The Elements of Bacteriological Technique / A Laboratory Guide for Medical, Dental, and Technical Students. Second Edition Rewritten and Enlarged.

Reagents Required:

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 Measure out 2 c.c. citrated human blood (collected at a surgical operation or a venesection, or withdrawn by venipuncture from the median basilic or median cephalic vein of a normal adult) into a centrifuge tube and centrifugalise thoroughly.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

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° C. for fifteen minutes (to inactivate) and test the serum quantitatively for hæmolytic properties in the following manner:

THE TITRATION OF HÆMOLYTIC SERUM.

Apparatus Required:

Reagents Required and Method of Preparation:

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 hæmolytic 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.

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°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° C. for further period of one hour.

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

10. Then examine the tubes.

The smallest amount of hæmolytic serum that has caused complete hæmolysis is known as the minimal hæmolytic dose (M. H. D.) and if hæmolysis has occurred in all the tubes down to No. 7—the m. h. d. of this particular serum is .005 c.c. = 200 minimal hæmolytic doses per cubic centimetre. Such a serum is strong enough for experimental work; indeed, for many purposes, complete hæmolysis 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 hæmolysed, this is an indication that the rabbit should receive further injections in order to raise the hæmolytic power to a sufficiently high level.

STORAGE OF HÆMOLYSIN.

If, and when the hæmolysin 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° 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° 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 hæmolytic power.

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

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 septicæmia, 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-à-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 anæsthesia, 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 anæsthesia 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 anæsthetic. 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:

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 potassæ. 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, fæces, 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 Tænia 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 septicæmia due to B. cuniculicida.

The Mouse and Rat suffer from septicæmia, and from the cysticercus form of Tænia 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 (Œsophagostoma 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 Hæmosporidia, 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 septicæmia—infections due to members of the hæmorrhagic septicæmia 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.

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.

(c) Toxins.—Prepared by previously described methods (vide page 318), are manipulated in a similar manner to cultivations in fluid media.

(d) Pathological Products.—Fluid secretions, excretions, etc., such as serous exudation, pus, blood, etc., are treated as fluid cultivations; but if the material is very thick or viscous, a small quantity of sterile bouillon or normal saline solution may be used to dilute it, and thorough incorporation effected by the help of a sterile platinum rod.

Solid tissues, such as spleen, lymph glands, etc., may be divided into small pieces by sterile instruments and rubbed up in a sterilised agate mortar (using an agate pestle), with a small quantity of sterile bouillon, and the syringe filled from the resulting emulsion.

If it is desired to inoculate tissue en masse, remove from the material a small cube of 1 or 2 mm. and introduce it into a wound made by sterile instruments in a suitable situation, and occlude the wound by means of Michel's steel clips and a sealed dressing.

Method of Securing Animals During Inoculation.—

For the majority of inoculations, especially when no anæsthetic is administered, it is customary to employ an assistant to hold the animal (see Fig. 175).

If working single handed Voge's holder for guinea-pigs, is a useful piece of apparatus the method of using which is readily seen from the accompanying figures (Figs. 176, 177).

The instrument itself consists of a hollow copper cylinder, one end of which is turned over a ring of stout copper wire, and from this open end a slot is cut extending about half way along one side of the cylinder. The opposite end is closed by a "pull-off" cap and is perforated around its edge by a row of ventilating holes, which correspond with holes cut in the rim of the cap. In the event of the animal resisting attempts to remove it from the holder backwards, this cap is taken off and the holder placed on the table and the guinea-pig allowed to walk out.

To provide for different-sized animals, two sizes of this holder will be found useful:

1. Length, 16 cm.; breadth, 6 cm.; size of slot, 8 cm. by 2.5 cm.

2. Length, 20 cm.; breadth, 8 cm.; size of slot, 10 cm. by 2.5 cm.

A convenient holder for mice and even small rats is shown in figure 178, the tail being securely held by the spring clip. Needless to say, the holder should be entirely of metal, and the wire cage detachable and easily renewed.

When the animal is anæsthetised, it is more convenient to secure it firmly to some simple form of operating table, such as Tatin's (Fig. 179), which will accommodate rabbits, guinea-pigs, and rats: or to the more elaborate table devised by the author (Fig. 180).

Operation Table.—This is a table of the "aseptic" type, composed of steel tubing, nickel-plated or enamelled. The table-top frame is sufficiently large to accommodate rabbits, dogs and monkeys; and is supported upon telescopic uprights, so that it is adjustable as to height; in its long axis it can be inclined (at either end) to 45° from the horizontal. Further it can be completely rotated about its long axis. The table-top itself is composed of a sheet of copper wire gauze loosely suspended from the long sides of the tubular frame. The slackness of the gauze bed permits of an india rubber hot water bottle, or an electrotherm being placed under the animal, and if during the course of an experiment it is necessary to reverse the animal, the table-top frame is completely rotated, the device adopted for suspending the gauze is detached and the gauze reversed also, so that it again supports the animal from below.

METHODS OF INOCULATION.

The following methods of inoculation apply more particularly to the rabbit, but from them it will readily be seen what modifications in technique, if any, are necessary in the case of the other experimental animals.

1. Cutaneous Inoculation.—(Anæsthetic, none.)

1. Have the animal firmly held by an assistant (or secured to the operating table).

2. Apply the liquid soap to the fur, over the area selected for inoculation, with a wad of cotton-wool, and lather freely by the aid of warm water; shave carefully and thoroughly; or apply the depilatory powder.

3. Wash the denuded area of skin thoroughly with 2 per cent. lysol solution.

4. Wash off the lysol with ether and allow the latter to evaporate.

5. Make numerous short, parallel, superficial incisions with the point of a sterile scalpel.

6. When the oozing from the incisions has ceased, rub the inoculum into the scarifications by means of the flat of a scalpel blade, or a sterile platinum spatula.

7. Cover the inoculated area with a pad of sterile gauze secured in situ by strips of adhesive plaster or by sealing down the edges of the gauze with collodion.

8. Release the animal, place it in its cage, and affix a label upon which is written: