CHAPTER III SCREENING, ISOLATION AND DETERMINATION OF ANTIMICROBIAL SPECTRA OF ACTINOMYCETES (a) Collection of Samples: A total of nine samples were collected in sterile containers for the systematic screening of actinomycetes. The samples included soil samples and fresh water reservoir sediments, in and around the Tirumala Hills. stored at 4 o C. About 50 g of each sample at a depth of 2 inches was collected and The source, place and description of each sample was presented in the following Table 9. Table 9: Source, place and description of samples Sample No Source Place Characters A Vishnu gundam Tirupati Reddish brown muddy soil B Ramakrishna Theertham Tirumala Hills Dry, blackish soil C Dheva Theertham Tirumala Hills Blackish brown soil D Thumbara Theertham Tirumala Hills Dark blackish soil E Kangumadugu Tirumala Hills Dry, stony reddish soil F Rudragala Theertham Tirumala Hills Dry, stony blackish soil G Markendeya Theertham Tirumala Hills Dry, stony brown soil H Kailasa Theertham Tirumala Hills Dry, greyish soil I Gunjan Jalapatham Tirumala Hills Muddy water sample 62
(b) Screening: Actinomycetes are isolated from soils and other natural substrates by plating out such materials in proper dilutions on suitable agar media. Actinomycete colonies can easily be distinguished on the plate from those of fungi and true bacteria. They are compact, often leathery giving a conical appearance, and have a dry surface. In the literature several media have been suggested for the isolation of actinomycetes such as Czapek s agar (Lawrence, 1956), egg albumin agar (Lawrence, 1961), glucose-asparagine agar (Lawrence, 1961), Starch casein agar medium (Kuster and Williams, 1964), and various selective media supplemented with or without antibiotics like potassium tellurite agar medium (Willoughby, 1971), humicacid-vitamin agar medium (Lacey and Goodfellow, 1975), Benedict s modification of Lindenbein s medium (Osman and Abou-zeid, 1968),Gauze agarized medium No. 1(Kuznetsova et al., 1988), Czapek Dox yeast extract casamino acid (CYC) agar medium (Jackson and Ball 1994), Nutrient Agar medium (Jackson and Ball 1994) and raffinose histamine agar (Williams et al., 1984). For the isolation of actinomycetes several methods have been suggested in the literature, such as the soil-dilution-plate technique. Several differential media for suppressing the growth of fungi and bacteria have been described. The simplest and the best method employed in the present work was the crowdedplate technique and the best antibiotic producer has been isolated in this way. 63
(c) Isolation of Actinomycetes The following media were used for the isolation of actinomycetes 1. Starch casein agar medium 2. Potassium tellurite agar medium 3. Half strength nutrient agar medium 4. Oatmeal agar medium 5. Yeast extract-malt extract agar medium 6. Inorganic salt starch agar medium 7. Glycerol asparagine agar medium Fifty ml quantities of media in 250 ml flasks were sterilized at 121 o C for 20 minutes by autoclaving. (d) Screening of soil samples for isolation of Actinomycetes: About 4 g of each of the above soil samples was suspended in 25 ml of sterile water in 100 ml conical flask and stirred for half an hour on a rotary shaker. The suspension was serially diluted, 1 ml of each of these dilutions was added to 50 ml of each of the above sterile molten agar medium maintained at 40-45 o C, thoroughly mixed and poured into sterile Petri plates (6 dia). Nystatin (25µg/ml) and rifampicin (5µg/ml) were added to the media to suppress the growth of fungi and bacteria respectively. Antifungal and antibacterial antibiotics were sterilized by filtration and added aseptically to the sterile medium before plating. All the plates were incubated at 28 o C for 7 to 14 days. After 7 days, the selected actinomycetes colonies were isolated from different plates and transferred to starch-casein agar slants. The slants were incubated at 28 o C for 7 to 10 days. 64
A total of five actinomycetes were isolated from the above samples. The isolates were pooled together and cultures which appeared identical to the naked eye were eliminated using the following criteria: Colour of the aerial mycelium, reverse colour and soluble pigment. The antimicrobial spectrum of these five isolates was determined by subjecting the organisms to submerged fermentation and assaying the broth for antimicrobial activity by cup plate assay. The following Table 10 shows the distribution of actinomycetes in various samples. Table 10: Distribution of actinomycetes in various samples Sample Number Place of collection Place Total number of actinomycetes C 1 Vishnu gundam Tirumala Hills Nil C 2 Ramkrishna Theertham Tirumala Hills 1 C 3 Dheva Theertham Tirumala Hills Nil C 4 Thumbara Theertham Tirumala Hills 1 C 5 Kangu madugu Tirumala Hills Nil C 6 Rudragala Theertham Tirumala Hills Nil C 7 Markendeya Theertham Tirumala Hills Nil C 8 Kailasa Theertham Tirumala Hills Nil C 9 Gunjana Jalapatham Tirumala Hills 3 65
(e) Evaluation of antimicrobial activity for broth: The five actinomycete isolates were screened for extra cellular antibacterial activity by submerged fermentation. The medium employed for this study was: Glucose 1.5% Soybean meal 1.5% NaCl 0.25% CaCO 3 0.1% ph 7.2 Procedure: Well sporulated 7 to 10 days isolates were taken and 5ml of sterile water was added to each slant and spore suspension was added to a 250ml shake flask containing 50ml of the inoculum medium and incubated at 28 o C on a rotary shaker (210-220 rpm) for 48 h. After 48 h 5ml of the inoculum medium was transferred to a 250 ml shake flask containing 45ml of the production medium. The flasks were incubated at 28 o C for 6 days on a rotary shaker. After 6 days, 10ml of the production medium was collected into sterile centrifuge tubes and centrifuged at 4000 rpm for 15 minutes to separate the fermented broth and the mycelium. The clear supernatant was used for the antibacterial assay by cupplate method (Grove and Randall, 1955). Nutrient agar (Himedia), potato dextrose agar (Himedia) were used as media. All the test organisms employed in the present study were supplied by the National collection of Industrial Microorganisms, National Chemical Laboratory, Pune, India. 66
The following test organisms were used for the determination of antibacterial activity. Bacillus subtilis (NCIM 2063) Bacillus megaterium (NCIM 2054) Staphylococcus aureus (NCIM 2079) Pseudomonas fluorescens (NCIM 2639) Klebsiella pneumoniae (NCIM 5082) Escherichia coli (NCIM 2685) Proteus vulgaris (NCIM 2027) Cup-Plate method for antibacterial studies (Grove and Randal, 1955) The molten sterile nutrient agar assay medium was cooled to 40-45 o C, inoculated with the test organisms, mixed thoroughly, poured into sterile Petri plates (4 dia) and allowed to settle. Cups were made using sterile borer; 50µl of clear broth supernatant was added to each cup. The plates were kept in a refrigerator for about 2 h to allow the diffusion of the bioactive metabolite. After 2 h, plates were incubated at 37 o C in an incubator. The inhibition zones were measured after 24 h using an antibiotic zone reader. Only one isolate from sample C 9 exhibited significant inhibitory activity against Gram positive and Gram negative organisms. The remaining isolates exhibited poor or no antimicrobial activity. The selected isolate was preserved by using cryopreservation technique. Hence this isolate designated as C 9 was selected for further studies. 67
Table 11: Antimicrobial spectrum of promising isolate C 9 by submerged fermentation in selected medium Test Organism Diameter of zone of Inhibition in mm Bacillus megaterium 11.0 13.0 15.0 17.0 Bacillus subtilis 30.0 32.0 33.0 35.0 Staphylococcus aureus 21.0 25.0 26.0 28.0 Klebsiella pneumoniae 32.0 33.0 34.0 35.0 Pseudomonas 24.0 24.0 25.0 28.0 fluorescens Proteus vulgaris 31.0 34.0 35.0 37.0 Escherichia coli 10.0 10.0 11.0 12.0 68
Figure 1: Antimicrobial spectra of promising isolate C 9 by submerged fermentation in selected medium 69
Figure 2: Antimicrobial spectrum of promising isolate C 9 by submerged fermentation in selected medium (f) Selection of suitable medium for antibiotic production: Selection of optimum medium for enhancing antibiotic production of isolate C 9 was carried by submerged fermentation in six different types of media. 70
Table 12: Composition of the various production media Composition of production medium No. 1 (PM 1 ) Composition of production Medium No. 2 (PM 2 ) Soybean meal 2.5 % Corn steep liquor 1.0 % Glucose 2.5 % Soluble starch 2.5% Na NO 3 0.4 % Diammonium sulphate (NH 4 ) 2 SO 4 0.5 % K 2 HPO 4 0.5 % Ca CO 3 0.5 % NaCl 0.25 % Distilled water 100 ml ZnSO 4 0.004% ph 7.2 Ca CO 3 0.04 % Distilled water 100 ml ph 7.0 Composition of production Medium No. III (PM 3 ) Composition of production Medium No. IV (PM 4 ) Glycerol 2.0 % Glucose 1.0 % Peptone 0.5 % Starch 1.0 % Yeast extract 0.3 % Peptone 0.75 % Malt extract 0.3 % Meat Extract 0.75 % Ca CO 3 0.25 % NaCl 0.3 % Distilled water 100 ml Distilled water 100 ml ph 6.8 ph 6.5 Composition of production medium No. V (PM 5 ) Composition of production medium No. VI (PM 6 ) Soybean meal 1.0 % Soybean meal 1.5 % Corn steep liquor 0.5 % Glucose 1.5 % Soluble starch 1.0 % Glycerol 0.25 % Dextrose 0.5 % NaCl 0.5 % CaCO 3 0.7 % CaCO 3 0.1 % Distilled water 100 ml Distilled water 100 ml ph 7.0 ph 7.0 71
Procedure: Well sporulated isolates (7 to 10 days old) were used for antibiotic production studies. Five ml of sterile water was transferred aseptically into each slant and the growth of the isolate on the surface of the medium was scrapped with sterile inoculating needle and transferred into 45 ml of production medium and incubated at 28 o C on a rotary shaker for 7 days. Then samples were collected into sterile centrifuge tubes and centrifuged at 2000 rpm for 15 minutes. The clear supernatant broth was used for antibiotic assay using cup-plate method (Grove and Randall, 1955). The antibacterial activity was tested on nutrient agar medium and antifungal activity was tested on potato dextrose agar medium. The molten sterile medium was cooled to 40-45 o C, inoculated with test organism, mixed thoroughly, poured into sterile Petri plates and allowed to settle. Four cups were made using sterile cork borer per plate. The clear supernatant fermentation broth was added to each cup. The following test organisms were used Bacillus subtilis, Bacillus megaterium, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas fluorescens, Escherichia coli, Proteus vulgaris and Aspergillus niger. The plates were kept in the refrigerator for about 2 h for antibiotic diffusion, and then plates were incubated at 37 o C for bacteria and 28 o C for fungi and yeast. The inhibition zones were measured after 24 h for bacteria. In case of fungi and yeast, inhibition zones were recorded after 48 h. 72
Antimicrobial spectrum of promising isolate C 9 by submerged cultivation in six types of media (PM 1, PM 2, PM 3, PM 4, PM 5 and PM 6 ) against seven test organisms Table 13(a): Antimicribial activity in several production media Production media Antimicrobial activity (Diameter of zone of Inhibition in mm) Test organism: Staphylococcus aureus 20 µl 30 µl 40 µl 50 µl PM 1 23.0 24.0 24.0 2.4 PM 2 23.0 26.0 27.0 28.0 PM 3 23.0 23.0 25.0 27.0 PM 5 25.0 27.0 27.0 27.0 Table 13(b): Antimicribial activity in several production media Production media Antimicrobial activity (Diameter of zone of Inhibition in mm) Test organism: Bacillus megaterium 20 µl 30 µl 40 µl 50 µl PM 1 20.0 21.0 21.0 23.0 PM 2 23.0 27.0 27.0 27.0 PM 3 22.0 23.0 23.0 24.0 PM 5 21.0 22.0 22.0 23.0 73
Table 13(c): Antimicribial activity in several production media Production media Antimicrobial activity (Diameter of zone of Inhibition in mm) Test organism: Bacillus subtilis 20 µl 30 µl 40 µl 50 µl PM 1 15.0 17.0 18.0 19.0 PM 2 24.0 26.0 28.0 29.0 PM 3 21.0 21.0 22.0 25.0 PM 5 20.0 20.0 20.0 24.0 Table 13(d): Antimicribial activity in several production media Production media Antimicrobial activity (Diameter of zone of Inhibition in mm) Test organism: Proteus vulgaris 20 µl 30 µl 40 µl 50 µl PM 1 24.0 27.0 27.0 28.0 PM 2 25.0 26.0 28.0 28.0 PM 3 23.0 23.0 25.0 26.0 PM 5 20.0 22.0 25.0 27.0 Table 13(e): Antimicribial activity in several production media Production media Antimicrobial activity (Diameter of zone of Inhibition in mm) Test organism: Klebsiella pneumoniae 20 µl 30 µl 40 µl 50 µl PM 1 20.6 20.0 21.0 23.0 PM 2 26.0 26.0 24.0 24.0 PM 3 23.0 23.0 24.0 25.0 PM 5 22.0 22.0 24.0 24.0 74
Table 13(f): Antimicribial activity in several production media Production media Antimicrobial activity (Diameter of zone of Inhibition in mm) Test organism: Pseudomonas fluorescens 20 µl 30 µl 40 µl 50 µl PM 1 21.0 22.0 24.0 25.0 PM 2 26.0 31.0 33.0 33.0 PM 3 22.0 25.0 25.0 25.0 PM 5 22.0 25.0 25.0 26.0 Table 13(g): Antimicribial activity in several production media Production media Antimicrobial activity (Diameter of zone of Inhibition in mm) Test organism: Escherichia coli 20 µl 30 µl 40 µl 50 µl PM 1 20.0 20.0 20.0 22.0 PM 2 21.0 21.0 24.0 26.0 PM 3 20.0 20.0 21.0 23.0 PM 5 22.0 22.0 22.0 23.0 Studies on antibiotic production by the selected isolate C 9 in six different production media. (PM 1 to PM 6 ) indicated that antibiotic activity was maximum against the seven test organisms in production media PM 2 which was inferred from the size of the inhibition zones observed whereas no antibiotic activity was observed in production media PM 4 and PM 6. Hence it was decided to determine the optimum concentration of the constituents present in the medium PM 2. The major ingredients present in the medium PM 2 are soybean meal, glucose, NaNO 3 and K 2 HPO 4. The optimum concentration of these ingredients was determined by carrying out antibiotic fermentations with varying concentrations of soybean meal, glucose, NaNO 3 and K 2 H PO 4. 75