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 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 C. for two hours--then centrifugalize thoroughly.

Pipette off the clear serum, and fill in quant.i.ties of 1 c.c., into small gla.s.s 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 haemolytic power.

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

FOOTNOTES:

[10] The quant.i.ties here given are not absolutely correct. If exact.i.tude 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 a.s.sociated 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 a.s.sociation 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 a.s.sume appearances so different from those regarded as typical or normal as to throw doubt on its ident.i.ty. 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, a.s.ses 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 s.e.x 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 s.p.a.ce for notes on the course of the resulting infection, and carries on the reverse the weight and temperature chart (Figs. 164 and 165).

[Ill.u.s.tration: Fig. 164.--Front of inoculation card.]

~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.

[Ill.u.s.tration: FIG. 165.--Back of inoculation card.]

~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 sc.r.a.pings of the skin, treated with liquor pota.s.sae. Another form of scabies (due to Psoroptes _communis cuniculi_) commences at the bottom of the concha, which is filled with whitish-yellow ma.s.ses 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 Pota.s.sium 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.

cra.s.sicollis_ 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.