216 MANUAL OF MICKOBIOLOGICAL METHODS 



If a sheep is not available, sheep blood may be obtained commercially; 

 however, the erythrocytes are likely not to survive shipment through the 

 mail. Freshly collected blood should be stored 48 hr before use. 



In the preparation of erythrocyte suspensions for testing, the blood in 

 Alsever's solution is diluted with 2 vol of 0.9 per cent saline solution 

 placed in a 50-ml centrifuge tube and centrifuged at approximately 

 500 X gravity for 5-10 min. The supernate is siphoned off and dis- 

 carded, and the cells resuspended in 35-40 ml of saline solution. The 

 cells are washed in this manner two additional times. If the last super- 

 nate is not colorless, the cells are too fragile and the blood should be dis- 

 carded. If the erythrocytes are satisfactory, dilute 1 vol of erythrocytes 

 with 49 vol of saline to produce a 2 per cent suspension. This suspension 

 should be filtered through a small wad of absorbent cotton to remove any 

 clumps. The suspension should be kept in the refrigerator when not in 

 use. Fresh suspension should be prepared each day. 



The red-cell counts of different batches prepared in this way may vary 

 somewhat, and therefore for work of precise nature it is preferable to 

 standardize by colorimetric determination of hemoglobin. 



For the initial determination, 5 per cent red-cell suspension may be 

 diluted to yield several concentrations ranging from 0.5 through 3 per 

 cent. One milliliter of each suspension is lysed with 9 ml of distilled 

 water, and the optical densities of the hemoglobin solutions read at 

 5,500 A in a spectrophotometer. If a spectrophotometer is not available, 

 a Klett-Summerson colorimeter with a No. 54 filter may be used. With 

 the values obtained, a calibration curve is plotted, and future 2 per cent 

 suspensions can be standardized against the curve. The same instrument 

 and cuvette should be used for all determinations. 



Sensitization of erythrocytes. The treatment of sheep erythrocytes 

 with their homologous antibody is spoken of as sensitization. The anti- 

 body, called hemolysin or amboceptor, may be prepared by immunizing 

 rabbits with sheep red cells ; however, it is usually preferable to purchase 

 commercial hemolysin. 



Before the proper concentration of hemolysin to use can be known, it 

 is necessary to perform a titration of the hemolysin. From a stock 

 1 : 100 dilution, prepare dilutions of 1 : 1,000 and 1 : 1,500. Arrange eight 

 15- by 85-mm test tubes in a wire rack and pipet 0.5 ml of 1 : 1,000 into 

 the first tube and 0.5 ml of 1 : 1,500 into the second. Into the remaining 

 tubes, place 0.5 ml of saline and prepare serial twofold dilutions of the 

 1:1,000 and 1:1,500 hemolysin to yield dilutions of 1:2,000, 1:3,000, 

 1 : 4,000, 1 : 6,000, 1 : 8,000, and 1 : 12,000. After 0.5 ml is discarded from 

 the last tube, each tube should contain 0.5 ml of diluted hemolysin. 

 Since the complement has not yet been titrated, a constant but excess 

 amount is added to each tube. In this case, add 0.3 ml of 1 :30 normal 



