These sera are collected and treated as already described (see page 379), and the portions of the blood pipettes containing them are arranged in the remaining space in plasticine block. [Illustration: FIG. 194.--Plasticine block with materials arranged for opsonin estimations.] The plasticine block now presents the appearances shown in Fig. 194. METHOD FOR DETERMINING THE OPSONIC INDEX.-- 1. Take a capillary pipette fitted with a teat, cut the distal end _square_ and make a pencil mark about 2 cm. from the end. 2. Aspirate into the pipette one volume of washed cells, air index, one volume of bacterial emulsion, air index, and one volume of specific serum (see Fig. 195). [Illustration: FIG. 195. Opsonin pipette.] 3. Mix thoroughly on a 3 by 1 slide by compressing the teat and ejecting the contents of the pipette on to the surface of the slide, relaxing the pressure and so drawing the fluid up into the pipette again. These two processes should be repeated several times; finally take up the mixture in an unbroken column to the central portion of the capillary stem. 4. Seal the point of the pipette in the peep flame of the bunsen burner and remove teat. 5. Mark the pipette (with the grease pencil) with the distinctive number of the serum and place it in the glass box or tray. 6. Take another similarly prepared pipette and aspirate into it equal volumes of washed cells, bacterial emulsion and pooled serum. Treat precisely as in 3 and 4, label it "control" or "N.S." (normal serum) and place in the box by the side of the specific serum preparation. 7. Place the box with the pipettes in the incubator and set the signal clock to ring at 15 minutes (or start the stop watch). 8. At the expiration of the incubation time remove the pipettes from the incubator. 9. Cut off the sealed end of the specific serum preparation. Mix its contents thoroughly as in step 3, and then divide the mixture between two 3 by 1 slips and carefully spread a blood film (_vide_ page 376) on each in such a way that only one-half of the surface of each slide is covered with blood--the free edge of the blood film approximating to the longitudinal axis of the slide. Allow films to dry and label the slides with writing diamond. 10. Treat the contents of the control pipette in similar fashion. 11. Select the better film from each pair for fixing and staining. 12. Fixing and staining must be carried out under strictly comparable conditions, and to this end the slides are best handled by placing in a glass staining rack which can be lowered in turn into each of a series of glass troughs containing the various reagents (Fig. 196). Place the rack in the first trough which contains the alcoholic solution of Leishman's stain for two minutes to fix. Transfer to the second trough containing the diluted stain for ten minutes.