9. Label, etc.

_B. Rats and Mice (Mark's Method)._

1. Secure the animal in the vertical position.

(a) _Rat._--Take a pair of catch sinus forceps about 22 cm. in length and seize the animal by the loose skin of the head as far forward as possible--fix the forceps, and holding the instrument vertically upward, transfer to the left hand of an a.s.sistant who secures the animal's tail between the fingers grasping the handle of the forceps. (See Fig. 191.)

[Ill.u.s.tration: FIG. 191.--Intragastric inoculation of rat.]

(b) _Mouse._--An a.s.sistant grasps the loose skin between the ears as far forwards as possible between the forefinger and thumb of the left hand.

He now grasps the tail with the right hand, draws the mouse straight and pa.s.ses the tail between the fourth and little fingers of the left hand and secures it there.

2. The a.s.sistant takes a closed pair of thin-bladed forceps in his right hand, pa.s.ses the ends into the animal's mouth, then allows the blades to separate. This opens the animal's jaw and serves as a gag.

3. Moisten the sterilised oesophageal tube with sterile water. (This tube is of silk rubber, 6.5 cm. in length, with the distal end rounded, the proximal end mounted in a syringe needle head, which fits the nozzles of the two sterile syringes to be used.)

4. Grasp the tube about its middle and pa.s.s it into the animal's mouth, downwards and a little to one side or the other until its length is lost in the digestive tract and mouth. Gentle guidance is alone necessary. Do not use any force.

5. Take up the required dose of inoculum into the syringe; insert the nozzle of the syringe into the needle-mount, and force the piston down.

6. Steadying the needle-mount with the left hand, detach the syringe.

7. Draw up some sterile water in the second (sterile) syringe, and inserting its nozzle into the needle-mount force a few drops of water through the tube to wash it out.

8. With one quick upward movement remove the tube from the animal's mouth.

9. Label, etc.

One other method of inoculation remains to be described, which does not require operative interference.

~11. Feeding.~--

1. _Fluid Inoculum._--Small pieces of sterilised bread or sop (sterilised in the steamer at 100 C.) are soaked in the fluid inoculum and offered to the animals in a sterile Petri dish or capsule.

2. _Solid Inoculum._--Small pieces of tissue are placed in sterile vessels and offered to the animals.

FOOTNOTES:

[12] This table is made by Messrs. Down Bros., St. Thomas's Street, London, S. E.

[13] This modification is made for the author by Messrs. Down Bros., St.

Thomas's Street, London, S. E.

[14] Manufactured by Messrs. Francis Lepper, 56, Great Marlborough Street, London, W.

XVIII. THE STUDY OF EXPERIMENTAL INFECTIONS DURING LIFE.

The possession of pathogenetic properties by an organism under study is indicated by the "infection" of the experimental animal--a term which is employed to summarise the condition resulting from the successful invasion of the tissues of the experimental animal by the micro-organisms inoculated and by their multiplication therein.

Infection is considered to have taken place:

1. When the death of the animal is produced as a direct consequence of the inoculation.

2. When without necessarily producing death the inoculation causes local or general changes of a pathological character.

3. When either with or without death, or local or general changes occurring, certain substances make their appearance in the body fluids, which can be shown (_in vitro_ or _in vivo_) to exert some profound and specific effect when brought into contact with subcultivations of the organism originally inoculated.

The important factors in the production of infection are:

A. Seed. Virulence of organism.

Dose of organism.

B. Soil. Resistance offered by the cells of the experimental animal.

The first two factors, although variable, are to a certain extent under the control of the experimenter. Thus by suitable means the virulence of an organism can be exalted or attenuated, whilst the size of the dose may be increased or diminished. The third factor also varies, not only amongst different species of animals, but also amongst different individuals of the same species. The essential causes of this variation are not so obvious, so that beyond selecting the animals intended for similar experiments with regard to such points as age, size or s.e.x, but little can be done to standardise cell resistance.

Immediately an animal has been inoculated a period of clinical observation must be entered upon, which should only terminate with the death of the animal. The general observations should at first and if the infection is an acute one, be made daily--later, and if the animal appears to be unaffected or if the infection is chronic, both general and special observations should be carried out at weekly intervals. If the animal appears to be still unaffected, it should be killed with chloroform vapour at the end of two or three months and a complete post-mortem carried out.

A. The ~general observations~ should take cognisance of:

1. _General appearance._ The experimental animal should be inspected daily, not only with a view to detecting symptoms due to the experimental infection, but also to prevent any intercurrent infection, naturally acquired, from escaping notice (_vide_ page 337).

2. _The weight_ of the inoculated animal should be observed and recorded each day during the course of an experimental infection at precisely the same hour, preferably just before the morning feed.

3. _The temperature_ should similarly be recorded daily, if not more frequently, during the whole period the animal is under observation, and carefully charted--individual variations will at once become apparent.

It should be borne in mind that the temperature regarded as normal for man (37.5 C.) is not the normal average temperature of any of the lower animals save the rat and mouse. The accompanying table of normal averages for the animals usually employed in bacteriological research may be of use in preventing the erroneous a.s.sumption that pyrexia is present in an animal, which merely shows its own normal temperature.

NORMAL AVERAGES.

---------------------------------------------------- | Rectal | Pulse. | Respirations.

Animal. | Temp. C.|------------------------ | | Rate per minute.

---------------------------------------------------- | | | Frog | 8.9-17.2 | 80 | 12 Mouse | 37.4 | 120 | ...

Rat | 37.5 | ... | 210 Guinea pig | 38.6 | 150 | 80 Rabbit | 38.7 | 135 | 55 Cat | 38.7 | 130 | 24 Dog | 38.6 | 95 | 15 Goat | 40.0 | 75 | 16 Ox | 38.8 | 45 | ..

Horse | 37.9 | 38 | 11 Monkey (Rhesus) | 38.4 | 100 | 19 Pigeon | 40.9 | 136 | 30 Fowl | 41.6 | 140 | 12 | | | ----------------------------------------------------

B. ~Special observations~ comprise some or all of the following, according to the method of inoculation and the character of the virus.

1. _The site of inoculation_ should be minutely examined at least at weekly intervals, and the neighbouring lymphatic glands palpated.

2. Any _local reaction_ at the site of inoculation and any other readily accessible lesion should be carefully investigated. Any suppurative process which may occur, whether in the subcutaneous tissues or in joints, should be explored and the pus carefully examined both microscopically and culturally.

Fluid secretions and excretions, such as pus or serous exudates when accessible are collected direct from the body in sterile capillary pipettes (_vide_ Fig. 13a,) in the following manner:

1. Open the case containing the pipettes, grasp one by the plugged end, remove it from the case, and replace the lid of the latter.

2. Attach a rubber teat (_vide_ page 10) to the plugged end of the pipette and use the teat as the handle of the pipette.