To measure the resistance of the patient to such invasion, or to find out his opsonic index, special technique has been developed, which may be briefly described as follows:
Twenty-four-hour or younger growths of the rapid-growing bacteria, as Staphylococci, Streptococci, Pneumococci, Gonococci and Colon bacilli, upon inclined agar are washed off with normal saline solution. After the mixture has sedimented, the upper, whitish layer composed of fluid and bacteria is removed with a pipette, and the finer clumps of bacteria precipitated by placing the fluid in a rapidly rotated centrifuge for a few minutes. The supernatant layer, which is still opalescent and is called a bacterial emulsion, should if suitable for work contain the germs in a well-separated condition.
Cultures of tubercle germs are heated, and ground in a mortar with salt solution until the mass is well broken up, and then centrifugated. In case glycerin cultures are used, such as are left in themanufacture of Koch’s old tuberculin, the glycerin must be removed by repeated washing with water and finally with 1·5 per cent. salt solution. The washed culture is worked up in a mortar and centrifugated until the clumps are practically all thrown down, and the cloudy layer or emulsion is removed.
The emulsions must be of uniform density. Wright computed the number of germs in a given volume by counting, but McFarland and L’Engle devised an apparatus which is called a nephelometer, consisting essentially of mixtures of BaSO4, put up in sealed tubes, which correspond to solutions containing from 1 to 10 per cent. of BaCl2, which serve as standards. The turbidity of the emulsion is compared in similar layer with the standard tubes of BaSO4. They found that the tube containing “5 per cent. of BaCl2corresponds to the most useful bacterial suspension.” The permanency of the emulsions varies a good deal. Suspensions of the gonococci should be used at once, staphylococci within two days, etc., while the emulsion of tubercle germs may be employed indefinitely.
The finger or lobe of the ear is punctured as in making a blood count, and the blood is allowed to drop directly into normal saline, ·85 per cent. solution, containing about 1 per cent. sodium citrate; this decalcifies the blood and prevents clotting. The corpuscles are completely precipitated in the centrifuge, and then repeatedly washed with ·85 per cent. saline and centrifugated, until all traces of the citrate and serum are removed. After the final precipitation the saline solution is withdrawn, and the thin upper grayish layer of the sediment, the leucocytic cream, consisting for the most part of washed white blood cells, is removed.
Blood is obtained in the usual way, but is collected in small, bent, glass tubes, which can be readily held in the centrifuge and the serum separated. In obtaining normal serum, care must be exercised in selecting a healthy subject, or, what is better, obtain serum from the mixed blood of several normal persons’ “Pool.”
With the exception of Malta fever and tubercle bacilli, Leishman’s stain, consisting of eosin and methylene-blue, in combination,or Jenner’s stain, is employed. Carbol-fuchsin is employed for the tubercle bacilli.