210 PHAGOCYTOSIS. OPSONINS AND BACTERIOTROPINS 



prepared capillary pipette. The suspension of bacteria is placed in a 

 sterile test-tube, the end of this tube drawn out in the blow-pipe flame and 

 sealed. The drawn-out portion should be about 2 inches in length and as 

 strong as possible. The emulsion is now vigorously shaken for fifteen 

 minutes. The extremity of the drawn-out tube is then cut and a few 

 drops of the emulsion expelled into a clean watch glass, or a small part of 

 the drawn end is cut off so that a portion of the emulsion is still contained 

 within it. The tube is resealed, and then submerged in water kept at 60 

 C. for one hour. This usually suffices to kill the bacteria. 



The small amount placed in the watch glass or in the capillary test- 

 tube serves for the standardization, which is carried out as follows: A 

 pipette with rubber bulb, as prepared for the opsonic-index test, is also used 

 here. A volume of freshly drawn blood of known corpuscular content, 

 best taken from the worker's own finger, and an equal volume of 

 bacterial emulsion are mixed thoroughly with six or seven volumes of i 1/2 

 per cent, citrate solution; several even films (which may be fairly thick), 

 are then made by means of the ordinary edge of a slide, and stained with 

 carbol-thionin, Leishmann's or Jenner's stain. 



The entire smear is divided up (with a blue skin pencil) into eight 

 equal subdivisions, by one transverse line drawn parallel to the long diam- 

 eter of the slide at its middle and five vertical lines, one at each edge of the 

 smear, one in the center and one equally distant between the edge and the 

 central line. It is also advantageous to employ an eye piece, the field of 

 which has been divided or made very much smaller by the insertion of a 

 paper screen with a small central opening representing the size of the 

 desired field. Five or six fields are then counted in each of the eight sub- 

 divided areas. The number of red blood cells seen in each field are enumer- 

 ated in one vertical column, the number of organisms in the same field in 

 another column. In this manner an average of the entire slide is obtained. 



By means of a simple proportional sum, the number of bacteria per 

 cubic centimeter of emulsion is estimated, e.g., the number of red blood cells 

 counted is 850 and the number of bacteria 1020. The red blood corpuscles 

 used in the standardization are known to number 5,000,000 to a cubic 

 millimeter or 5,000 million to a cubic centimeter; therefore the number of 

 bacteria to a cubic centimeter of the unknown emulsion is expressed as 

 follows. 



850 : 1020 : : 5,000,000,000 : No. of bacteria per c.cm. of emulsion, 



.*. 6,000,000,000 = the number of bacteria per c.cm. of emulsion. 



After the emulsion has been heated for one hour, the tube is opened 

 and a drop is expressed into an agar culture tube which is incubated for 

 twenty-four hours to demonstrate whether the emulsion is sterile or not. 

 At the end of this time, if a growth is observed, the emulsion must be heated 

 again for one hour at 60 C/and its sterility again tested for. 



