276 THE PRINCIPLES OF IMMUNOLOGY 



sterile test-tube. In case of scanty growth the emulsion is directly transferred 

 to another surface culture, the growth in this tube suspended and the process 

 repeated with additional growths until a satisfactory 'emulsion is obtained. 

 Instead of filtering the emulsion one may shake the emulsion in a test tube 

 containing glass beads to break up the clumps. It is of great importance to have 

 a homogeneous suspension. Because of the presence of pepton or proteins 

 from the culture media some authors advise washing of the organisms until the 

 supernatant fluid gives a negative biuret reaction. The next step in the prepara- 

 tion is the counting of the 'emulsion. This can be done by the hemocytometer 

 method, by Wright's method and other methods. 



Hemocytometer Method. (From Zinsser, Hopkins and Ottenberg, " A 

 Laboratory Course in Serum Study.") A staining solution is prepared by adding 

 to 20 c.c. of i per cent, phenol I c.c. of a saturated alcoholic solution of thionin. 

 A small amount of the carefully shaken bacterial suspension is removed to a 

 watch glass. A dilution of i-ioo is prepared in a red cell pipette with the staining 

 solution as diluent to the 101 mark. After carefully shaking and after blowing 

 out the portion of the fluid in the capillary end of the pipette a small drop is 

 placed in a counting chamber and covered with a flat coverslip. After allowing 

 fifteen minutes for the bacteria to settle a count is made, with 4. num. objective, 

 of a number of squares until 200 or more bacteria have been counted. It is best 

 to take this count from different portions of the ruled surface and from two 

 separate drops of the mixture. The small squares have an area of 1/400 of a 

 square mm., the depth of the chamber is o.i mm., the dilution is I IOO. The 

 number of bacteria may be estimated by the following formula : 



Number of bacteria counted X 400 X 10 X 100 X 1000 



; ; = number of bacteria in r.o c.c. 



Number of squares counted 



Wright's Method. A drawn-out capillary pipette is prepared and marked 

 with a grease pencil about 2. cm. from the tip. A small puncture is made in the 

 tip of the finger and a fresh drop of blood obtained. Three units of salt solution 

 are then drawn up in the pipette, admitting a bubble of air between each unit of 

 salt solution. The unit is the amount that is drawn up to the mark on the 

 pipette. Blood from the finger-tip is then drawn up to the mark, a bubble of air 

 admitted and the bacterial suspension drawn up to the mark. The mixture is then 

 blown out on a clean slide and drawn in and out of the pipette several times to 

 ensure even mixing of the blood and bacteria. A drop of this mixture is placed 

 on a second slide and carefully spread across the slide in the manner of making 

 blood smears. It is important that the film be thin and even, so that the red 

 cells are not piled in masses in any portion of the film. This film is stained 

 with Wright's stain, or by any other simple method, and a differential count of the 

 number of bacteria and red cells in a number of fields in different parts of the 

 slide is made. For this a rule scale to be inserted in the eyepiece of the micro- 

 scope is very helpful. Fields are counted until 200 red cells have been counted. 

 The number of bacteria in the suspension may then be estimated from the number 

 of bacteria counted, using the following formula (assuming that the blood of the 

 worker contains 5.000,000 red cells per cmm.) : 



Number of bacteria X 5,000,000 X 1,000 XT 



^ r r j T; ? \ = Number of bacteria per c.c. 



Number of red cells (200) 



Other Methods. Among the other methods of standardization of the sus- 

 pension are the comparison of the emulsion with a known standard emulsion, the 

 estimate of the average number of organisms per slope grown in, say, eighteen 

 hours, or an estimate of the number of germs per loopful (Kolle's method). 

 Hopkins centrifugalized his suspension at high speed in a special tube with 

 graduated tip until the supernatant fluid was clear. The number of organisms 

 for a number of species in such a closely-packed sediment has been determined 

 and is as follows : 



Staphylococcus aureus o.oi c.c. equals 10 billion 



Streptococcus hemolyticus o.oi c.c. equals 8 billion 



Gonococcus o.oi c.c. equals 8 billion 



Pneumococcus (capsulated) o.oi c.c. equals 2.5 billion 



B. typhosus o.oi c.c. equals 8 billion 



B. coli o.oi c.c. equals 4 billion 



