal operations.

Surgical needles.

Needle holder.

Soft rubber catheters, various sizes.

Gum elastic oesophageal bougies with connection to fit
syringe.

5. Anaesthetic.

(a) General: The safest general anaesthetic for animals is an A. C. E. mixture, freshly prepared, containing by volume alcohol 1 part, chloroform 2 parts, ether 6 parts, and should be administered on a "cone" formed by twisting up one corner of a towel and placing a wad of cotton-wool inside it, or from a saturated cotton-wool pad packed into the bottom of a small beaker.

(b) Local:

1. Cocaine hydrochloride, 2 per cent. in adrenalin 1 per mille
solution.
2. Beta-eucaine, 2 per cent. in adrenalin, 1 per mille solution.
3. Ethyl chloride jet.

6. Sterile glass capsules of various sizes.

7. Cases of sterile pipettes { 10 c.c. (in tenths of a cubic centimetre).
{ 1 c.c. (in hundredths of a cubic
centimetre).

8. Flasks (75 c.c.) containing sterilised normal saline solution (or sterile bouillon).

9. Sterilised cotton-wool. Cotton-wool (absorbent) is packed loosely in a copper cylinder similar to that used for storing capsules, and sterilised in the hot-air oven.

10. Sterilised gauze. Gauze is sterilised in the same way as cotton-wool.

11. Sterilised silk and catgut for sutures. These are sterilised, as required, by boiling for some ten minutes in the water steriliser.

12. Flexible collodion (or compound tincture of benzoin).

13. Grease pencil.

14. Tie-on celluloid labels, to affix to the cages.

15. Razor.

16. Small pot of warm water.

17. Liquid soap. Liquid soap is prepared as follows: Measure out 100 grammes of soft soap and add to 500 c.c. of 2 per cent. lysol solution in a large glass beaker; dissolve by heating in a water-bath at about 90 deg. C. Bottle and label "Liquid Soap."

18. In place of the liquid soap and razor it is sometimes convenient to use a Depilatory powder.

Barium sulphide 1 part
Rice starch 3 parts

Dust the powder thickly over the area to be denuded of hair, sprinkle with water and mix into a thin paste _in situ_; allow the paste to act for three minutes, then scrape off with a bone spatula--the hair comes away with the paste and leaves a perfectly bare patch. This process is preferably carried out, the day previous to the operation.

~Material Utilised for Inoculation.~--The material inoculated may be either--

1. Cultures of bacteria--grown in fluid media, or on solid media.

2. Metabolic products of bacterial activity--e. g., toxins in solution.

3. Pathological products (fluid secretions and excretions, solid tissues).

~The Preparation of the Inoculum.~--

(a) _Cultivations in Fluid Media._--

1. Flame the plug of the culture tube.

2. Remove the plug and flame the mouth of the tube.

3. Slightly raise the lid of a sterile capsule, insert the mouth of the culture tube into the aperture and pour some of the cultivation into the capsule.

4. Remove the mouth of the culture tube from the capsule, replace the lid of the latter, flame the mouth of the tube, and replug.

5. Remove the syringe from the steriliser, squirt out the water from its interior, and allow to cool.

6. Raise the lid of the capsule sufficiently to admit the needle of the syringe and draw the required amount of the cultivation into the barrel of the syringe.

(Or, remove a definite measured quantity of the cultivation directly from the tube or flask by means of a sterile graduated pipette, discharge the measured amount into a sterile capsule, and fill into the syringe; or take up the required quantity of the cultivation directly into the graduated syringe from the tube or flask.)

If it is necessary to introduce a large bulk of fluid into the animal, the cultivation should be transferred with aseptic precautions, to a sterile separatory funnel, preferably of the shape shown in figure 173, and graduated if necessary. This is supported on a retort stand and raised sufficiently above the level of the animal to be injected, so as to secure a good "fall." A piece of sterilised rubber tubing of suitable length, fitted with an injection needle and provided with a screw clamp, is now attached to the nozzle of the funnel and the operation completed according to the requirements of the particular case.

This method is quite satisfactory when the injection is made into the pleural or abdominal cavities or directly into a vein but if the injection has to be made into the subcutaneous tissue the "fall" may not be sufficient to force the fluid in. In this case it will be necessary to transfer the culture to a sterile wash-bottle and fasten a rubber hand bellows to the air inlet tube (interposing an air filter) and attach the tubing with the injection needle to the outlet tube (Fig. 174). By careful use sufficient force can be obtained to drive the injection in.

(b) _Cultivations on Solid Media (e. g., Sloped Agar)._--

1. By means of a sterile graduated pipette introduce a suitable small quantity of sterile bouillon (or sterile normal saline solution) into the culture tube.

2. With a sterile platinum loop or spatula scrape the bacterial growth off the surface of the medium, and emulsify it with the bouillon. It then becomes to all intents and purposes a fluid inoculum.

3. Pour the emulsion into a sterile capsule and fill the syringe therefrom.