200 MANUAL OF MICROBIOLOGICAL METHODS 



able animal. The dosage will vary depending on the organism under 

 study. While viable cells may be used also in studying pathogenic bac- 

 teria, particularly where one wishes to study antibody response during 

 infection, it is more common to employ killed vaccines. Killing is accom- 

 plished by heating the microbial suspension at a temperature high enough 

 to kill yet not so high as to cause drastic changes in the antigens, usually 

 60°C for 1 hr for nonsporulating organisms. An alternative and some- 

 times preferable choice is to add 0.3-1.0 per cent formalin to the culture 

 and allow it to stand at 37° C for 12 hr. 



The medium used for growth of the antigen will vary with the organism 

 and in complexity from the simple synthetic media entirely suitable for 

 certain bacteria, yeasts, and molds to the living host tissues necessary for 

 viruses. The more complex media contain antigenic materials which 

 may interfere with the system under study. This is particularly true 

 with soluble extracellular antigens. Where possible, nitrogen should be 

 supplied by inorganic salts or amino acids. In other instances it may be 

 possible to replace a complex medium with casein hydrolysate or an ultra- 

 filtrate of peptone plus accessory growth factors which are not antigenic. 

 It should be noted that with some pathogenic bacteria, the antigenic 

 structure may be atypical unless the strain has recently been passed 

 through a suitable animal. 



After the antigen has been grown, harvested, and killed, it is centri- 

 fuged and washed several times with 0.9 per cent sodium chloride solution 

 (''saline")- Following this, the antigen is resuspended in saline and 

 standardized so that the dose used for injection will be contained in 1 or 

 2 ml. Standardization may be accomplished by direct microscopic count, 

 nephelometry, or Kjeldahl nitrogen determination. If the antigen is 

 toxic, this will be a factor in determining the density; however, for many 

 bacteria and yeasts, the arbitrary selection of 10^ to 10^ cells per milliliter 

 will yield satisfactory results. After the vaccine has been standardized, 

 it should be checked for sterility by inoculating a tube of thioglycollate 

 broth and a blood agar slant with 0.1 ml of the vaccine. A preservative 

 like phenol, 0.5 per cent, or merthiolate, 0.01 per cent final concentration, 

 should then be added to the vaccine. 



Soluble antigens such as toxins, toxoids, or various protein solutions 

 may often be sterilized by filtration through a Seitz or Berkefeld filter. 

 If the antigen fails to pass through such a filter, sterilization with ethylene 

 oxide may be tried (Reddish, 1954). Sterility tests should be conducted 

 as described for particulate antigens. Soluble antigens may be standard- 

 ized on a weight basis, 1-2 mg of protein per milliliter of saline usually 

 being satisfactory. In other instances, it may be desirable to standardize 

 by nitrogen content using a micro-Kjeldahl procedure. 



Antigens, whether particulate or soluble, should be dispensed in sterile 



