The micro serological technique developed by G. Takatsy (Acta Microbiol. Acad. Sci. Hung. 3:191, 1955) permits a considerable savings in reagents and in time. This report describes a micro antistreptolysin 0 (ASO) test which is simple, rapid, and reliable. The Microtiter equipment (Cooke Engineering Co., Alexandria, Va.) consists of six 0.05-ml microdiluters, one 0.05-ml and two 0.025-ml calibrated dropper pipettes, centrifuge carriers, and a reading mirror. Mark off disposable U plates (Linbro

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... Chemical Co., New Haven, Conn., or Cooke Engineering Co.) so that each specimen is assigned two rows with six wells per row. Include a serum standard of known titer in each run to serve as a positive control. Label wells at the bottom of the plate for streptolysin, cell, and serum controls. Each plate will contain six specimens plus controls. Prepare 1:60 and 1:85 dilutions from a 1:10 dilution of serum in buffered diluent, pH 7.4 (barbital, 0.64 g; sodium barbital, 0.31 g; sodium chloride, 8.5 g; gelatin, 1 g; and distilled water, to 1.0 liter). Add 0.1 ml of the 1:60 dilution to the first well in the first row of the U plate. Add 0.1 ml of the 1: 85 dilution to the first well of the second row. Add 0.05 ml of the 1: 85 dilution to the serum control well. Add diluent to the remaining wells as follows: 0.05 ml to the specimen wells; 0.025 ml to the serum control; 0.05 ml to the streptolysin control; and 0.075 ml to the cell control. Use six microdiluters to make serial dilutions simultaneously in six rows. Transfer 0.05 ml of the serum dilution from the first well in each row to the next well, mix, and transfer serially through the sixth well in each row. Each specimen will contain the following dilutions: first row, 1:60, 1:120, 1:240, 1:480, 1:960, 1: 1,920; second row, 1: 85, 1:170, 1: 340, 1:680, 1:1,360, 1:2,720. Add 0.025 ml of cold streptolysin 0 to all wells except the cell and serum controls. Mix, cover the plates, and incubate in a moist atmosphere at 37 C for 15 min. Add 0.025 ml of 2% sheep or rabbit red blood cells (washed three times in buffered diluent) to all wells. Mix and incubate at 37 C for 15 min. Remove from the incubator, mix thoroughly to resuspend the cells, and incubate for an additional 30 min. Centrifuge at 250 x g for 3 min. Use the reading mirror to determine the highest serum dilution showing no hemolysis. The ASO titer is the reciprocal of this dilution. There should be complete lysis in the streptolysin control and no lysis in the cell or serum controls. Advantages of the micro over the macro ASO techniques are: the savings in reagents amounts to approximately $65 per 100 tests, and twice as many tests can be carried out in a given period of time. Reproducibility of the micro test was determined by an analysis of 18 replicate blind determinations on each of 11 pools of sera. Duplicate determinations yielded titers within one dilution step 98% of the time. 184 on May 7, 2020 by guest http://aem.asm.org/ Downloaded from