Blood agar preparation

To prepare plate cultures, frozen stocks are placed on dry ice and are stabbed with a hot wire loop. Material adhering to the loop is then plated onto the blood agar plates. Frozen stocks may require several passages to regain growth characteristics. 

To prepare a seeding culture, fill a 25 pL universal with approximately 10 pL of sterile Mueller-Hinton broth and seed these with 1-2 colonies from the blood agar plate. This is then incubated at 37°C overnight with vigorous shaking at approximately 200 rpm. 

Before using heparin with antibiotics, blood agar plates with various organisms were cultured. The plates were prepared in the ordinary fashion. In a second set of cultures, disks with antibiotics were soaked in heparin, and finally heparin was incorporated into the blood agar alone. Examination of the plates made it obvious that heparin did not inhibit the action of the antibiotics and often seemed to enhance it. It is not known whether heparin is truly an antibiotic. 

Preparation of culture supernatants - R haemolytica biotype A1 was grown as previously described (17). Briefly, colonies from a blood agar plate wCTe used to inoculate tryptic soy broth. This culture was incubated at 37 C for 4.5 hours. This broth was used to inoculate fresh tryptic soy broth which was incubated for 1 hour at 370 C with 5% CO2 bubbled throu it. The broth was stirred as rapidly as possible without foaming. After 1 hour of incubation bacteria were removed from the culture by ultrafiltration (Pellicon, Millipore, Inc.). Culture supernatants free of bacteria were lyophilized and stored dessicated at -20 C until loaded into hydrogels. 

This medium was prepared by adding sterile defribrinated sheep blood (10%) to sterile molten nutrient agar (Oxoid) at 55 °C. 

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