20 DIFGOMANUAL 



Repeated melting of solidified agar, or long holding of melted agar at high tem- 

 perature, may likewise cause a precipitate to form in the media. Media contain- 

 ing agar may also form a flocculent precipitate if the liquid medium is held in 

 the water bath at 43 to 45 °G. for longer than 30 minutes. This flocculent agar 

 precipitate, however, may be dispersed by reheating the medium. 



Excessive heating of media also results in an increase in acidity. The reaction 

 of the media will become more acid as heating is prolonged. Some media which 

 are acid, such as Wort Agar (pH 4.8) will, upon prolonged heating, cause de- 

 struction of the agar. It is possible to destroy completely the jellifying properties 

 of agar by prolonged heating, and this destruction is hastened as the acidity in- 

 creases. 



Culture media which may be injured by autoclaving are sometimes sterilized 

 by the discontinuous or intermittent method. This procedure consists of heating 

 the medium in a chamber of flowing steam for a period of 20 or 30 minutes, or 

 longer, on several successive days. Body fluids and sera are sometimes sterilized 

 in the inspissator at 53° to 70° C. for one hour on six successive days. Liquid 

 media may be sterilized by filtration through unglazed porcelain or earthenware 

 candles, or through a sterilizing pad. 



External contamination of culture media is prevented by plugging the tubes or 

 flasks with nonabsorbent cotton before sterilization. Plugs should fit neither too 

 loosely nor too tightly and should protect the lip of the container against the ac- 

 cumulation of dust. Screw cap tops or metal covers may also be used to close the 

 tubes or flasks. Marcus and Greaves^ have called attention to the fact that atypi- 

 cal cultural reactions may be obtained in sealed tubes of media used to test 

 biochemical activities due to anaerobic conditions. Tubes of Kligler Iron Agar 

 and Russell Double Sugar Agar, for example, gave aberrant reactions in tubes 

 sealed with screw caps or rubber stoppers. The same medium with tubes loosely 

 capped, or caps replaced with cotton plugs, showed typical reactions. 



Media should always be stored in a cool moist atmosphere to prevent evapora- 

 tion. Prolonged storage of sterile media cannot, however, be recommended. If 

 tubes of media have been kept for any length of time they should be reheated 

 just before use. Liquid media should be heated in a boiling water bath or in flow- 

 ing steam for a few minutes, to drive off dissolved gases, and then cooled quickly 

 in cold water without agitation just prior to inoculation. Agar tubes should be 

 melted and allowed to solidify in order to secure a moist surface which is desired 

 by most microorganisms. These precautions for both liquid and solid media are 

 extremely important for the initiation of growth of highly parasitic organisms 

 such as those encountered in blood culture work. 



Blood or other body fluids to be cultured should always be taken prior to the 

 administration of the therapeutic agent. If drugs have been administered their 

 bactericidal effects should be neutralized, if possible. The addition of j!?-amino- 

 benzoic acid (PAB) in 0.5 mg. per cent to the medium will assure the inactiva- 

 tion of any sulfa drug carried over with the inoculum. Bacto-Penase, a concen- 

 trated purified penicillinase, should be added to the sterile cooled medium used 

 for blood culture if the patient is under penicillin therapy. Bacto-Brain Heart 

 Infusion with P.A.B. and Agar with added Bacto-Penase is an ideal medium for 

 blood culture work. The small amount of agar present will give all degrees of 

 anaerobiosis, permitting the development of aerobes as well as the strictest 

 anaerobes, the PAB will inactivate any sulfa drug, the added Bacto-Penase will 

 inactivate any penicillin in the inoculum, while 100 ml. of the medium itself will 

 inactivate up to 1000 units of streptomycin. 



'Am. J. Med. Tech., 14:214:1948. 

 2 Am. J. Pub. Health, 25:301:1935 

 •J. Lab. Clin. Med., 36:134:1950. 



