Basic HTML Version
Molecular Microbiology Research (Online) 2013, Vol.3 No.2 9-20
ISSN 1027-5595
http://mmr.sophiapublisher.com
16
plates spreaded with 10
-2
, 10
-3
and 10
-4
dilutions were
exposed to UV for 5, 10, 15 and 20 minutes. The work
was carried out under dim light (yellow or red). The
plates were incubated in dark (to prevent photo
reactivation) at 37 for 24 hours and to bacterial load
℃
in respective plates were enumerated.
3.5 NTG treatment
The overnight culture of
S. typhimurium
MTCC 98
broth was subcultured again in LB broth (1:50 ratio)
and incubated at 31 for 2
℃
-2 1/2 hours with agitation
to get a midlog phase growth. From this, 1.5 mL was
taken in eppendorf tubos and spun at 10 000 pm for 10
minutes. The pellet was washed twice with sterile
saline(1 mL). The pellet resuspended in saline (1 mL)
in three eppendorf tubes. Of the three eppendorf tubes
the first tube was taken and the 1 mL sample was
serially diluted to 10
-6
, 10
-7
and 10
-8
with sterile saline
and spreaded on air dried in plates (TVC). From
dilutions 10
-1
, 10
-2
, 10
-3
and 10
-4
, 0.1 was spread
plated on LB with selective antibiotic plates to
determine TSM. To the second and third tube, NTG
(50 μg/mL) was added and incubated at 37 for
℃
15~20 minutes. The tubes were spun at 10 000 rpm
and to pellet was washed thrice with saline.
The pellet was resuspended in 10 mL LB broth and
incubated at 37 for an overnight. Then it was
℃
serially diluted upto 10
-6
with sterile and from
dilutions 10
-4
, 10
-5
and 10
-6
, 0.1 mL was taken and
spread plated on air dried LB plates incorporated with
streptomycin (TIM).
3.6 Maintenance of
Salmonella typhimurium
MTCC 1251 and treated strains of MTCC 98
Auxotrophic strain maintenance is very important.
Being a mutant strain it could get easily reverted at
any condition and at any time. Hence maintenance
medium have the ability to maintain the character of
an auxotrophy. Here, ampicillin (8 mg/mL in 0.02 N
NaOH) / Tetracycline (8 g/mL in 0.02 N NaCl) and
histidine (2 g/400 mL) was added to the medium. The
lyophilized MTCC Cultrure 1251 and the treated
strains of MTCC 98 were inoculated in maintenance
medium and incubated at 37 for 24~48 hrs and after
℃
growth, it was stored in refrigerator.
3.7 Reverse mutation assay
9.9 mL, 9 mL, 7.5 mL and 95 mL of LB broths were
prepared in required test tubes/conical flask and 0.1
mL of the test cultures were inoculated into the broths
separately. To this, the selected samples (raw oils,
fried oil and reheated oil (0.1 mL, 1 mL, 2.5 mL and 5
mL) and other foods (as 1 gm, 5 gms and 10 gms)
were added in quantitative manner and incubated for
24~48 hrs at 37 with agitation. After incubation, 1
℃
mL of the test sample mixed culture and 0.1 mL of
histidine were added to the molten top agar and
vortexed for a homogenous mixture.
The top agar was then poured on E-minimal glucose
agar. The plates were incubated at 37 for 24 hrs.
℃
The growth was observed in the plates in which small
pin pointed colonies were not to taken into account
while large colonies were considered for enumeration
(Cappuccino and Sherman, 2002). The number of
chemically induced mutations were determinedby
substracting the number of colonies on the negative
control plate (representative of spontaneous mutations)
from the number of colonies on each test plate. The
relative mutagenicity of the test compounds were
determined on the basis of the number of induced
mutants. If below 10, (-); if greater than 10, (1+); if
greater than 100, (2+) and if greater than 500, (3+).
3.8 Ames test and spot test method
A widely used method of Ames test (1975) was
performed by spot test method.
3.9 Bacterial genetics of mutant strains
S. typhimurium
auxotrophic mutant strain (UV and
NTG) were taken up for the study of bacterial genetics
on amino acid markers. The procedure followed was:
From an overnight grown culture, one loopful was
taken and inoculated into three sets of thirteen tubes
containing 10 mL minimal broth and appropriate
amino acids were given as selected concentrations.
The tubes were incubated at 37 for 1 hr, 2
℃
hrs and 3
hrs. After one hour of incubation, the first set of tubes
was taken and a single set of tubes were taken and a
single streak was made on the minimal plates. The