358 BACTERIOLYSINS 



The serum and culture must now be mixed, being careful not to con- 

 taminate the broth. This is effected by blowing these two volumes out 

 into a sterile mixing tube and drawing up and blowing out the fluid 

 several times in succession. By starting with the highest dilution, the 

 same mixing tube may be used throughout the series. With an air- 

 bubble of at least 5 cm. between serum and broth, this can be quite easily 

 achieved without driving the sterile broth down from the bulb of the 

 pipet into the lower part of the capillary stem and contaminating it there. 



The column of mixed serum and culture is now drawn up into the 

 middle region of the capillary stem and the lower end of the tube is 

 sealed. The teat is then removed, and the dilution that has been used 

 is written on the barrel of the pipet. 



The series of pipets are now filled with the nine measuring dilutions 

 of the culture. 



Controls. The serum from a healthy individual, or, better still, the 

 pooled serums of several persons, may be used in precisely the same way, 

 with at least the second, fourth, sixth, and eighth dilution^ of culture. 



Several culture controls are advisable. The pipets are filled with 

 mannite broth in the usual manner, and then with one volume of at least 

 four different dilutions, usually 1 : 50, 1 : 5000, 1 : 100,000, and 1 : 1,000,- 

 000. The tubes are sealed, labeled, and incubated together with those 

 concerned in the test proper. 



One pipet is to contain the usual quantity of broth and one volume 

 of the patient's serum. This is a control on the sterility of the patient's 

 serum. A similar preparation is made with the control serum to serve 

 as a control on its sterility. 



When the whole series of tubes have been filled, these are placed 

 upright in a large test-tube or cylinder labeled with the date and the 

 source of the serum, and incubated at 37 C. for from eighteen to twenty- 

 four hours. 



Test for the Germicidal Activity of the Serum. The serum and culture 

 in the capillary portion of the tube must now be mixed with the broth in 

 the barrel. This is accomplished by taking each pipet in hand singly, 

 and heating the lower portion of the capillary stem in a " peep-flame" 

 and drawing out into a small thread with a pair of forceps. 



A collapsed teat is now fitted over the barrel, and the negative pres- 

 sure carefully regulated by keeping the finger and thumb in position on 

 the teat, and the finely drawn out end of the capillary stem gently 

 snapped across. The column of serum and culture will then be carried 

 up into the bulb of the pipet. The end of the tube is now sealed. 



