260 Journal oj Agricultural Research voi. 45, No. 5 



a flask containing 50 c c of sterile water and glass beads. After the 

 scales had softened in the water, the flask was shaken for one-halt 

 hour to insure complete maceration of the scales. The suspension 

 was then filtered through two thin layers of sterile absorbent cotton 

 into another sterile flask in order to remove any lumps or debris. 



In preparing the stock suspensions of spores, at first 75 to 100 

 scales were taken by counting. Later it was found that the average 

 American foulbrood scale weighs 0.0223 g. Therefore, the 100 scales 

 for the stock suspensions were obtained by weight, the scales bemg 

 •weighed in a sterile covered glass dish before they were deposited 

 in the flask of sterile water. 



After the suspension had been filtered and tested for contamma- 

 tion and was ready for use, the number of spores per cubic centimeter 

 was determined by the following method: By means of a blood- 

 diluting pipette giving a dilution of 1 to 20, the spore suspension was 

 diluted with a weak solution of carbol fuchsin and a drop placed in 

 the counting chamber of a Helber bacteria-counting cell 0.02 mm 

 deep and ruled in squares of 0.0025 mm^ each.* With the use of 

 two 15 X eyepieces in a binocular microscope and a 1.8-mm oil- 

 immersion objective, the spores in 25 squares of the Helber chamber 

 were counted. Then by means of the formula 



Total spores counted X dilution X 20,000 X 1,000 

 Number of squares counted 



the approximate number of spores per cubic centimeter in the sus- 

 pension was determined. 



Later this method was checked by the method of Breed and Brew 

 (2) for counting bacteria in milk. With the aid of a binocular micro- 

 scope having two 15 X eyepieces and a 1.8 mm oil-immersion ob- 

 jective, the area of a circle etched on an ocular micrometer disk was 

 determined by means of a stage micrometer. One one-hundredth 

 cubic centimeter of a 1 to 100 dilution of the stock suspension of 

 spores was placed on a glass slide on which 1 cm^ had been ruled 

 with a diamond pencil. This was mixed with a small loopful of 

 carbol fuchsin stain and the whole spread over the 1 cm ^ of surface * 

 and allowed to dry uniformly. The number of spores per cubic 

 centimeter of the stock suspension was determined according to the 

 formula 



Area 1 cm ^ total number of spores counted X dflution X 100. 



Area of circular field number of circular fields counted 



These two methods were found to check fairly closely within the 

 limits of the precision of the methods used in counting. Further- 

 more, by both methods it was found that in the majority of cases 100 

 scales in 50 c c of water give approximately 5,000,000,000 spores per 

 cubic centimeter for each suspension made up in this way. Therefore, 

 this number was used as a standard for making all dilutions. 



» Mm' and cm' are the abbreviations tor square millimeter and square centimeter, respectively, recently 

 adopted by the Style Manual for United States Government printing. 



