178 LABORATORY COURSE IN SERUM STUDY 



amount of the carefully shaken bacterial suspension is removed to a 

 watch glass. A 1-100 dilution 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 cover slip. After allowing 15 minutes for the bacteria to 

 settle, a count is made, with a No. 5 or a No. 6 lens, 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 ^rJ-fr of a square mm., the depth of the chamber is 0.1 mm., the dilu- 

 tion is 1-100. The number of bacteria may be estimated by the fol- 

 lowing formula : 



No. of bacteria counted X 400 X 10 X 100 X 1000 _ Dumber of bac- 



Number of squares counted 



tend in i c.c. 



(6) Wright's Method 



A Wright capillary pipette is prepared with a mark about one inch 

 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 two 

 portions of salt solution. Blood from the finger tip is then drawn up 

 to the mark, a bubble of air admitted, and 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 insure 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 Jenner's stain, or by any other simple method, and a differ- 

 ential 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 ruled scale to 

 be inserted in the eyepiece of the microscope is very helpful. A num- 

 ber of fields are counted, taken at random, 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 cubic mm.) : 



Number of bacteria X 5,000,000 X 1000 XT 



Number of red cells (200) " = Number f bactena per C ' C ' 



