Animal inoculation has been referred to (1) as a method of assisting in the preparation of pure cultures of pathogenic organisms; (2) as a means of testing the poisonous properties of substances produced in bacterial cultures; (3) in order to test the ability of an organism to cause a disease; (4) for the production of various antibodies; it may be added (5) that some bacteria produce in the smaller experimental animals lesions which do not occur in animals naturally infected, but which nevertheless are characteristic for the given organism. The best illustration is the testicular reaction of young male guinea-pigs to intraperitoneal injections of glanders bacilli. Experimental animals are also inoculated (6) to test the potency of various bacterial and other biological products, as toxins, antitoxins, etc.
Guinea-pigs are the most widely used experimental animals because they are easily kept and are susceptible to so many diseases on artificial inoculation. Rabbits are used very largely also, as are white mice. For special purposes white rats, pigeons, goats and swine are necessary. For commercial products horses (antitoxins) and cattle (smallpox vaccine) are employed. In the study of many human diseases the higher monkeys and even the anthropoid apes are necessary, since none of the lower animals are susceptible.
The commonest method of animal inoculation is undoubtedly the subcutaneous. This is accomplished most readily with the hypodermic needle. The skin at the point selected (usually in guinea-pigs the lateral posterior half of the abdominal surface, in mice the back near the root of the tail) is pinched up to avoid entering the muscles and the needle quickly inserted. Clipping the hairs and washing with an antiseptic solution should precede the inoculation as routine practice. Frequently a small “skin pocket” is all that is needed. The hair is clipped off, the skin pinched up with small forceps and a slight snip with sharp scissors is made. The material may be inserted into this pocket with a heavy platinum needle. Cutaneous inoculation is made by shaving the skin and rubbing the material onto the shaved surface or scratching with a scalpel or special scarifier, but without drawing blood, and then rubbing in the material to be inoculated.
Intravenous injections are made with larger animals. In rabbits the posterior external auricular is a convenient vein. In larger animals the external jugular is used.
Intraperitoneal, -thoracic, -cardiac, -ocular, -muscular injections, and injections into the parenchyma of internal organs are accomplished with the hypodermic needle. In the case of the first two, injury to contained organs should be carefully avoided. Intracardiac injection, or aspiration of the heart to secure blood, requires considerable practice to be successful without causing the death of the animal at once through internal hemorrhage. In subdural injections into the cranial cavity it is necessary to trephine the skull first, while such injections into the spinal canal may be accomplished between the vertebra with needles longer and stronger than the usual hypodermic needle. Occasionally animals are caused to inhale the organisms, or are fed cultures mixed with the feed.
If the site of the lesion is readily accessible from the exterior, material from the living animal should be collected with sterile instruments and kept in sterile utensils until the necessary tests can be made. Testing should be done on material as soon after collection as possible, in all cases, to avoid the effects of “decomposition” bacteria.
If the blood is to be investigated it may be aspirated from a peripheral vein with a sterile hypodermic syringe of appropriate size or allowed to flow through a sterile canula into sterile receptacles. The site of the puncture should be shaved and disinfected before the instrument is introduced.
Discharges of whatever kind should likewise be collected in sterile receptacles and examined as soon as may be.
If internal organs are to be examined it is best to kill a moribund animal than to wait for death, since after death, and in severe infections even sometimes before, the tissues are rapidly invaded by saprophytic bacteria from the alimentary and respiratory tracts which complicate greatly the isolation of the specific organism. Hence the search for specific bacteria in carcasses or organs several hours after death is frequently negative. Animal inoculation with such material is very often followed by sepsis or septicemia in a few hours, so that the specific organism has no opportunity to manifest itself.
In securing material for cultures from internal organs it is a good plan to burn the surface of the organ with a gas or alcohol flame, or to sear it with a hot instrument to kill surface organisms, then make the incision or puncture through the burned area and secure material from the interior of the organ. Such punctures made with a stiff platinum needle frequently give pure cultures of the organism sought. Slides may be made from such material and culture media inoculated at once.
Since a bacteriological diagnosis depends most commonly on growing the organisms, it is evident that material sent for examination must never be treated with an antiseptic or preservative. If decomposition is to be feared the only safe procedure is to pack the material in ice and forward in this way.
Tuberculous material from the parenchyma of internal organs may be forwarded in a preservative (not formalin, since this makes it very difficult to stain the bacteria) as in this special case a very positive diagnosis may be made by staining alone. Even here it is better to pack in ice in order that the diagnosis by staining may be confirmed by inoculating the living organisms into guinea-pigs.
In the case of material from a rabid animal and many protozoal diseases the rule against preservatives is not absolute, since staining is a reliable diagnostic means. Even in these cases it is often desirable to inoculate animals, hence, as before stated, it is best to make it a uniform practice to pack material for examination in ice and use no preservatives.