3. Fit a rubber teat to the barrel of each of the pipettes.

4. Make a mark with the grease pencil on the stem of one of the pipettes about 2 or 3 cm. from the open extremity.

[Ill.u.s.tration: FIG. 193.--Filling the capillary teat pipette.]

5. Compress the teat between the finger and thumb (Fig. 193) to such an extent as to drive out the greater part of the contained air.

6. Maintaining the pressure on the teat pa.s.s the stem of the pipette into the capsule holding the saline solution, until the open end of the pipette is below the level of the fluid.

7. Now cautiously relax the pressure on the teat and let the fluid enter the pipette and rise in the stem until it reaches the level of the grease pencil mark. As soon as this point is reached, check the movement of the column of fluid by maintaining the pressure on the teat, neither relaxing nor increasing it.

8. Withdraw the point of the pipette clear of the fluid, and again relax the pressure on the teat very slightly. The column of saline solution rises higher in the stem, and a column of air will now enter the pipette and serve as an index to separate the first volume of fluid drawn into the stem from the next succeeding one.

9. Again introduce the end of the pipette into the fluid and draw up a second volume of saline to the level of the grease pencil mark, and follow this with a second air index.

10. In like manner take up seven more equal volumes of saline solution and their following air bubbles. There are now nine equal volumes of normal saline in the pipette.

11. Now pa.s.s the point of the pipette into the blood tube and dip the open end below the surface of the serum. Proceeding as before, aspirate a volume of serum into the capillary stem up to the level of the pencil mark.

12. Eject the contents of the pipette into the small tube marked 10 per cent. by compressing the rubber teat between thumb and finger.

13. Mix the one volume of serum with the nine volumes of saline solution very thoroughly by repeatedly drawing up the whole of the fluid into the pipette and driving it out again into the test-tube.

14. Now take a clean pipette and proceed precisely as before, 4 to 10.

15. Having aspirated nine equal volumes of saline into this second pipette, now take up one similar volume of the fluid in the "10 per cent. tube."

16. Eject the contents of this pipette into the second tube marked 1 per cent. and mix thoroughly as before.

17. In similar fashion make the 0.1 per cent. solution and transfer to the third tube.

18. Further dilutions in multiples of ten can be prepared in the same way, and by varying the number of volumes of diluting fluid or serum any required dilution can be made (see Appendix, Dilution Tables).

NOTE.--The saline diluting fluid _must always_ be taken into the pipette first, otherwise if the serum contains a very large amount of agglutinin the traces of this serum added to the saline solution may be sufficient to entirely vitiate the subsequent observations--whilst if more than one sample of serum is diluted from the same saline solution serious errors may be introduced into the experiments.

~The Microscopical Reaction:~

_Apparatus Required:_

Five hanging-drop slides (or preferably two slide), with two cells mounted side by side on each (Fig. 62, a), and one slide with one cell only.

Vaseline.

Cover-slips.

Platinum loop.

Grease pencil.

Eighteen to twenty-four-hour-old bouillon cultivation of the organism to be tested (e. g., Bacillus typhi abdominalis)

Pipette end with the remainder of the specific serum labelled ~s.s.~

Tubes containing the three solutions of the specific serum, 10, 1, and 0.1 per cent. respectively.

Pipette end with pooled normal serum labelled ~p.s.~

METHOD.--

1. Make five hanging-drop preparations, thus:

(a) One loopful of bouillon cultivation + one loopful pooled serum; label "Control."

(b) One loopful culture + one loopful undiluted specific serum; label 50 per cent.

Mount these two cover-slips on a double-celled slide.

(c) One loopful bouillon culture + one loopful 10 per cent. serum; label 5 per cent.

Mount this on single-cell slide.

(d) One loopful bouillon culture + one loopful 1 per cent. serum; label 0.5 per cent.

(e) One loopful bouillon culture + one loopful 0.1 per cent. serum; label 0.05 per cent.

Mount these two cover-slips on a double-celled slide.

2. Note the time: Examine the control to determine that the bacilli are motile and uniformly scattered over the field--not collected into ma.s.ses.

3. Next examine the 50 per cent. serum preparation.

If agglutinin is present and the test is giving a positive reaction, the bacilli _will_ be collected in large clumps.

If the test is giving a negative reaction, the bacilli _may_ be collected in large clumps owing to the viscosity of the concentrated serum.

4. Observe the 5 per cent. preparation microscopically.

If the bacilli are aggregated into clumps, positive reaction.

If the bacilli are _not_ aggregated into clumps, observe until thirty minutes from the time of preparation before recording a negative reaction.

5. Examine the 0.5 and 0.05 per cent. preparations.

These may or may not show agglutination when the result of the examination of the 5 per cent. preparation is positive, according to the potency of the specific serum; and by the examination of a series of dilutions a quant.i.tative comparison of the valency of specific sera from different sources, or of serum from the same animal at different periods during the course of active immunisation may be obtained.

NOTE.--The graduated pipettes supplied with Thoma's haematocytometer (intended for the collection of the specimen of blood required for the enumeration of leucocytes), giving a dilution of 1 in 10--i. e., 10 per cent.--may be subst.i.tuted for the graduated capillary pipettes referred to above, if the vessel in which the serum has been separated is of sufficiently large diameter to permit of their use.