Isolation and biochemical characterization of thermophilic bacteria from hot spring of Bakreshwar, West Bengal

1. Presented By,
Asit Kumar Saha,
M.Sc. 4th semester,
Department of Biotechnology,
Registration no – 02173 of 2020-2021
ROLL - PG/VUOGP57/BIT –IVS No- 035
Oriental Institute of Science and Technology
(Affiliated to VIDYASAGAR UNIVERSITY)

2. INTRODUCTION
Prokaryotes
Single-celled organisms
Ubiquitous
2
Bacteria are
 Can grow at temperatures
of 55°C or higher.
 Have more stable Cell membrane.
 Have more heat-stable enzymes
and protein synthesis systems.
 Found in composts, hot springs,
deep-sea hydrothermal vents,
geo-thermally heated oil and
volcanic regions.

3. AIMS & OBJECTIVE
3
 To isolate the bacterial strains from the Bakreshwar hot spring.
 To characterize their morphological, biochemical properties.

4. Isolation of Bacteria
Sample Collection
Enrichment Culture
Serial Dilution
Plating
Colony isolation
Culturing in Nutrient
Media
4
Water Sample Enrichment
Broth
Serial dilution
Plating
TSB
media

5. Culture Media
5
Effect of pH and Temperature on Bacterial growth:
The different tubes were adjusted with pH
range 4.5 to 11.0 respectively.
Inoculate each tube with the loopful of
culture.
Incubate the tubes in shaking incubator at 37°
C temperature for 24hrs.
In separated tubes (with optimized pH)
isolated strain was also inoculated.
 Incubated at different temperatures (range
45˚C to 90˚C) for 24hrs.
After incubation observe the growth of
culture in each tube.
Soyabean Casine Digest Medium (SCDM)
Tryptone soya broth
Composition gm / lit
Papaic digest of soyabean meal 3.00
Dipotassium phosphate 2.50
Pancreatic digest of casein 17.00
Sodium chloride 5.00
Glucose 2.50 Fig.- Bacterial growth in TSB

6. Morphological Characteristic
Developed in 1884
by the Danish
Physician Christian
Gram.
6
Cells stain
purple.
Cells remain
purple.
Gram Positive cells
remain purple;
Gram Negative cells
become colorless.
Gram Positive cells
remain purple
Gram Negative cells
appear red.
Gram-stain Procedure
(a) Crystal violet; 30 seconds
(b) Rinse for 5 seconds
(c) Cover with Gram's iodine for 1 minute
(d) Rinse with water for 5 seconds
(e) Decolorize for 15–30 seconds
(f) Rinse with water for 5 seconds
(g) Counterstain with safranin
(h) Rinse for 5 seconds for about 60–80
seconds
It divides bacteria into
two groups-
A. Gram Positive.
B. Gram Negative

7. Biochemical Characterization
7
Indole test-
To detect the production of
indole from the amino acid
tryptophan.
SIM medium is used.
Presence of a red layer indicates
bacteria are indole Positive.

8. Biochemical Characterization
8
MR-VP TEST
MR reaction –
To determine whether the
bacterium carries out mixed acid
fermentation.
Methyl red is a pH indicator.
VP reaction –
•VP (Voges-Proskauer) detects
the production of acetoin.
•Barritt’s reagent detects the
presence of acetoin.

9. Biochemical Characterization
9
Lipid Hydrolysis-
Detects the presence of lipase.
Lipase, lipids into simple fatty acids
and glycerol.
Media- Tributyrin Agar Medium.
Starch Hydrolysis-
Detects the presence of the
enzyme amylase, which
hydrolyzes starch.
Media- Starch Agar medium.
Gram’s Iodine used as
Indicators.
Starch + iodine Starch-iodine complex
(colorless) (Blue/Deep Blue)
Triglyceride Glycerol + Fatty acids

10. Biochemical Characterization
10
Gelatin Hydrolysis-
Detects whether or not a bacterium can
produce gelatinase that hydrolyze gelatin
and liquify solid gelatin medium.
Media- Nutrient broth with 12% gelatin
Liquefies gelatin shows positive result.
Catalase Activity-
Detects the presence of catalase, which
converts hydrogen peroxide to water and O2.
Media- tryptonc soya agar slant .
After adding the substrate H2O2, bubbles of
O2 represent a positive catalase test.
Casein Hydrolysis-
Detects the presence of caseinase, an
enzyme able to hydrolyze milk protein
casein.
Media- SM agar media.
Bacteria that use casein appear as colonies
surrounded by a clear zone.

11. Biochemical Characterization
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Urease Activity Test-
Detects the enzyme that splits urea to
NH3 and CO2.
Media- Urea Agar Base slant.
Development of pink colour indicates
positive test.
Citrate Utilization Test-
When citrate is used as the sole carbon
source, this results in alkalinization of the
medium.
Medium- Simmon's Citrate Agar Slant.
Indicator- Bromothymol blue.
Development of blue from green,
indicates positive test.

12. RESULT
Effect of pH and Temperature on Bacterial growth
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Isolated
Bacterial
Strain
Under
Microscope
Rod in shape
Purple in color
Inoculated Media at Different pH
4.
0
5.
0
5.
5
6.0 6.5 7.0 7.5 8.0 9.0 10.
0
11.0
Cont
rol
- - - - - - - - - - -
Sam
ple
- - - - + + ++ +++ + + -
Incubated Media at Different Temperature
45°C 50°C 55°C 60°C 70°C 80°C 90°C
Control - - - - - - -
Sample + +++ +++ ++ + + -

13. Cot…
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No. Biochemical tests Result
1 Indole Test - Positive
2 MR Test - Positive
3 VP Test - Negative
4 Starch Hydrolysis- Positive
5 Lipid Hydrolysis- Negative
6 Casein Hydrolysis- Negative
7 Gelatin Hydrolysis- Negative
8 Catalase Activity- Positive
9 Urease Activity Test- Positive
10 Citrate Utilization Test- Negative

14. CONCLUSION
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 Thermophilic microorganisms are of special interest as a source of novel
thermostable enzymes.
 Thermozymes have faster reaction rate, increased solubility of substrates,
decreased viscosity and elimination of microbial contamination than
mesophiles.
 Microbial thermozymes have been extensively used in waste management,
biofuel, food, paper, detergent, medicinal and pharmaceutical industries.

15. FUTURE STUDY
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Further the study may get a shape with the following works;
 Screening and transmission electron microscopy to understand both the
surface and intercellular morphology of the isolate
 Identification of bacterial strain by 16S rRNA gene sequencing
 Quantitative analysis and characterization of enzymes from the isolates

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THANK YOU