The actinomycetes has been found to be the most promising group of microorganisms particularly for their industrial uses for the production of antibiotics, enzymes and related compounds. Among the antibiotic producing actinomycetes, the genus Streptomyces, is well known since the discovery of streptomycin. Later on several other antibiotics have been reported from various strains of this genus. However, species of this genus differ greatly in their response to various physical and chemical conditions around them. The characterization of morphological and biochemical properties, and the conditions favouring production of antibiotic like substances by this group of organisms is an important aspect for should be worked out in detail for their practical application.
MATERIALS AND METHODS
More that hundred actinomycetes were isolated from soil samples collected from places of Sagar District (Madhya Pradesh) by using the method of Williams and Cross. Out of these isolates one strain, i.e., Ac43 which showed promising antagonistic activity during screening was selected for further detailed study.
Antagonistic activity
Antagonistic activity of the above strain was tested against 14 pathogens by using the cross streak method as suggested by Casida.
Cultural characteristics
The test strain Ac43 was grown on the following three media and the cultural characteristics were noted:
(i) Yeast Extract Malt Extract Agar
Yeast Extract – 4.0 gm, Malt extract – 10.0 gm, Dextrose – 4.0 gm, Distilled Water – 1000 ml, Agar-agar – 20.0 gm, pH adjusted to 7.3.
(ii) Inorganic Salts – Starch Agar
Solution (A). 10.0 gm. starch was dissolved in distilled
water and then final volume of the solution was made to 500
ml. *
Solution (B). K2HP04 – 1.0 gm, MgSo4.7H20 – 1.0 gm, NaCI -1.0 gm, (NH4)2S04 – 2.0 gm, CaC03 – 2.0 gm, were dissolved in 400 ml distilled water and to this 1.0 ml trace salt solution (FeS04.7H20 – 0.1 gm, MnCI2.4H20- 0.1 gm, ZnS04.7H20 – 0.1 gm, Distilled water – 1000.00 ml) was added to this. Its pH was adjusted to 7.0 before raising its. final volume to 500 ml.
Both solution A and B were fixed together and then to this, 20 gm Agar-agar was added before sterilization.
(iii) Glycerol – Asparagine Agar
L – asparagine (anhydrous basis ) – 1.0 gm, Glycerol -10.0 gm, K2HP04 (anhydrous basis) – 1.0.gm.
Trace salts solution – 1.0 ml were dissolved in distilled water. The pH was adjusted to 7.2 and then the volume finally raised to 1000 ml with distilled water. To this 20 gm agar was added.
The morphological details of the mycelium, spore bearing structures, etc. were made by using microscopic methods. The colour of the colony, mycelium, etc. were recorded with the help of Rayner’s Mycological Colour Chart (CMI, Kew) while the melanin formation was studied following the method of Shirling and Gottlieb.
Biochemical Characterization
Tests were made to evaluate the production of H2S gas and acid by using standard methods (Hi-Media). For the study of catalase activity and starch hydrolysing capacity the method described by Waksman was used. The utilization of carbohydrates was determined by the method as given by Pridham and Gottlieb.
Antibiotic Production
The production of antibiotic was tested in the starch peptone broth having pH 7.2 . For this the organism was grown in 150 ml flask containing 30 ml of above medium on a reciprocating shaker (160 rpm) at 28°±2°C for 10 days. After incubation all the flasks were taken out and the content of each flask was filtered and to obtain a cell free extract the filtrate was then centrifuged at 6000 rpm for 20 minutes. The supernatant was condensed to 2 ml and purified by using silica gel column.
The purified sample was then studied by using thin layer chromatography. For chromatograpic analysis three solvent systems i.e., ethyl acetate : methanol (95:5), acetone : water (9:4) and butanol saturated with water were used as suggested by Stahl. The developed chromatograms were observed under ultraviolet Tight. Samples indicating the flourescent spot at or above Rf 0.80 were considered positive for production of polyene antibiotics. To confirm antibiotic production the spots were eluated by using ethyl acelate: methanol and the absorption spectrum of the eluate was obtained using Shimadzu, Double – beam spectrophotometer, UV-190. The spectrum was obtained at a wave length of 250 – 340 nm.
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