International Research Journal of Engineering and Technology (IRJET)
e-ISSN: 2395 -0056
Volume: 04 Issue: 04 | Apr -2017
p-ISSN: 2395-0072
www.irjet.net
Identification of Protease Producing Bacteria from Different Samples V.P.Mohan Babu, R.Kanakadurga, R.Hema Kalai Rani Dept. of Biotechnology, Adhiyamaan College of Engineering, Tamilnadu, India ---------------------------------------------------------------------***---------------------------------------------------------------------
Abstract - This study is about the isolation and
were made up to
identification of protease producing bacteria Pseudomonas fluorescens from three different samples soil, water and milk by performing antibiotic sensitivity assay and the bacterial activity zone was measured using the antibiotic sensitivity scale for serially diluted microbes(bacteria). The number of colonies formed for soil, milk and tap water were noted. Similarly the inhibition zone near the streptomycin added well was measured.
serially diluted up to a concentration of
Nutrient agar were prepared and autoclaved at 121 . 1 drop of clotrimazole (antifungal agent) was added to the medium in lukewarm condition. Then the medium was poured into six petriplates under aseptic conditions. After solidification of agar, concentrations of and of the samples were taken from all the three samples and poured onto the petriplates. The petriplates were incubated for 24 hrs at a temperature of 37 . After incubation, bacterial colonies were counted and were expressed in CFU/ml.
1.INTRODUCTION Protease is a digestive enzyme responsible for proteolysis by hydrolysis of peptide bonds in proteins. Nowa-days due to its vast applications, it is essential to produce protease by microbial techniques. Serial dilution is a technique that is used for counting the number of bacteria that is present in the given sample based on geometric progression of decreasing concentration. The process of counting the number of bacteria present in the sample after serial dilution is called colony counting and it is measured by CFU/ml (Colony Forming Unit). The inhibition zone produced by bacteria is measured by antimicrobial activity by agar well diffusion method. The formation of clear zone by hydrolysis was observed by enzymatic activity.
Table -1: Number of colonies present in the samples Colonies of bacteria
concentration
concentration
2. Materials And Methods 2.1 Sample Collection
Tap water
Milk
54 X
22 X
69 X
CFU/ml
CFU/ml
CFU/ml
6 X
34 X
65 X
CFU/ml
CFU/ml
CFU/ml
The three agar plates were spread with equal volumes of the three samples of concentration . 4 wells were created in each petriplate using cork borer (or) tip aseptically. The wells were named as E, S, A, B. In the wells A and B, 50 μl and 100 μl of bacterial culture were added. Well E was used as blank. In the well S, 20 μl of Streptomycin was added which acted as the Standard. After incubation at 37 for 24 hours, the zones of inhibitions were measured.
2.2. Serial Dilution 1 g soil was taken in a test tube with 10 ml of distilled water and concentration was noted as . Then the test tube was shaken well. 1 ml of solution from the above test tube was pipetted out to a new test tube. Then distilled water was added to make up the solution to 10 ml and concentration was noted as . Similarly concentrations
Impact Factor value: 5.181
Soil
2.4. Antimicrobial Activity
The Soil sample was collected from the road side. Tap water was collected from the Jayagen Biologics Lab, Chennai. Milk sample was collected from a shop at Chennai.
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2.3. Colony Counting Method
Key Words: Protease, Pseudomonas fluorescens, soil, water, milk, antibiotic sensitivity.
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. Similarly, all the three samples were
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