d to 7.0 before autoclaving using 0.1N NaOH or 0.1 N HCl solution. Fifty-ml aliquots of this medium were dispensed in 250 ml Erlenmeyer flasks. The medium was adjusted to pH 7.0 and sterilised at 121o C for 20 min. Each flask was inoculated with 1.0 ml S. violatus spore suspension obtained from a 6-day-old slant culture. The flasks were then incubated under static conditions at 30o C for 7 days. The antibiotic bioassay was carried out at the end of the incubation period.of dry weight: The cells were separated from the culture filtrate by at 5,000 rpm for 15 minutes, washed twice with distilled water and then dried at 70o C until reaching a constant weight. Preparation of the crude antibiotic: Following 7 day incubation period, S. Violatus cells were separated from the culture by centrifugation at 5,000 rpm for 15 minutes in a cooling centrifuge at 4o C (Chilspin centrifuge MSE Fisons). The blue-coloured clear supernatant was then tested for its antibiotic activity.bioassay: This was carried out using the paper-disc diffusion method, Hinton agar as an assay medium and S. aureus as a test organism. The Mueller-Hinton agar (45o C) was poured into sterile Petri-dishes (9 cm diameter) and allowed to solidify. 0.1 ml bacterial suspension (3 x 106 cells) of the test organism was inoculated into the agar surface. Sterile paper discs (6.0 mm diameter Whatman antibiotic assay discs) were placed on the dried surface of the medium using alcohol-flame-sterilised forceps. Each disc received 20 ?l of the culture filtrate. Petri-dishes were kept in a refrigerator for 2 hours to allow for the diffusion of the antibiotic. Petri-dishes were then incubated inverted for 18-24 hours at 37o C. The inhibition zone diameter was measured in mm (Amade et al. 1994). The antibiotic concentration (?g/ml) was determined using a standard calibration curve using the purified antimicrobial substance (MSW2000) produced by S. violatus (Said 2001). estimation: The blue pigment concentration in the culture broth was estimated colorimetrically at 566 nm. This wave length was selected since it showed a maximum absorption of the coloured supernatant measured in UV VIS 4B Spectrophotometer. Each experiment in this work was repeated three times and the average of the three replicates was taken.
. 2 Results and discusion
of some locally isolated actinomycetes for the production of antibiotic (s). A survey of four locally isolated Streptomyces strains for antibiotic production was carried out in static and shaken cultures (Table 1). It was generally observed that the growth and antibacterial activity obtained in static cultutres were higher than shaken cultures. Streptomyces astreogriseus showed the longest incubation time (12 days) needed to obtain maximum antibacterial activity, while Streptomyces violatus showed a relatively short time (7-days) and produced the highest activity among the tested strains. Streptomyces violatus was also characterised by its broader antibacterial activity, because it affected the growth of all the tested bacteria, showing a stronger activity on S. aureus and B. subtilis. Accordingly, S. violatus was selected for further investigation.
1 - Screening for the antibacterial activity of Streptomyces strains in static (St) and shaken (Sh) cultures.
growth of S. violatus and the production of antibiotic in a starch-nitrate medium were monitored over a period of 14 days (Fig. 1). The antibiotic production by S. violatus occurred in a growth-phase dependent manner and the highest antibiotic yield was obtained in the late exponential phase and the stationary phase, indicating that it is mainly a product of secondary metabolism. Similar results were observed for streptomycin production in batch cultures of S. griseus [29] when grown in a mineral medium and for the production of candicidin in liquid grown cultures of S. Griseus.The results also showed that S. violatus produced a blue pigment associated with the antibiotic appearance in the culture. It was noticed that a direct tight relationship occurred between the antibiotic production and the intensity of the blue colour formed in the culture (r=0.95). These results may suggest the production of a pigmented antibiotic in S. violatus cultures. The production of the blue-pigmented antibiotic actinorhodin and its physiology are known in S. coelicolor cultures.
1. Effect of different incubation periods on the production of antibiotic by Streptomyces violatus.
2.2.1 Influence of some cultivation factors on the production of antibioticof antibiotic production in batch cultures of S. violatus was carried out. This strain was able to grow in all the tested carbon sources (Table 2). However, maximum antibiotic production was obtained in cultures supplemented with glycerol as a sole ...
