(Continued from PREVIOUS  PAGE )

• Week 8.  Liquid cultures (50 ml in 250 ml erlenmyer flasks)  of Streptomyces Broth Medium are inoculated with one to two ml of Streptomyces spore suspension and incubated at 28 C with vigorous aeration for a minimum of two weeks.

Figure  5.  (above) Sephadex G25 sizing gel column apparatus.  Columns were loaded with approximately 1.0 ml of filtered culture supernatant and eluted with 10 mM Tris-HCl (pH 7.4).

• Week 11 or 12.  The sterile filter disks are allowed to dry and applied to plates of Mueller Hinton Agar inoculated with a lawn of test bacteria in the  Kirby Bauer Assay.  After 24 hr incubation at 37 C the zones of inhibition are recorded. The pattern of elution of the antibiotic activity can be correlated with the antibiotics size and charge properties.

Figure 6.  (left) Modified Kirby Bauer Assay (versus E. coli) on Anion Exchange column fractions of the culture supernatant from Streptomyces isolate ALD13.  Column fractions were added to sterile 13 mm filter disks which were applied plied to  Mueller Hinton Agar plates inoculated with E. coli.

Figure 7 (above) Recovery of antibiotic activity from an Anion Exchange column loaded with Streptomyces Ald13 culture supernatant and eluted with 2 M NaCl in 10 mM Tris (pH 7.4 ). The column fractions were tested against  four bacteria  COLOR KEY: BLUE is Staphylococcus  aureus,  RED is E. coli,  YELLOW is a tetracycline resistant E. coli , and AQUA is Pseudomonas aeruginosa