Nội dung

Int.J.Curr.Microbiol.App.Sci (2020) 9(2): 584-591 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 9 Number 2 (2020) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2020.902.073 Screening of Bacteria Present in Cow Dung Ayush Tomar, Shalu Choudhary*, Lalit Kumar, Megha Singh, Nisha Dhillon and Sonam Arya College of Applied Education and Health Sciences, Meerut, India *Corresponding author ABSTRACT Keywords Bacteria, Cow dung, Lactobacillus plantarum Article Info Accepted: 08 January 2020 Available Online: 10 February 2020 Cow dung has a wide range of micro-organisms, like- endospore forming Bacillus, Enterococcus & protozoa. In the present investigation attempts were made to isolate bacteria using nutrient agar media (NAM). The isolated bacteria were identified on the basis of their colony characteristics, morphology, Gram’s staining and biochemical test. It was observed that spore forming bacillus were the predominant type of organism, which possibly helped in maturation of manure forming. number of bacteria such as - Bacillus, like lactobacillus as central endospore forming bacillus some cocci, fungi and yeast such as Saccharomyces (Sharma & Singh, 2015). According to Ware et al., (1988), lower part of the gut of cow has probiotic acitivity due to presence of different types of microorganisms like Lactobacillus plantarum, Lactobacillus casei, Lactobacillus acidophilus, Bacillus subtilis, Entrococcus diactylactis, Bifido bacteirum and yeasts (Saccharomyces cerevisiae). There are several evidences to show that fresh cow dung and cow urine are antifungal and antiseptic in nature, which might be due to secretion of antimicrobial metabolites by cow dung micro-flora (Sharma & Singh, 2015). Introduction Cow dung is excreted by bovine animal species, which are herbivores. It consists of undigested residues of consumed matter which has passed through the cow's gastrointestinal system (Teo & Teoh, 2011). Cow dung is widely used in formation of manure which is used as bio-fertilizers. Manure of cow dung enhances the minerals of soil and also develops resistance power of plants against pests and plant diseases. It contains about 80% of water and has some part of undigested plants material that have high amount of organic substance. The microflora of cow dung contains a wide 584 Int.J.Curr.Microbiol.App.Sci (2020) 9(2): 584-591 Sample-1st (After 2 days) The aim and objective of this study was to isolate bacteria from cow dung, using nutrient agar media (NAM). The isolated bacteria were identified on the basis of their colony characteristics, morphology, Gram’s staining and biochemical test, so that we can identify which microorganism will possibly helped in maturation of manure formation. Cow dung sample was streaked on nutrient agar media with the help of sterilized inoculating loop. Upper surface sample and lower surface sample were streaked on nutrient agar media separately. The plates were incubated at 37⁰ C for 24 hours. After incubation, colony characteristics were studied. The colony was transformed to slants for further characterization. Materials and Methods In this study, cow dung was collected and left it for some days for the isolation of different types of bacteria. Using nutrient agar media (NAM), the bacteria from cow dung were characterized and identified on the basis of Gram’s staining method and biochemical analysis. Sample-2nd (After 7 days) After 7 days, sample was collected from upper surface and lower surface of cow dung separately. Inoculum was made from the upper surface on nutrient agar medium, after that sterilized the loop again over the flame, till it became hot red. The plate was turned and streaked over agar surface. Incubation of all the plates was done at 37⁰ C for 24 hours. Same procedure was repeated for lower surface sample. For fulfilling the requirement of objective, we required following materials and chemicals: Cow dung, Petri plates, test tubes, Nutrient agar media, Inoculating loop, sprit lamp, LAF, autoclave, Hot air oven, Sample container, Cotton, Foil paper, Slides, Distilled water, Cotton swab, Phosphate buffer saline solution (PBS solution), pH strip, Incubator, Microscope, Gram's staining kit and Normal saline solution. Sample-3rd (after 15 days) Sample of cow dung from upper surface and lower surface was collected after 15 days for isolation of bacteria. By applying the both pour plate method & streak plate method. Method Streak plate method Cow dung was Collected and placed at a place from where samples were collected from upper surface and lower surface in sterile containers at a specific intervals- 2nd day, 7th day, 15th day, 21st day and 31st day. In this process streak plate method and pour plate method were used. The sample was taken with sterilized inoculating loop and streaked at the medium of the plates. Then the plates were incubated at 37⁰ C for 24 hours. Pour plate method In pour plate method the sample was picked from upper surface and lower surface separately by the help of sterilized inoculating loop and transferred in Petri plates separately, after that the media (47⁰ C) was poured in the Isolation of bacteria Nutrient agar media was used for the isolation of bacteria from cow dung. 585 Int.J.Curr.Microbiol.App.Sci (2020) 9(2): 584-591 plates. The plates were rotated and left for solidification. All plates were incubated in incubator at 37⁰ C for 24 hours. Streak plate method Sample was taken by sterilized inoculating loop aseptically from upper surface and transferred over the sample by drawing straight line over agar surface. All the plates were incubated at 37⁰ C for 24hours. The same process was repeated for isolation of bacteria from lower surface. Sample-4th (after 21 days) After 21 days sample was collected from both upper and lower surface of cow dung. Both pour plate method and streak plate method were applied for isolation of bacteria from cow dung. Pour plate method The upper surface sample was transferred in the sterilized Petri plates by the help of sterilized inoculating loop, under aseptic condition. The nutrient agar media was poured in Petri-plates and mixed gently and the plate was left for solidification. After solidification all plates were incubated at 37⁰ C for 24 hours. The same process was repeated for lower surface sample. Streak plate method The sample was taken by sterilized inoculating loop and inoculum was made on nutrient agar media and line was drawn over agar surface, (streak plate method) for both upper and lower surface sample separately. All the plates were incubated for 24 hours at 37⁰ C temperature, and then single colony was isolated and transferred this colony in nutrient agar slant. Characterization micro-organisms and Identification of Pour plate method After isolation pure colonies of bacteria were identified and characterized on the basis of morphological analysis such as color, size and shape. These colonies of bacteria were identified by the Gram's staining method. The sample was taken from upper surface by the help of sterilized inoculating loop and transferred on Petri plates separately and the media (nutrient agar media) was poured, sample was mixed gently and left the plates for solidification. Effect of Different pH on the growth of bacteria After solidification, all the plates were incubated at 37⁰ C for 24hours and then single colony was transferred on nutrient agar slant, after that same process was repeated for lower surface sample. The bacterial culture was maintained on nutrient agar media. Isolated bacteria were grown on media with different pH values. Preparation of Different pH media Media of different pH was prepared by addition of different pH buffer solution named according to pH as pH-2, pH-4, pH-6, pH-7 & pH-8. Two plates of each pH buffer was prepared and the pure culture of isolated bacteria was transferred by the help of sterilize inoculating loop aseptically on each nutrient agar media done by streak plate th Sample-5 (after 31 days) After 31 days sample was collected from upper and lower surface. Both method (pour plate & streak plate method) applied for isolation of bacteria. 586 Int.J.Curr.Microbiol.App.Sci (2020) 9(2): 584-591 method. Among the streaked plates, one plate from each pH was incubated at 37⁰ C for 24 hours, and second streaked plate of different pH plates was incubate at room temperature for 24 hours. Enterococcus was dominant on other bacterial growth. These bacteria were characterized on the basis of morphological and biochemical analysis as per Table 2. On the basis of gram's staining, bacteria were differentiated as gram positive and gram negative bacteria shown in Figure 10. Bacillus, Diplococcus, Enterococcus were gram positive bacteria and show purple colour after gram's staining. Pseudomonas show pink colour, rod shape after gram's staining. Isolated bacillus was central endospore forming bacteria. Results and Discussion In this study, cow dung was collected and left it for 31st days. The sample was taken at different time interval of days- 2, 7, 15, 21, 31st days. Sample was taken from upper and lower surface in a sterilized container. Different bacteria were isolated from cow dung on nutrient agar media from upper and lower surface and identified by Gram’s Staining. Effect of different pH level on Bacillus From the isolated bacteria by gram staining the bacillus was found in high amount than other bacteria so we check the effect of different pH level on growth of bacillus. There was no effect of pH on the growth of bacteria, optimal temperature for growth was 37oC. Among the isolated bacteria the Bacillus was present in high amount as compared to other bacteria, such as Enterococcus, Diplococcus, Pseudomonas and thread like bacteria. Table.1 Composition of NAM (Nutrient agar media) S.No 1. 2. 3. 4. 5. 6. Chemicals Peptone NaCl Beef extract Yeast extract Agar Distilled water Amount Required 5.0gm 5.0gm 3.0gm 3.0gm 15.0gm 1000ml Table.2 Growth of bacteria at different surface of cow dung S.No. Sample/Time duration 1. 2. Cow dung sample (after 2nd days) Cow dung sample (after 7days) 3. Cow dung sample (after 15days) 4. 5. Cow dung sample (after 21st days) Cow dung sample (after 31st days) Gram's staining Purple color Purple Colour Purple & Pink colour Purple colour Purple colour 587 Lower surface Bacillus Bacillus Upper surface Bacillus Bacillus Bacillus & pseudomonas Bacillus Thread like bacteria Diplococcus Bacillus Enterococcus Int.J.Curr.Microbiol.App.Sci (2020) 9(2): 584-591 Table.3 Growth of different pH level and temperature Different Temperature Different pH level pH-2 pH-4 pH-6 pH-7 pH-8 + + ++ ++ ++ Room Temperature o ++ ++ ++ ++ ++ Incubator temperature (37 C) * + Sign represents moderant growth; ++ Represents good growth Figure.1 Sample 1st after 2 days Figure.2 Sample 2nd after 7 days Figure.3 Sample 3 rd after 15 days 588 Int.J.Curr.Microbiol.App.Sci (2020) 9(2): 584-591 Figure.4 Sample 3rd after 15 days (Pour Plate Method) Figure.5 Sample 4th after 21 days (Streak plate method) Figure.6 Sample 4th after 21 days (Spread Plate Method) Figure.7 Sample 5th after 31 days (Streak plate method) 589 Int.J.Curr.Microbiol.App.Sci (2020) 9(2): 584-591 Figure.8 Sample 5th after 31 days (Pour Plate Method) Figure.9 Isolated Pure Culture Plates of Different pH by Streak Plate Method (A- at Room temperature; B- at Incubator Temperature) Figure.10 Different types of bacteria were identified such as A: Bacillus; B: Pseudomonas; C: Endospore forming Bacillus; D: Enterococcus; E: Thread like Structure; F: Diplococcus In conclusion, it was found that various species of gram positive bacteria were present in cow dung such as - Bacillus spp., Enterococcus, Diploccocus and gram negative bacteria such as - pseudomonas. Enterococcus was present in dormant condition which affects the growth of other bacteria. According to time, number of Bacillus species was increasing in cow dung and present in motile condition. It is concluded that the micro-flora of cow dung shows the isolated bacteria is endospore forming bacillus. The Enterococcus show dormant nature on nutrient agar media and inhibit the growth of other bacteria. Hence, the Enterococcus cultures work as a bacteriostatic for the growth of other bacteria. 590 Int.J.Curr.Microbiol.App.Sci (2020) 9(2): 584-591 Sharma, B. and Singh, M. (2015). Isolation and charactreization of bacteria from cow dung of desi cow breed on different morpho-biochemical parameters in Dehradun. International journal of Advances in Pharmacy, Biology and Chemistry, Vol:4(2). Singh, A. and Hayashi, K. (1995). Microbial cellulases, protein architechure, molecular properties and biosyhthesis. Advance Applied Microbial, 40:1-4. Teo, K.C. and Teoh, S.M. (2011). 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How to cite this article: Ayush Tomar, Shalu Choudhary, Lalit Kumar, Megha Singh, Nisha Dhillon and Sonam Arya. 2020. Screening of Bacteria Present in Cow Dung. Int.J.Curr.Microbiol.App.Sci. 9(02): 584591. doi: https://doi.org/10.20546/ijcmas.2020.902.073 591