MATERIALS AND METHODS. All the chemicals used in this study were of analytical grade. The L-Asparaginase activity was determined by detection of

Transcription

1 57 CHAPTER II

2 58 Chemicals MATERIALS AND METHODS All the chemicals used in this study were of analytical grade. Determination of L-Asparaginase activity The L-Asparaginase activity was determined by detection of ammonia or L-aspartic acid. The assay procedure is based on direct Nesslerization of ammonia. Ammonia is estimated by detecting the optical density at 425nm. The activity was determined in 0.05 M-borate buffer (ph 8.5) at 37ºC in the presence of 10mM-substrate (asparagines). Two methods were used: the first one is test tube method using direct Nesslerization. The optical density was measured at 400nm. The other method is an automated technicon method. Both took advantage of the observation that the incidence of turbidity which is often encountered in the Nessler s reaction is lower in the presence of EDTA and when alkali is added immediately prior to Nesslerization. The 0.05M borate buffer (ph 8.5) was used for bacteria 116. By following assay method proposed by Wade et al. 1974, we faced several difficulties. Hence the method was modified to the extent of existing facilities and situations available in our laboratory. Construction of standard graph: Standard graph was prepared by treating 1ml of 0.25, 0.5, 0.75 and 1mM ammonium sulphate with trichloroacetic acid, NaOH and Nessler s reagent.

3 59 Stock solution of 10 mm: It was prepared by dissolving 132 mg of ammonium sulphate in 100 ml distilled water in a volumetric flask. Working solution of 1mM: It was prepared by taking 5 ml of stock solution and made upto 50 ml with distilled water. From this 0.25 and 0.5 mm were prepared. Procedure: Into a series of test tubes, 2 ml of (0.05M) borate buffer was taken. To this 1 ml of each 1 mm, 0.75 mm, 0.5 mm and 0.25 mm of working solutions were added. From this 1 ml sample was withdrawn and delivered into 2.5 ml of 0.1N trichloroacetic acid. To this 1 ml of 1N NaOH was added. 0.2 ml of EDTA (0.1M) was added to each sample to overcome the encountered turbidity. After 2 min., 0.5 ml of Nessler reagent was added. The OD was measured after 5 min. at 425 nm.blank was prepared by adding 1 ml of water instead of ammonium sulphate solutions. The data is shown in Table 3.1. A standard curve was constructed by taking ammonium sulphate (µm/ml) on X-axis and corresponding optical density on Y-axis and the data is shown in Fig Unit: One unit (IU) is defined as the amount of enzyme that released 1 mole of ammonia from L-asparagine per minute at ph 8.5 at 37ºC.

4 60 ESTIMATION OF TOTAL PROTEIN Estimation of extracellular protein: Extracellular protein was estimated by Lowry method (Lowry et al., 1951).The protein reacts with the Folin Ciocalteau reagent to give a coloured complex. The colour formed is due to the reaction of alkaline copper with protein as in the Biuret reaction and the reduction of the phosphomolybdate by tyrosine and tryptophan present in the protein. The intensity of the colour depends on the amount of these aromatic amino acids and thus varies with different proteins. Preparation of reagents Solution A (alkaline sodium carbonate): 2% Na2CO3 in 0.1 N NaOH. Solution B (copper sulphate solution): 1% CuSO4. 5H2O in distilled water. Solution C (Rochelle s salt): 2% sodium potassium tartarate. Alkaline solution (working solution): 1 ml CuSO4. 5H2O solution + 1ml Rochelle s salt + 98 ml alkaline Na2CO3 solution Folin and Ciocalteau s reagent: The commercial reagent (LOBA) was used after diluting with equal volume of water on the day of use. Standard protein solution: Five mg of Bovine serum albumin (BSA) was dissolved in 50 ml of distilled water to get a final concentration of 0.1 mg/ml (100 g/ml). Procedure: To 1 ml properly diluted protein sample (10-60 g/ml), 5 ml alkaline solution (working solution) was added, mixed well and incubated at 37ºC for 10 min. To the above mixture 0.5 ml Folin Ciocalteau reagent

5 61 was added, mixed well and incubated at 37ºC for 30 min. The absorbency of the colour was measured at 280 nm in a spectrophotometer. The results are presented in Table 3.2 and Fig The amount of protein present in the sample was calculated from the standard curve. Experimental SCREENING AND ISOLATION OF L-ASPARAGINASE PRODUCING BACTERIA Chemicals All chemicals and medium constituents used in this study were of analytical grade. Screening programme The following samples were collected at various places of Andhra Pradesh with a view to isolate potent L-Asparaginase producing microorganisms. Sample I: This soil sample was collected from the farms of Agricultural College, Bapatla (Guntur District), Andhra Pradesh. It is a clay loam black cotton soil with neutral to slightly alkaline in reaction. It is rich in lime and potash. Sample II: The soil sample was collected from the sea coast of Machilipatnam (Krishna District). It is a black clay loamy or sandy and saline soil containing excess of natural soluble salts dominated by chlorides and sulphates, which affects plant growth. Sample III: The soil sample was collected from Hindupur, Anantapur District. It is red in colour which is due to various oxides of iron, with a ph of 7.2. It is light textured with porous structure. Sample IV: The sample was collected from Alamuru, East Godavari District and is black silty clay soil with a ph of 5.5. It contained more humus. Sample V: This soil sample was collected from the farm of Acharya N.G.Ranga Agricultural University, Rajendra nagar, Hyderabad. It was red sandy soil.

6 62 Sample VI: The soil sample was collected from Araku Valley which was black clay. It was sandy loam to clay loam with light grey to dark colour, structure is loose and more fertile..sample VII: The soil sample was collected from the dumping ground of University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, where college canteen waste was dumped, which was rich in organic matter and loamy in nature. Sample VIII: This sample was collected from the Visakha Port area, (fishing harbor), Visakhapatnam, Andhra Pradesh, where debris of fishes and others are dumped, which was black in colour and rich in organic matter. Sample IX: This sample was collected from Visakha Dairy, Visakhapatnam, Andhra Pradesh, where unsealed and leaked milk pouches were dumped, which was gray in colour and rich in proteinaceous material. All the samples were collected in sterile screw capped tubes and care was taken to the points of collection had as widely varying characteristics as possible with regard to the organic matter, particle size, colour of soil and geographical distribution. Isolation of L-Asparaginase producing microbial strains from soil samples About 1 gm of each of the above samples was taken into separate conical flasks each containing 100 ml of sterile water. The suspension was kept on rotary shaker for 30 min and kept aside to settle the suspending matter. One ml of the supernatant was serially diluted with sterile water. One ml each, of these dilutions were added to 20 ml of sterile modified M9 medium (as described below) maintained at 45ºC, mixed thoroughly and plated in 10 cm dia. sterile petridishes and incubated at 37ºC. Antifungal agents (Fluconazole-75 μg/ml,

7 63 Ketoconazole-75 µg/ml) were incorporated to control the fungal contamination. After 24 h of incubation, the selected bacterial colonies with pink zones around them were picked up and transferred onto M-9 medium slants (Gulati et al., 1991). The composition of M-9 medium is (g/l): Na2HPO4.2H20, 6; KH2PO4, 3; NaCl 0.5; L-asparagine, 5; 1M MgSO4.7H20, 2 ml; 0.1M CaCl2 2H20, 1 ml; 20% Glucose stock solution 10 ml, Agar, 20 and ph 7.0 in distilled water to 1 L with phenol red (2.5%): ml indicator. A total of 148 colonies were selected and isolated from all the samples based on L-asparaginase enzyme producing microbial strains were identified by their pink colour zones around the colonies (Fig 3.3). The selected isolates were transferred onto nutrient agar slants and incubated for 24 h. The number of isolates from each sample is given in Table3.3. Out of 148 isolates, 46 were selected based on their macroscopic characters, eliminating those that appeared close to each other. SECONDARY SCREENING: Detection of L-asparaginase positive cultures: In the present investigation, a novel and semi-quantitative plate assay for screening L-asparaginase producing microorganisms is reported. It is generally observed that L-asparaginase production is accompanied by an increase in ph of the culture filtrates (De Jong, 1972). The plate assay was devised using this principle by incorporating the ph indicator phenol red in medium containing asparagines (as sole

8 64 nitrogen source). Phenol red at acidic ph is yellow and at alkaline ph turns pink, thus a pink zone is formed around microbial colonies producing L-asparaginase. M-9 medium was supplemented with different concentrations of the dye. A 2.5% stock of the dye was prepared in ethanol and the ph was adjusted to 7.0 using 1 mol/l NaOH. The stock solution of the dye, ranging from 0.04 ml to 0.36 ml, was added to 1000 ml of M-9 medium giving a final dye concentration of % respectively. Different concentrations of the dye were used to standardize the optimum concentration of the dye. The media were autoclaved and plates were prepared. Control plates contained M-9 medium without dye and without asparagines (instead containing NaNO3 as nitrogen source). The plates were inoculated with a 24 h culture of Serratia macerans and Erwinia Carotovora as test organisms and incubated. The colony and zone diameters were measured after 18, 24 and 48 h incubation. After standardizing the dye concentration, the isolates were tested by the plate assay on appropriate medium at 37 C. Zone diameters were measured after 18h. Submerged fermentation studies were also carried out in order to compare the results obtained with those of plate assay. Quantitative estimations of enzyme activities were carried out using M-9 medium. Erlenmeyer flasks (250 ml) containing 50 ml of the M-9 medium were inoculated with each of the test organisms. The flasks were incubated at 37ºC at 250 rpm for 48 h, in a controlled environment shaker-incubator. Uninoculated medium served as control. The flask

9 65 contents were centrifuged at 4000 rpm for 15 min. The enzyme activities were estimated in culture filtrates by Nesslerization (Imada et al., 1973) and expressed as IU/ml. One international unit of L-asparaginase activity is defined as that amount of enzyme which catalyses the formation of 1µm of ammonia per min under the conditions of the assay. Results were statistically evaluated by calculating the correlation coefficient, r (Lewis, 1984), between the zone size (cm) and enzyme activities (IU/ml) in culture broths. After secondary screening, better enzyme producing isolates (5 numbers) were selected and they are designated as MS-3, MS-6, MS-8, MS-11 and MS-15. Among them, the strain MS-6 showed the maximum enzyme production, hence further studies were focused on this strain. To perform further investigation, the selected MS-6 strain was grown on M-9 medium and incubated at 37ºC for 18 h and stored until use at 4ºC in refrigerator. Shake flask fermentation Procedure for production and assay of L-asparaginase The selective promising isolate MS-6 were subcultured onto M-9 medium and incubated at 37ºC for 24 h. Here, the selective wild strain compared with test organisms Serratia marceans and Erwinia carotovora. The growth contents of each slant was suspended in 5 ml of sterile water and transferred into 250 ml EM flask containing 50 ml M-9 medium.

10 66 The selected isolates were subcultured on M-9 solid medium before they inoculated to M-9 broth for production. All the production flasks were incubated on rotary shaker for 24 h. At the end of incubation period, 10 ml of the cell suspension was taken and centrifuged. Quantitative detection was carried out by a Nesslerization method (Imada et al., 1973). A 0.5 ml sample of cell suspension, 1.0 ml of 0.1M sodium borate buffer (ph 8.5) and 0.5 ml of 0.04M L-asparagine solution were mixed and incubated at 37ºC for 10 min. The reaction was then stopped by the addition of 0.5 ml of 0.1N trichloroacetic acid. The precipitated protein was removed by centrifugation and the liberated ammonia was determined by direct nesslerization. Each sample was individually mixed with 1ml of 1N NaOH and 0.2 ml of 0.1M EDTA was added. After 2 min. 0.5 ml of nessler s reagent was added and mixed. Suitable blanks of substrate and enzyme containing sample were included in all assays. After 5 min. from the addition of Nessler s reagent to the sample, the yellow colour was read on 117 UV-Visible spectrophotometer (systronics) and the optical density of the sample was read at 450nm. The results are presented in Table 3.4. IDENTIFICATION OF THE ISOLATE MS-6 For the identification of the promising isolate MS-6, the preliminary morphological, physiological, biochemical tests, utilization of carbon and nitrogen sources were done in our laboratory. The isolate was also sent to MTCC for its identification. The MTCC has done all the relevant tests for its identification and the isolate was identified as a Bacillus cereus strain

11 67 and it was deposited in the MTCC with accession No The detailed data supplied by the MTCC are presented in Tables 3.5 to Taxonomic Studies Media used for characterization: Identification and characterization of the selected isolate Proper identification and characterization of microorganisms is very important because it expands the scope for exploitation of industrially important products. To establish the novelty or otherwise of the present isolate with those reported in the literature, the various morphological, cultural and biochemical characteristics of the isolated organism compared with the descriptions of the numerous bacterial species cited in the literature. The literature survey includes: Bergey s Manual of Determinative Bacteriology (Buchanan and Gibbons, 1974), Bergey s Manual of Systematic Bacteriology (Williams et al., 1989). The promising isolate MS-6 was subjected to taxonomic studies. The following taxonomic properties were investigated. Identification of the isolate MS-6 The isolate exhibited good growth on all media. The cultural characteristics, physiological and biochemical properties, carbon source utilization pattern, utilization of nitrogen sources, resistance or sensitivity to various antibiotics etc were conducted and the data are shown in Tables. Different microbial identification tests were performed as per Bergeys manual for selected microbial species.the following biochemical

12 68 reactions were determined employing the prescribed media: gelatin hydrolysis, coagulation and peptization of milk, casein hydrolysis, starch hydrolysis, nitrate reduction, carbon source utilization, sodium chloride tolerance, effect of inhibitory compounds on growth, effect of various nitrogen sources on growth, resistance to various antibiotics, growth temperature range, chemical tolerance and cell wall composition. Physiological Characteristics Gram staining To study the Gram s reaction of the culture, a 24 h culture was used. The experimental protocol as described by Salle (1948) was followed. Gelatin hydrolysis (Gordon and Mihm, 1957) This represents the ability of microorganisms to hydrolyze or liquefy gelatin. Most of the bacterial species bring about liquefaction of gelatin but the rapidity of liquefaction varies with the species. The nonpigmented forms are most active while the pigmented forms are least active. For this test, the isolates were grown on gelatin agar plates for 24 h at 37 C. At the end of incubation period, the plates were flooded with 1 ml of the following solution. Mercuric chloride Conc. HCl Distilled water : 15 g : 20 ml : 100 ml

13 69 The extent of hydrolysis was noted by comparing the width of the clear zone around the growth. The width of the hydrolyzed zone and growth zone were measured and the ratio of hydrolyzed zone and growth zone was calculated. Casein hydrolysis (Salle, 1948) The enzyme caseinase is excreted out of the cells (an exo-enzyme) into the surrounding media, catalyzing the breaking down of milk protein called casein, into peptides and amino acids. This reaction causes the milk agar, normally the opacity of real milk to clear around the growth surrounded by a clear zone. This test is used to differentiate aerobic organisms based on casein utilization. The proteolytic activity was studied with milk-casein agar medium by measuring the hydrolyzed zones after incubating the inoculated plates at 37 C for 24 h. The extracellular protease (caseinase) activity of the isolates was determined qualitatively as the ratio of the diameter of the hydrolyzed zone to that of the growth on the milk-casein agar medium. Indole and Hydrogen production The medium used for Indole and hydrogen sulphide production tests is, SIM (Sulfide, Indole, Motility) medium. This medium contains a sulfur source, an iron salt, the amino acid tryptophan, and is semi-solid (agar content, 0.3%). It can be used to detect hydrogen sulfide production, indole production and motility. The SIM medium tube was stabbed with the isolate and incubated at 37ºC for 24 h. After incubation period Kovac s reagent to each tube to detect indole production.

14 70 Catalase activity: This test detects the presence of catalase, which converts hydrogen peroxide to water. H2O > H2O + O2 This test is useful in differentiating the given strain is Streptococcus (-) from that of the Streptococcus (+) and Bacillus (+) from Clostridium. H2O2 when present in the organisms is harmful, in the process of evolution organism have developed a unique mechanism to break the hydrogen peroxide into water and oxygen thus reducing its toxicity activity. Add a few drops of 3% hydrogen peroxide to each culture and look for the release of oxygen as a result of hydrogen peroxide breakdown. This appears as foaming. Some bacteria and macrophages can reduce diatomic oxygen to hydrogen peroxide or super oxide. Both of these molecules are toxic to bacteria. These resistant bacteria use two enzymes to catalyze the conversion of hydrogen peroxide and super oxide back into diatomic oxygen and water. One of these enzymes is catalase and its presence can be detected by simple test that peroxide test which produces oxygen gas, if it is catalase positive. The evolution of gas causes bubbles to form and is indicative of a positive test. Starch hydrolysis (Salle, 1948) Diastatic activity (Lyons and Pridham, 1962) This test detects the presence of the enzyme amylase, which hydrolyses starch, used to identify typical starch hydrolyser by the action of amylolytic enzymes. For this test, starch agar plates were inoculated with a loop-full of organism at the center and after 24 h of incubation,

15 71 the plates were flooded with 2.0 ml of Lugol s Iodine solution (3 g of potassium iodide and 2 g of iodine dissolved in 300 ml of water). Hydrolysis of starch was noted by the formation of clear zone around the growth. The width of the hydrolyzed zone around the growth versus the width of the growth was measured and the ratio was recorded. Nitrate reduction test (Salle, 1948) The reduction of nitrate to nitrite has been universally used among the criteria for species differentiation. The reduction is the result of the use of NaNO3 or KNO3 as an electron acceptor with some organisms. Nitrate (NO3) and nitrite (NO2)) serve as sources of nitrogen for the synthesis of organic nitrogen compounds or they may function as H + acceptors in reactions concerned with the organisms energy metabolism. The first step in the reduction of NO3 involves its conversion to NO2 by an enzyme system, which is adaptive in nature and is known as nitratase. 5ml of nitrate broth medium was inoculated with a loop-full of organism and incubated at 37 C for 24 h. Controls were also run without inoculation. After 24 h, the clear broth was tested for the presence of nitrite in the following way. Reagents: a) -Naphthylamine test solution: -Naphthylamine 5.0 g Conc.H2SO4 Distilled water 8.0 ml 1 L

16 72 To the diluted sulphuric acid, -naphthylamine was added and stirred until solution was affected. b) Sulphanilic acid test solution: Sulphanilic acid Conc. H2SO4 8.0 g 48 ml Distilled water to 1 L Sulphuric acid was added to 500 ml of water. Then sulphanilic acid was added followed by distilled water to make up the volume. Procedure: To 1 ml of the broth under examination and 1 ml of control, two drops of sulphanilic acid solution followed by two drops of - naphthylamine solution were added. The presence of nitrite was indicated by appearance of a pink, red or orange colour and absence of colour change was considered as nitrite negative. In the later case presence or absence of nitrate in the broth under examination was confirmed by adding a pinch of zinc dust, after the addition of the reagents, when the unreduced nitrate, if present, gave a pink, red or orange colour. Growth temperature (Williams et al., 1989) Ability of the isolates to grow at different temperatures was studied at 4, 10, 15, 25, 30, 37, 42, 50, and 60 C. The selected isolates were inoculated on Nutrient agar medium and Minimal agar medium slants

17 73 and incubated at the different temperatures as mentioned above. Results were recorded after 24 h. Chemical tolerance (Williams et al., 1989) 10 ml quantities of glycerol-nutrient broth with ph levels of 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0 and 13.0 were inoculated and incubated for 2 to 4 days. Then the tubes were examined for the extent of growth of the organism. Sodium chloride tolerance (Pridham et al., 1956) The ability of certain types of bacterial isolates to tolerate and to adapt to high concentrations of sodium chloride is well known. This adaptability is particularly marked in organisms found in marine water and salt lake mud. For the determination of sodium chloride tolerance, Bennett s agar medium was supplemented with graded amounts of sodium chloride (1, 4, 7, 10 and 13%). The above medium was inoculated with suspension of the organism. After incubation for 24 h, the maximum salt concentration supporting growth was recorded. Growth in the presence of different nitrogen sources (Williams et al., 1989) The ability of isolates to use different nitrogen sources was studied by the following method. Each nitrogen source was incorporated into the basal medium at 0.1% level. The prepared slants were inoculated and incubated at 37 C. Results were recorded after 24 h. The growth on each source was compared with that on the un-supplemented basal medium

18 74 and on a positive control containing L-asparagine. The following nitrogen sources were used in the present study: L-asparagine (positive control), L-arginine, L-cysteine HCl, L-histidine, L-valine, phenyl alanine, threonine, hydroxy proline, methionine, serine and potassium nitrate. Resistance to antibiotics (Williams et al., 1989) The ability of the isolates to resist various antibiotics was one of the criteria for identification of new isolates. This was carried out on Bennet s agar medium. The antibiotic solutions were sterilized by filtration and added to the molten Bennet s agar medium, mixed thoroughly and poured into sterile petriplates. The isolates were streaked on the agar surface and incubated at 37 C for 24 h and observed for the presence or absence of growth. The resistance was scored as (+). The following six different antibiotics at a chosen concentration ( g/ml) were tested: Streptomycin (100), Tetracycline (50), Cephalexin (100), Gentamicin (100), Rifampicin (50) and Penicillin (10 IU). The diluents (The Indian Pharmacopoeia, 1985 and 1996) used for the preparation of standard antibiotic solutions are given in Appendix II. Discussion: The most significant characteristics of our isolate MS-6 are summarized below. The isolate grew well on most of the media. The isolate was nonchromogenic and it did not produce any other soluble pigment.

19 75 Gram staining was done to know the morphology of the strain under the microscope. The strain retained violet colour after gram staining indicating the selected isolate is gram-positive. The external morphological features indicated that it may be Bacillus species. Further, different biochemical tests were performed to confirm the nature of the bacteria. Starch hydrolysis: this test detects the presence of the enzyme amylase, which hydrolyses starch, used to identify typical starch hydrolyser. Our isolate MS-6 gave positive results for starch hydrolysis. Casein hydrolysis: The enzyme caseinase is excreted out of the cells (an exoenzyme) into the surrounding medium, catalyzing the breakdown of casein, into peptides and amino acids. Bacteria that produce caseinase, hydrolyzes casein with growth surrounded by a clear zone. This test is used to differentiate aerobic bacteria based on casein utilization. The organism gave positive result for casein hydrolysis. Gelatin hydrolysis: Gelatin is a protein that is solid at room temperature. For this test, the isolates were grown on gelatin agar plates for 37 o C about 18 h. At the end of incubation period, the plates were flooded with 1 ml of the following solution. Mercuric chloride Conc. HCl Distilled water : 15 g : 20 ml : 100 ml

20 76 The extent of hydrolysis was noted by comparing the width of the clear zone around the growth. The widths of the hydrolyzed zone and growth zone were measured and the ratio of hydrolyzed zone and growth zone was calculated. The organism gave positive result for gelatin hydrolysis. Indole test: The ability to degrade amino acids to identifiable end products is often used to differentiate bacteria. The indole test detects the production of indole from the amino acid tryptophan. Indole is a component of the amino acid tryptophan. Some bacteria have the ability to break down tryptophan for nutritional needs using the enzyme tryptophanase. When tryptophan is broken down, the presence of indole can be detected through the use of Kovacs reagent. Kovac s reagent, which is yellow, reacts with indole and produces a red colour on the surface of the test tube. This test is used to differentiate Escherichia from Enterobacter, also used to characterize members of genus Bacillus. The organism tested MS-6 gave the positive result for indole test. H2S production test: Some bacteria are capable of breaking down sulfur containing amino acids (cystine, methionine) or reducing inorganic sulfur containing compounds (such as sulfite, sulfate or thiosulfate) to produce hydrogen sulfide (H2S). This reduced sulfur may then be incorporated into other cellular amino acids, or perhaps into co-enzymes. The ability of an organism to reduce sulfur-containing compounds to hydrogen sulfide can be another test for identifying unknown organisms such as certain Proteus and Salmonella. Our isolate MS-6 gave negative results for hydrogen sulphide production.

21 77 Catalase activity: This test detects the presence of catalase, which converts hydrogen peroxide to water. Some bacteria and macrophages can reduce diatomic oxygen to hydrogen peroxide or super oxide. Both of these molecules are toxic to bacteria. These resistant bacteria use two enzymes to catalyze the conversion of hydrogen peroxide and super oxide back into diatomic oxygen and water. One of these enzymes is catalase and its presence can be detected by simple test that peroxide test which produces oxygen gas, if it is catalase positive. The evolution of gas causes bubbles to form and is indicative of a positive test. Our isolate MS-6 gave positive result for catalase tests. Methyl red and Voges-Proskauer test: Methy red is a ph indicator dye to determine weather the bacterium has the ability to produce acid. Voges-Proskauer test detects the presence of acetoin citrate. Methyl red is a dye which turns red at below ph 4.4. Methyl red test determines the use of glucose, with the subsequent production of acid which is tested by the ph indicator methyl red. The Voges-Proskauer test also determines glucose use, but for a different end product i.e., not acid but a neutral product called acetoin (or acetylmethylcarbinol). The isolated MS-6 was found to be negative for both tests Methyl red and Voges-Proskauer. The overall data of the isolate MS-6 are as follows: The isolate was H2S production is positive. It exhibited slight tyrosinase activity. It could hydrolyze starch casein and gelatin. It could not reduce nitrite. It exhibited good growth at 25 C. It could tolerate the ph levels between 5.0 and 12. It could not grow above 1% NaCl level.

22 78 It exhibited moderate growth on valine, methionine, hydroxy proline, threonine & cysteine-hcl. It showed sensitivity to penicillin G, ampicilin, tetracycline, chloramphenicol, gentamicin, rifampicin Amoxicillin and Cloxacillin, all the concentrations which are described in Table A detailed screening of the literature indicated that our isolate is closely related to B. cereus. Hence, the cultural properties of our isolate was compared with reported properties of B. cereus. In view of the general agreement and more similarities and a few differences, our isolate MS-6 can be considered to be as a new strain of cereus.

23 79 Table 2.1: Construction of standard graph for Ammonium Sulphate (NH4)2SO4 conc. (µm/ml) Optical Density (nm)

24 O.D. at 425nm Conc. of ammonia in µm/ml Fig.2.1: Standard graph for Ammonia

25 81 Table 2.2: Construction of standard graph for protein estimation Con. of BSA OD (at 680 nm) ( g/ml) Blank

26 O>D. at 680 nm Conc. of BSA (u/g/ml) Fig.2.2: Standard graph for protein estimation

27 83 Table 2.3: L-asparaginase producing strains` from various samples Sample No. No. of cells per gm. of the sample No. of selected isolates I II III IV V VI VII VIII IX

28 84 Table 2.4: Comparision of MS-06 with Erwinia carotovora and Serratia macerans Organism MS Erwinia carotovora 1.46 Serratia macerans 1.22 Activity(IU/ml)

29 85 Table 2.5: Morphological Characters of the isolate MS-6 Tests Colony Morphology Observation Configuration Circular or irregular Margin Entire Elevation Flat Surface Dry and Granular Pigment No pigmentation Opacity Opaque Gram s reaction Gram positive Cell shape Rods Size (µm) Length-4µ, Width-1µ Arrangement Short chains or pairs Spore(s) Endospore Endospores present Position Central Shape Oval Motility Positive

30 86 Table2.6: Physiological Tests of the isolate MS-6 Tests Growth at temperatures Result 4ºC - 10ºC - 15ºC + 25ºC + 30ºC + 37ºC + 42ºC + 50ºC - 60ºC - Growth at ph ph ph5.0 + ph ph ph ph ph ph Growth on NaCl (%) Growth under anaerobic condition +

31 87 Table 2.7: Biochemical Tests of the isolate MS-6 Tests Result Growth on MacConkey agar - Indole test + Methyl red test - Voges Proskuer test + Citrate utilization - Gas production from glucose - Casein hydrolysis + Esculin hydrolysis + Gelatin hydrolysis + Starch hydrolysis + Urea hydrolysis - Nitrate reduction + Nitrite reduction - H 2S production - Catalase test + Oxidase test + Arginine dihydrolase + Ornithine decarboxylase - Lysine decarboxylase - Tween 20 hydrolysis + Tween 40 hydrolysis + ONPG test - Lysozyme resistance test + Phosphatase test + OF test - Haemolysis on Blood agar Beta-Haemolysis

32 88 Table 2.8: Acid production from carbohydrates by the isolate MS-6 Tests Result Salicin + Arabinose - Cellobiose + Dextrose + Mannitol - Lactose - Maltose - Sucrose + Xylose -

33 89 Table 2.9: Growth of isolate MS-6 in the presence of various nitrogen sources Nitrogen source (0.1% w/v) Growth response L-asparagine (positive control) +++ Methionine, Hydroxy proline, ++ Valine, Threonine & Cysteine HCl Phenylalanine, Serine, Arginine, - Histidine, Potassium nitrate - : No growth; ++ : Moderate growth;

34 90 Table 2.10: Resistance of isolate MS-6 to various antibiotics Antibiotic ( g/ml) Growth response Result Penicillin G (10 IU) - Sensitive Ampicillin (100) - Sensitive Tetracycline (50) - Sensitive Chloramphenicol (100) - Sensitive Gentamicin (100) - Sensitive Rifampicin (50) - Sensitive Amoxicillin (50) - Sensitive Cloxacillin (50) - Sensitive

35 91

36 92 Fig.2.3: Agar Plate Showing Microbial colonies and inhibition zone

37 93 Fig.2.4: Detection for isolated microbial strain