3. Pa.s.s the entire length of the pipette twice or thrice through the flame of the Bunsen burner.

4. Snap off the sealed end of the pipette with a pair of sterile forceps.

5. Compress the india-rubber teat, thrust the point of the pipette into the secretion; now relax the pressure on the teat and allow the pipette to fill.

6. Remove the point of the pipette from the secretion, allow the fluid to run a short distance up the capillary stem and seal the point of the pipette in the flame. (If using a pipette with a constriction below the plugged mouthpiece (Fig 13b), this portion of the pipette may also be sealed in the flame.)

When ready to examine the morbid material snap off the sealed end of the pipette with sterile forceps and eject the contents of the pipette into a sterile capsule. The material can now be utilized for cover-slip preparations, cultivations and inoculation experiment.

3. _The peripheral blood_ should be examined from time to time for from this tissue is often obtained the fullest information as to the course and progress of an infection.

a. The ~histological examination of the blood~ should be directed chiefly to observations on the number and kind of white cells; and since but few bacteriologists are at the same time expert comparative haematologists, some notes on the normal characters of the blood of the commoner laboratory animals, contrasted with those of man, are inserted for reference. These have been very kindly compiled for me by my friend and one time colleague Dr. Cecil Price Jones.

COMPARATIVE HaeMOCYTOLOGY OF LABORATORY ANIMALS.

-------------------------------------------------------------------- | Totals | Percentages |------------------------------------------------------------ Animal | | | Hb, |Lympho-|Large |Poly- |Eosin-| Mast |Red cells |White | per | cytes,|monos,|morph,| oph, |cells, | | cells|cent.| per | per | per | per | per | | | | cent. | cent.| cent.|cent. |cent.

-------------------------------------------------------------------- Frog | 490,000| 8,000| 58 | 40 | 10.0 | 22.0 |15 | 13 Mouse | 8,700,000| 8,000| 78 | 60 | 21.5 | 17.0 | 1.4 | 0.1 Rat | 9,000,000| 9,000| 85 | 54 | 7.0 | 37.5 | 1.3 | 0.2 Guinea-| | | | | | | | pig | 5,700,000|10,000| 99 | 55 | 9.0 | 32.8 | 3.0 | 0.2 Rabbit | 6,000,000| 7,000| 70 | 50 | 2.0 | 46.0 | 0.6 | 1.4 Rhesus | 4,500,000|13,000| 77 | 43 | 5.0 | 50.0 | 1.3 | 0.7 Goat |14,600,000|15,000| 58 | 35 | 6.3 | 56.7 | 1.25 | 0.75 Fowl | 3,500,000|30,000| 100 | 49 | 3.0 | 42.0 | 1.0 | 5.0 Pigeon | 3,500,000|20,000| 101 | 43 | 9.0 | 43.0 | 3.0 | 2.0 -------------------------------------------------------------------- Man | | | | | | | | (adult)| 5,000,000| 7,500| 100 | 25 | 5.5 | 65 | 4.0 | 0.5 Normal | (4.5-5) | (7-9)|(95- |(20-30)| (4-8)|(55- |(3-5) |(0.5-2) limits.| millions.| thou-| 101)| | | 68) | | | |sands.| | | | | | --------------------------------------------------------------------

The above table represents in each case the average of a large number of counts.

REMARKS.

_Frog._--The _red cells_ are large oval nucleated (20-25 by 12-15) discs, the nucleus relatively small and irregularly elongated or oval, about 10 in length. Many primitive and developing forms are usually observed--also free nuclei and many cells in various stages of degeneration. Haemoglobin estimation is difficult owing to turbidity of the blood after dilution with water. The _polymorphonuclear_ leucocytes are large cells, about 20; no definite granules can be observed. The _eosinophile_ cells contain large deeply staining coccal-shaped granules.

_Mouse._--The granules of the _polymorphonuclear_ leucocytes are usually not stained, or only very faintly so. The nucleus of the _eosinophile cell_ is ring-shaped or much divided, and the granules are coccal and stain oxyphile. The remarkable character of the blood is the high percentage of large _mononuclear_ cells.

_Rat._--The fine rod-shaped granules of the _polymorphonuclear_ leucocytes are usually very faintly stained. The granules of _eosinophile_ cells are well stained and coccal-shaped, the nucleus is often ring shaped. The _basophile_ granular cells are few--but the granules are large, and stain deeply basophile.

_Guinea-pig._--Polychromasia and punctate basophilia of _red cells_ are very commonly observed--nucleated red cells are also frequent. The large _mononuclear_ cells often contain vacuoles--"Kurlow cells"--possibly of a parasitic nature.

_Rabbit._--It is not uncommon to find nucleated _red cells_ in films from quite healthy animals. The granules of the _polymorphonuclear_ leucocytes stain oxyphile. The coa.r.s.e granules of the _eosinophile_ cells appear to stain less deeply oxyphile, probably owing to the basophile staining of the cytoplasm.

_Rhesus monkey._--The blood cells resemble those met with in human blood. The minute neutrophile granules of the _polymorphonuclear_ leucocytes are often very scanty, and sometimes apparently absent. The _eosinophile_ cells are not so densely packed with coa.r.s.e oxpyhile granules as in the human eosinophile, and the nuclei of these cells are usually much divided, or polymorphous.

_Goat._--The _red cells_ are small, nonnucleated discs, only about 4.5 diameter, not much more than half that of the human red cell. The _polymorphonuclear_ leucocytes have only a few very minute coccal-shaped oxyphile granules, the nucleus is polymorphous. The _eosinophile_ cells are large cells up to 20, the cytoplasm is basophile and contains coa.r.s.e coccal-shaped oxyphile granules, and the nucleus is often much divided.

_Fowl._--The _red cells_ are oval nucleated discs about 12 by 6, the nucleus being relatively small (about 4 long), irregularly elongated or oval; round, more deeply stained cells with round or diffuse nuclei, also free nuclei and degenerated forms of red cells are often present.

The granules of the cells corresponding to the _polymorphonuclear_ leucocytes are rod-shaped, often beaded or with clubbed ends. The nucleus is not polymorphous, but usually divided into two, though it may be single. The cells probably corresponding to _eosinophile_ leucocytes have fine coccal-shaped granules, faintly staining eosinophile or neutrophile. The basophile granules of the "mast" cells are coccal-shaped, of various size--often quite powdery.

_Pigeon._--_Red cells_ resemble those of the fowl, and similar varieties of appearance may be noted. The granules of those cells which correspond to _polymorphonuclear_ leucocytes are rod-shaped, but smaller and finer than in the fowl, and do not show clubbed appearances. The nucleus is not polymorphous, and only occasionally divided. The coccal-shaped granules of the _eosinophile_ cells are stained more deeply oxyphile than those of the corresponding cells of the fowl.

_The preparation of dried films_ for this histological examination of the blood is carried out as follows:

1. Small samples of blood for the preparation of blood films are most conveniently obtained from the veins of the ear in most of the ordinary laboratory animals, viz., monkey, goat, dog, cat, rabbit, guinea-pig; in the pigeon and fowl the axillary vein should be punctured; in the rat and mouse either a vein in the ear or preferably by wounding the tip of the tail; in the frog, the web of the foot should be selected.

2. Puncture the selected vein with a sharp needle. A flat Hagedorn needle (size No. 8) with a cutting edge is the most useful for this purpose. If the vein cannot be distended by proximal compression, vigourous friction with a piece of dry lint may have the desired effect--or a test-tube full of water at about 40C. may be placed close to the vein. Failing these methods, a drop or two of xylol may be dropped on the skin just over the vein, left on for a few seconds and then wiped off with a piece of dry lint.

3. One of the short ends of a 3 by 1 gla.s.s slip is brought into contact with the exuding drop of blood, so that it picks up a small drop.

4. The slide is then lowered transversely on to the surface of a second 3 by 1 slip, which rests on the bench near to one end at an angle of about 45, and retained in this position for a few seconds, while the drop of blood spreads along the whole of the line of contact (see also Fig. 69).

5. Draw the first slide firmly and evenly along the entire length of the lower slide, leaving a thin regular film which will probably show the blood cells only one layer thick.

6. Allow the film to dry in the air.

7. Stain with one of the polychrome blood stains (see page 97).

8. Examine microscopically.

b. The ~bacteriological examination of the blood~ is directed solely to the demonstration of the presence in the circulating blood of the organisms previously injected into the animal. For this purpose several cubic centimetres of blood should be taken in an all-gla.s.s syringe from an accessible vein corresponding to one of those suggested as the site of intravenous inoculation--and under similar aseptic precautions.

1. Sterilise an all-gla.s.s syringe of suitable size, and when cool draw into the syringe some sterile sodium citrate solution and moisten the whole of the interior of the barrel; then eject all the citrate solution if less than 5 c.c. blood are to be withdrawn; if more than 5 c.c. are required retain about half a cubic centimetre of the fluid in the syringe. This prevents coagulation of the blood.

The sodium citrate solution is prepared by dissolving:

Sodium citrate 10 gramme.

Sodium chloride 0.75 grammes.

In distilled water 100 c.c.

Sterilise by boiling.

2. Prepare the animal as for intravenous inoculation (see page 363) and introduce the syringe needle into the lumen of the selected vein.

3. Slowly withdraw the piston of the syringe. When sufficient blood has been collected direct the a.s.sistant to release the proximal compression of the vein; and withdraw the needle.

4. Remove the needle from the nozzle of the syringe and deliver the citrated blood into a small Ehlenmeyer flask containing about 250 c.c.

of nutrient broth.

5. Label, incubate and examine daily until growth occurs or until the expiration of ten days.

c. The ~serological examination of the blood~ is directed to the demonstration of the presence of certain specific antibodies in the sera of experimentally infected animals, and within certain limits to an estimation of their amounts.

The chief of these bodies are:

Ant.i.toxin.

Agglutinin.

Precipitin.

Opsonin.

Immune body or Bacteriolysin.

None of these substances are capable of isolation in a state of purity apart from the blood serum, consequently special methods have been elaborated to permit of their recognition. In every instance the behaviour of serum from the experimental animal, which may be termed "specific" serum, is studied in comparison with that of serum from an uninoculated animal of the same species, and which is termed "normal"

serum. In view of minor differences in const.i.tution exhibited by the serum of various individuals of the same series, it is usual to employ a mixture of sera obtained from several different normal animals of the same species as the inoculated animal, under the term "pooled serum."

The method of collecting blood (e. g., from the rabbit) for serological tests is as follows:

~Collection of Serum.~

_Apparatus required:_