Agglutinin

Agglutinin is the name given to a substance present in the blood-serum of an animal that has successfully resisted inoculation with a certain micro-organism. This substance possesses the power of collecting together in clumps and masses, or agglutinating watery suspensions of that particular microbe.

Dilution of the Specific Serum:

Apparatus required:

Sterile graduated capillary pipettes to contain 10 c. mm. (Fig. 17).

Sterile graduated capillary pipettes to contain 90 c. mm. (Fig. 17).

Small sterile test-tubes 5 × 0.5 cm.

Normal saline solution in flask or test-tube.

Pipette of specific serum.

Glass cutting knife, or three-square file.

Glass capsule, nearly full of dry silver sand, or roll of plasticine.

Grease pencil.

Method.—

1. Take three sterile test-tubes and number them 1, 2 and 3.

2. Pipette 0.9 c.c. sterile normal saline solution into each tube, and stand tubes upright in the sand in the capsule, or in the plasticine block.

3. Make a scratch with the glass cutting knife on the blood pipette above the upper level of the clear serum, and snap off and discard the empty portion of the tube.

4. Remove 0.1 c.c. of the serum from the blood pipette tube, and mix it thoroughly with the fluid in tube No. 1; and label s.s., (specific serum), 10 per cent.

5. Remove 0.1 c.c. of the solution from tube No. 1 by means of a fresh pipette, and mix it with the contents of tube No. 2; and label s.s., 1 per cent.

6. Remove 0.1 c.c. of the solution from tube No. 2 by means of a fresh pipette, and mix it with the contents of tube No. 3; and label s.s., 0.1 per cent.

When the yield of serum from the specimen of blood which has been collected, or is available, is small, the above method of diluting is not practicable, and the dilution should be carried out by Wright's method in a capillary teat pipette.

Dilution of Serum by Means of a Teat Pipette.

Materials required:

Blood pipette containing sample of specific serum after centrifugalisation.
Capsule of diluting fluid—normal saline solution.
Supply of Pasteur pipettes (Fig. 13a).
India-rubber teats.
Small test-tubes.
A block of plasticine to act as a test-tube stand.
Grease pencil.

Method:

1. Mark three small test-tubes 10 per cent., 1 per cent. and 0.1 per cent. respectively, and stand them upright in the plasticine block.

2. Take a Pasteur pipette, nick the capillary stem just above the sealed end with a glass cutting knife, and snap off the sealed end with a quick movement so that the fracture is clean cut and at right angles to the long axis of the capillary stem—cut "square", in fact. Prepare several, say a dozen, in this manner.

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.

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 pass 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 pass 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 masses.

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 quantitative 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 hæmatocytometer (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 substituted 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.

The Macroscopical Reaction:

Sterile graduated capillary pipettes to contain 90 c. mm.

Eighteen to twenty-four-hours-old bouillon cultivation of the organism to be tested.

Three test-tubes containing the 10, 1, and 0.1 per cent. solutions of specific serum (about 90 c. mm. remaining in each).

Tube containing 50 per cent. solution of pooled serum.

Sedimentation pipettes (vide page 17) or teat pipettes.

Method.

1. Pipette 90 c. mm. of the bouillon culture into each of the tubes containing the diluted serum; and the same quantity into the tube containing the pooled serum.

2. Fill a sedimentation tube (by aspirating) or a teat pipette from the contents of each tube. Seal off the lower ends of the sedimentation tubes in the Bunsen flame.

3. Label each tube with the dilution of serum that it contains—viz., 5, 0.5, and 0.05 per cent.

4. Place the pipettes in a vertical position, in a beaker, in the incubator at 37°C., for one or two hours.

5. Observe the granular precipitate which is thrown down when the reaction is positive, and the uniform turbidity of the negative reaction as compared with the appearances in the control pooled serum.