XVI. METHODS OF IDENTIFICATION AND STUDY. (continued 2)
Reading edition
Pipette into tube No. 4 0.3 c.c. = 0.03 c.c. hæmolytic serum}
Pipette into tube No. 5 0.2 c.c. = 0.02 c.c. hæmolytic serum}
pipette into tube No. 6 0.1 c.c. = 0.01 c.c. hæmolytic serum}
Pipette into tube No. 7 0.5 c.c. = 0.005 c.c. hæmolytic serum} From tube 2.
Pipette into tube No. 8 0.3 c.c. = 0.003 c.c. hæmolytic serum}
Pipette into tube No. 9 0.2 c.c. = 0.002 c.c. hæmolytic serum}
Pipette into tube No. 10 0.1 c.c. = 0.001 c.c. hæmolytic serum}
5. To each tube add 1 c.c. of erythrocyte solution.
6. When necessary (that is to say in tubes 2, 4, 5, 6, 8, 9 and 10) add normal saline solution to the mixture in the test-tubes till the column of fluid in each reaches to the same level.
7. Shake each tube in turn, so as to thoroughly mix its contents. Plug the mouth of each tube with cotton wool, and place entire set in the incubator at 37°C. for one hour.
8. Remove the tubes from the incubator and into each tube pipette 0.1 c.c. complement (guinea-pig's serum) and replace tubes in incubator at 37° C. for further period of one hour.
9. Remove the tubes from the incubator, and if complete hæmolysis has not taken place in every tube, stand on one side, preferably in the ice chest, for an hour.
10. Then examine the tubes.
Complete hæmolysis is indicated by a clear red solution, with no deposit of red cells at the bottom of the test-tube.
Absence of hæmolysis is indicated by a clear or turbid colourless fluid, with a deposit of red cells at the bottom of the test-tubes.
The smallest amount of hæmolytic serum that has caused complete hæmolysis is known as the minimal hæmolytic dose (M. H. D.) and if hæmolysis has occurred in all the tubes down to No. 7—the m. h. d. of this particular serum is .005 c.c. = 200 minimal hæmolytic doses per cubic centimetre. Such a serum is strong enough for experimental work; indeed, for many purposes, complete hæmolysis down to tube 6 will indicate a serum sufficiently strong(= 100 m. h. d. per cubic centimetre). If, however, only the first one or two tubes are completely hæmolysed, this is an indication that the rabbit should receive further injections in order to raise the hæmolytic power to a sufficiently high level.
STORAGE OF HÆMOLYSIN.
If, and when the hæmolysin content of the rabbit's serum is found to be sufficient, destroy the animal by chloroform vapour.
Remove as much of its blood as possible from the heart under aseptic precautions into sterilized centrifuge tubes.
Transfer the tubes of blood to the incubator at 37° C. for two hours—then centrifugalize thoroughly.
Pipette off the clear serum, and fill in quantities of 1 c.c., into small glass ampoules or pipettes, and hermetically seal in the blowpipe flame, care being taken to avoid scorching the serum.
Place the ampoules when filled with serum and sealed, in a water-bath at 56° C. for 30 minutes. This destroys the complement, i. e., inactivates the serum, and at the same time, provided the various operations have been carried out under aseptic precautions, ensures its sterility. A longer exposure reduces the hæmolytic power.
Place the ampoules in a closed metal box and store in the ice chest for future use.