I2 4 



Scientific Proceedings (106). 



needle point is transferred to 10 c.c. of Nageli solution in a sterile 

 test tube and the tube shaken from to 2 hours on a shaking 

 machine. At the end of that time the uniformity of the emulsion 

 is tested by removing ten units of the suspension with the pipette, 

 blowing them out on a microscope slide, staining and counting. 

 If the counts are uniform the suspension is used, if not it is shaken 

 further. This uniformity of the emulsion is extremely important 

 in comparing results. Having secured a uniform emulsion the 

 pipettes are prepared as follows : Two units of a sterile extract of 

 vitamine are first drawn up into the pipette (the unit we have 

 used is about 1/800 c.c). We have prepared our extracts in 

 various ways but in all cases they have been sterilized for two 

 30-minute intervals in the Arnold Sterilizer with a 24-hour inter- 

 val. Next two units of yeast suspension are drawn up. By 

 manipulating the bulb a bubble of air separates the yeast and 

 vitamine in the tube and permits accurate measurement. When 

 both materials are in the tube they are drawn up to the large part 

 of the tube and mixed. The tube is then sealed as described 

 above. The control tube contains the yeast cells in the Nageli 

 solution (two units) but no vitamine. Our Nageli solution is 

 the same as used by Dr. Bachman, being a sterilized solution of the 

 following components: 100 c.c. distilled water; 10 gms. dextrose; 

 1 gm. ammonium nitrate 0.05 gms. calcium phosphate, 0.5 gms. 

 potassium acid phosphate; 0.25 gms. magnesium sulfate. 



After the tubes are filled and sealed they are incubated at 35 0 

 Cent, for the time necessary, usually 18-24 hours. At the end of 

 that time the ends are broken off, the rubber bulb adjusted and 

 the contents blown out on a specially prepared counting slide, 

 fixed and stained and can then be counted at leisure. For this 

 purpose we prepare common microscope slides by etching 7 mm. 

 squares on them, experience having shown that such a square 

 will hold the contents of a tube. In this way it is possible to get a 

 series of the contents of ten or twelve pipettes on one slide. Count- 

 ing is done under the high power with the aid of a mechanical 

 stage. 



Results with the Method. — The methodology reported above has 

 been arrived at by experiment. The necessity for accurate 

 calibration, the need for uniform suspensions of yeast, the size of 



