Morphological mcg), ceftazidime (30), Ciprofloxacin (5 mcg), Co-Trimoxazole (25

Morphological & Biochemical characterisation

In order to
study colonial morphology, Isolates were grown on solidified LB agar plates. Gram
staining and motility were determined by bright field microscopy.

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For
biochemical characterization the isolates were tested for catalase activity,
oxidase activity, utilization of citrate, carbohydrate fermentation,
Voges-Proskauer test. (James G. Cappuccino 1987). Biochemical
properties of these isolates were tested according to Bergey’s Manual of
systematic Bacteriology. (krieg 1984).

 

Evaluation of Chromium Tolerance:

For the
screening of chromium tolerant bacteria, 1 OD fresh
culture of the isolates were inoculated in 30 ml of LB Broth containing
different concentration of Potassium dichromate (K2CR2O7)
ranging from 100 ppm-1300 ppm. The growth of bacteria was tested by measuring
the Optical Density on spectrophotometer at 620 nm.

 

PCR amplification and sequencing:

For PCR amplification of chromium tolerant bacteria colony PCR
protocol was used. Single colony from the given sample
were scraped and resuspended in 20µl of 1X PBS in a fresh 1.5ml eppendorf tube.
Upon resuspension, the tube was allowed to stand at room temperature for 5 minutes.
1-2µl of this suspension was then added to the PCR cocktail to serve as the
template for amplification. The samples were analysed using Agarose gel
electrophoresis. Phylogenetic tree analysis was done using bioinformatics.

 

 

 

 

Antimicrobial susceptibility Test:

Antimicrobial
susceptibility Test was done by Disc Diffusion Method on Mueller Hinton Agar. (Oyetibo n.d.) Commercially
available antibiotic disk from HiMedia were use. The antibiotics discs were placed on Mueller Hinton agar plates
previously seeded with cell suspension with a turbidity of 0.5 McFarland
standards.
The plates were incubated at 37° C for 24 hrs and observed for zone of
inhibition. Selected bacterial isolate was tested for 20 different antibiotics.
(Owolabi 2014) The 20 different
antibiotics use were, Amikacin (30), Augmentin (30), Ampicillin (10), Aztreonam
(30 mcg), Chlorampenicol (30 mcg), ceftazidime (30), Ciprofloxacin (5
mcg), Co-Trimoxazole (25 mcg), ceftriaxone (30 mcg), Cefuroxime (30 mcg),
Cefuroxime (30 mcg), Fusidic Acid (30 mcg), Gentamicin (10 mcg), Imipenem (10
mcg), Linezolid (30 mcg), Meropenam (10 mcg), Oflaxin (5 mcg), Pperacillin (100
mcg), Rifampicin (5 mcg), Tetracycline (30 mcg), Vancomycin (30 mcg).

The sensitivity and
resistance profile was determined on the basis of diameter of zone of
inhibition and evaluation done according to National Committee for Clinical
laboratory standard’s (NCCLS) chart provided with the antibiotic kits by
Himedia.

 

Result and Discussion

Soil Characteristics:

Soil sampling was done
at 20°47’00” N, 79°39’00” E at Pauni and 20°40’00” N,
79°24’55” E  at Taka. Electronic
conductivity of Pauni sample 0.21 deci/m and 0.31 deci/m of Taka region.
Organic Carbon Content 0.50 % and 1.34%, Total Nitrogen (Kjeldahl Method) 100 kg/ha and 22.50 kg/ha, Available Phosphorous 65.69
kg/ha and 60.02 kg/ha, Available Potassium 40.32 kg/ha and 470.40 kg/ha for
Pauni sample and Taka sample respectively. As per XRF analysis soil samples
shows 34.82 and 0.34 mg/100 g.

Isolation and Characterization of Strains:

14 different
isolates were isolated from soil samples were isolated. Out of  LB 1, LB 2 200, LB 3 200,
LB 5 big, LB
6, LB 8, LB 9,
LB 10, LB 11,
M9+ LB ,
and NA+LB were isolated from Pauni soil Sample
by and LB 3.7, LB 4.2, LB 4.7 were
isolated from Taka region soil sample by serial dilution method and enrichment
culture technique.

 

Study of
Colonial Morphology

Isolated colonies of bacterial strain grown on LB agar plates were
observed and data were recorded as form (Circular, filamentous and
irregular); elevation (flat, convex,); margin (entire, undulate, erose and filamentous),
and optical feature (opaque, translucent, and transparent) of the colonies  (MJJ. Pelczar 1958)

All bacteria
were Motile except LB 9, NA+LB & LB
4.7 were Non-Motile.

 

 

 

Biochemical Characterization.

Biochemical
characterization were performed included Sugar fermentation, indole test,
methyl red and Voges Proskauer test, citrate utilization test, H2S production
test other tests to detect  the presence
of enzymes namely oxidase, catalase.

 

Table. Shows the results of Biochemical
characterization of all 14 isolates. (+ve= Positive, W +ve= Weak Positive, -ve=
Negative)

 

Sr. No

Strain

Dextose

sucrose

Mannitol

Maltose

 

 

Acid

Gas

Acid

Gas

Acid

Gas

Acid

Gas

1

LB 1. 1

+ve

-ve

-ve

-ve

+ve

+ve

+ve

-ve

2

LB 2. 200
 

-ve

-ve

+ve

-ve

W +ve

-ve

+ve

-ve

3

LB 3 200

-ve

-ve

+ve

-ve

-ve

-ve

+ve

-ve

4

LB 3. 7

– ve

– ve

+ ve

– ve

– ve

-ve

+ ve

– ve

5

LB 4. 2

+ ve

– ve

+ ve

– ve

– ve

– ve

– ve

– ve

6

LB 4. 7

+ ve

– ve

+ ve

– ve

– ve

– ve

+ ve

-+ve

7

LB 5  Big 1

+ve

W +ve

+ve

-ve

W +ve

-ve

+ve

-ve

8

LB Type 6

+ ve

+ ve

+ ve

+ ve

– ve

– ve

– ve

– ve

9

LB 8

+ ve

– ve

+ ve

– ve

+ ve

-ve

    – ve

– ve

10

LB 9

+ve

+ve

+ve

+ve

-ve

-ve

+ve

-ve

11

LB 10

+ve

+ve

+ve

+ve

W +ve

-ve

-ve

-ve

12

LB 11

+ve

-ve

+ ve

-ve

+ ve

– ve

– ve

– ve

13

NA+LB 20

W +ve

-ve

+ve

-ve

-ve

-ve

+ve

+ve

14

M9+ LB 14

+ve

-ve

+ve

-ve

-ve

-ve

-ve

-ve