Dixit et al.

Physiological and Biochemical Characterization

motility test: Motility was examined following hanging drop method. One small

drop of exponentially grown broth culture of strains was dropped in the center of a

cover glass which was hanging in cavity slide. The cavity was sealed with paraffin

wax and observed for bacterial motility under 100 X objective of compound light

microscope.

generation time: A loopful log phase culture of each isolate was inoculated

separately in flasks containing respective media at 28±2°C and 150 rpm. Bacterial

growth of each isolates was assessed by measuring the optical density at 600

nm after every 6 h in UV-VIS spectrophotometer (Labtronics-model LT 2800,

India). Generation time was calculated by using the formula: generation time =

, where

= time interval taken at any two points in the log phase

of growth and

= difference between the

value of OD at time T ₂ h to

value of OD at time T ₁ h (Dubey

and Maheshwari, 2012).

temperature tolerance: All the isolates were streaked onto the respective medium

and incubated at different temperature regime viz., 5, 15, 25, 35, 45 and 55

for

72h to examine the temperature tolerance ability of the isolates (Jida and Assefa,

2011).

pH tolerance: The ability of the bacterial isolates to grow in alkaline or acid

media was assessed using respective agar medium at different pH viz., 3, 4, 5, 6,

7, 8, 9 and 10 (by using HCL or NaOH) and incubated at 28±2

for the 72h

(Jida and Assefa, 2011).

Salt tolerance: Bacterial isolates were tested for their salt tolerance on respective

medium supplemented with 0.5, 1, 2, 3, 4, 5, 6 and 7 % (w/v) NaCl (Ben Romdhane

et al ., 2006).

catalase activity: Drops of hydrogen peroxide (3%) were added over to 48 h old

bacterial cultures. The plates were observed for the liberation of effervescence of

oxygen around the bacterial colonies (Graham and Parker, 1964). Bacterial isolates

exhibiting oxygen evolution confirmed the presence of catalase activity.

gelatin Hydrolysis: Culture tubes containing sterilized agar medium amended

with 0.4% (w/v) gelatin were separately stabbed by exponentially grown culture

of bacterial isolates and incubated at 28±2°C for the 7 days. The tubes were

observed for liquefaction of gelatin. Culture tubes showing liquefaction of gelatin

confirmed for gelatin hydrolysis (Sadowsky et al ., 1983).

Hydrolysis of urea: Log phase cultures of isolates were inoculated in the tubes

containing broth amended with 2% (w/v) urea and 0.012% phenol red and

98

Int. J. Bioinformatics and Biological Sci.: v.2 n.1&2, p. 95-109. March & June 2014