156 CLINICAL BACTERIOLOGY AND HEMATOLOGY 



a vaccine prepared from one variety may have little action on 

 another. The culture should be made on agar (or blood-agar m 

 the case of gonococci, etc.), and should be one or two days old. 

 To each culture-tube about 2 to 5 c.c. of sterile normal saline 

 solution are added, and the bacteria scraped off by means of a 

 platinum needle and the tube gently shaken, so that the organisms 

 are emulsified. The number of bacteria in this emulsion must 

 next be counted. Wright's method is usually employed, and is 

 carried out by mixing the bacterial suspension with blood in known 

 proportions, making films, and counting the red corpuscles and 

 bacteria in the same field of the microscope, so as to obtain the 

 relative proportions of the two. The number of red corpuscles 

 per cubic millimetre is known (or can be counted), and from that 

 the number of organisms can be calculated. Proceed as follows : 

 Take an opsonin pipette, and draw up one volume of blood from 

 a healthy person, then a bubble of air, then one volume of citrate 

 solution (p. 150), then one volume of the emulsion. Mix them 

 together on a slide, make films on two cover-glasses, dry, and 

 stain by Jenner's method. Proceed to count the red corpuscles 

 and bacteria on several fields of the microscope : this will be easier 

 if you rule four ink lines enclosing a square on the lower lens of 

 your eyepiece, and count the objects lying therein. When you 

 have done this, move the slide so as to get a fresh film, and count 

 again. Do this a large number of times, and add the totals of the 

 corpuscles and of the bacteria, and calculate the ratio between the 

 two. (Thus in one case the total red corpuscles on the various 

 fields were 18, 15, 20, 21, 14, 17, 10, and 15 ; and the bacteria 37, 

 26, 31, 40, 25, 30, 32, 36 : the totals were 130 and 257, or, roughly, 

 I to 2.) The calculation of the number of bacteria per cubic 

 millimetre is then easy, and that multiplied by 1,000 gives the 

 number per c.c. 



Next the emulsion has to be sterilized by heat. It is placed in 

 a test-tube, which is drawn out and sealed in a blow-pipe flame 

 and completely immersed in a water-bath at 60°. After one hour it 

 is removed, and a small amount placed on a suitable culture 

 medium and incubated (in order to ascertain its sterility), and the 

 tube re-sealed. If sterile, it is now to be diluted with a 0-25 per 

 cent, solution of lysol or carbolic acid in sterile normal saline 

 solution to such an extent that the dose required is made 

 I c.c. Thus an emulsion of staphylococci was found to contain 

 2,500,000,000 cocci per c.c. The dose required was 500,000,000, 



