TECHNIC FOR PREPARING BACTERIAL VACCINES 211 



then proceed to mix together the whole contents of the pipet, aspirating 

 and reexpelling these a dozen times. Then make two or three micro- 

 scopic films from the mixture, spreading these out on slides that have 

 been roughened with emery. 



The films are dried in the air, fixed by immersing them for two min- 

 utes in a saturated solution of corrosive sublimate, washed thoroughly, 

 and stained for a minute with carbolfuchsin diluted 1 : 10 or carbolthionin 

 for two to five minutes, and then washed and dried. 



The films are now given a preliminary examination. If red cor- 

 puscles and bacteria are found in approximately the same numbers and 

 the suspension is free from bacterial aggregates, the count may be made 

 (Fig. 60). If either the bacteria or the corpuscles are largely in excess, 

 new mixtures and new films must be made. In case the bacteria are 

 gathered in clumps, the suspension should be shaken again and new 

 films prepared (Fig. 61). 



When satisfactory films have been obtained, the actual counting 

 may be done. This is carried out with an oil-immersion lens, and in 

 order to secure accuracy, it is necessary to restrict or divide the field by 

 a small square diaphragm made of paper or cardboard, or by inscribing 

 cross lines on a small clean cover-glass and dropping them on the dia- 

 phragm of the eye-piece. 



A field is now chosen at random, and the corpuscles and bacteria 

 are counted, the results being jotted down on a sheet of paper, keeping 

 each enumeration separate and writing the numbers in two columns. 

 Proceed at random from field to field, traversing every part of the 

 slide. Establish a rule for counting corpuscles that transgress or touch 

 the edge of the field. Eliminate from consideration any parts of the 

 films in which the preparation is unsatisfactory as regards the staining, 

 or with respect to the integrity of the red corpuscles. The examination 

 is continued until at least 500 corpuscles have been counted, half of the 

 count being made from the second slide. The number of microorganisms 

 counted is now totaled, and the approximate number per cubic centi- 

 meter estimated. Let us assume, for example, that 600 red cells and 

 1200 bacteria have been counted. Now, a cubic millimeter of blood 

 contains 5,500,000 red corpuscles, and equal volumes of blood and emul- 

 sion were taken. A cubic millimeter of the emulsion, therefore, contains 



5 500 000 ^ 1 200 



= 11,000,000 organisms per cubic millimeter, or 

 bOO 



11,000,000,000 per cubic centimeter. 



(b) The second method of counting is precisely similar to that em- 



