INTERNSHIP PROGRAM

ON
MICROBIOLOGY
TITLE : Screening and isolations of amylase or
protease producing bacteria from soil collected from
different areas

NAME : G SAILAJA
ID NUMBER : 160010022

BIO LIM
CENTRE FOR SCIENCE & TECHNOLOGY
CHENNAI
SCREENING & ISOLATE THE AMYLASE PRODUCING
BACTERIA IN THE SOIL COLLECTED FROM DIFFERENT
AREAS
AIM OF THE EXPERIMENT :-
Screening and isolating amylase producing bacteria from soil sample collected from
different areas. Estimating the amylase or protease bacteria producing in the soil sample
collected form the 4 different places

OBJECTIVE :-
The objective of present study was to screen and isolate amylase producing bacteria
from soil sample collected from different areas .soil sample were serially diluted and 0.1ml of
sample was spread on nutrient agar for 24hrs.skimmed milk to know the protease producing
bacteria is present in collected soil samples & starch agar to know the amylase producing
bacteria is present in collected soil samples at 37 ͦc for 24 hrs and streaking on both plates broth
culture and the protein estimation and then the enzyme plate assay and then enzyme tube assay
of the amylase and doing the both nitrogen and carbon source

NUMBER OF EXPERIMENTS HAS BEEN DONE TO KNOW THE

PROTEASE OR AMYLASE PRODUCING BACTERIA IN THE SOIL
SAMPLE WE TOOK
SERIAL DILUTION
Take the collected soil samples from the different areas and add the soil sample to the
10 ml of distill water . stock solution is prepared and take the 9 ml of distill water in all
10^-1 to 10^-10 and then take the 1 ml of stock solution and add to the 10^-1 and then
again take 1ml of 10^-1 solution and add to the 10^-2 and repeat it for all remaining
samples . when it reaches to 10^-10 the solution will be clear and then take only the
10^-5 , 10^-6 , 10^-7 , 10^-8

NUTRIENT AGAR PREPARATION

Prepare the nutrient agar composition ( by adding peptone , yeast extract and agar ,
Nacl in 100 ml of distill water and autoclave 121c for 15 – 20 mins and by using the
laminar air flow. pour the nutrient agar media in 4 petri-dish plates let it be half open
leave the petri-dish plates up to the nutrient agar solidified after the nutrient agar is
 solidified invert the petri-dish plates and pour the 1 ml of 4 samples (10^-5 , 10^-6 , 10^-
7,10^-8) in all four petri-dish plates and place it in incubator for 24 hrs

RESULT : growth is observed in all 4 samples we have take the 10^-6

1 STREAKING

Prepare the protease media (peptone of 0.3g and skimmed milk of 1g and nacl of 0.3g
and agar of 3g in 100 ml of distill water)and amylase media (peptone of 0.3g and starch
of 1g and nacl of 0.3g and agar of 3g in 100ml of distill water) autoclave it for 121c for
15-20 mins after the autoclave pour the protease and amylase in 2 petri-dish plates by
using laminar air flow allow it to solidified the media and invert it and 10^-6 culture is
used for streak plating on both protease and amylase media and place it in the incubator
for 24 hrs

RESULT : amylase bacteria is seen as a colony’s in streak plate

2 PREPARATION OF BROTH CULTURE

We have take the two production broth culture because we don’t know which bacteria is
producing in the soil sample we took so we prepared the protease and amylase production
broth

PROTEASE PRODUCTION BROTH PREPARATION

0.5 g of peptone and 0.2g of magnesium sulphate and 0.2 g of calcium chloride
dehydrate and 1 g of glucose is dissolved in 100ml of distill water

AMYLASE PRODUCTION BROTH PREPARATION

0.5g of peptone and 0.2g of calcium chloride dehydrate and 1g of glucose and
0.2g of potassium dehydrate and 0.2g of potassium hydrogen phosphate is
dissolved in 100ml of distill water

Both the protease and amylase broth is place it in the autoclave(to make the
solution sterial ) at 121c for 15 -20 mins let it be cool
Take the 10^-6 colony culture bacteria in the both protease and amylase broth and place it
in the incubator for 24hrs. after 24hrs to know the growth in broth . we use the U.V
spectroscopy at 600nm and adjust the water (blank) as 0
RESULT : we have observed OD of amylase =0.078 and protease =0.028 according to
the OD values amylase broth is have more growth when compared to the protease

PROTEIN ESTIMATION BY LOWRIS METHOD :

0.2 ml of BSA working standard in 5 test tubes and make up to 1ml using distilled
water The test tube with 1 ml distilled water serve as blank. Add 4.5 ml of reagent 1(
48ml of 2% Na2CO3 in 0.1 N NaOH and 1ml of 1% Na-K Tartrate in H2O and 1 ml of
0.5% CuSO4.5 H2O in H2O) and incubate for 10 minutes. After incubation add 0.5 ml
of reagent II(Folin-Phenol [2 N]) and incubate for 30 minutes • Measure the absorbance
at 660 nm

RESULT : OD values of amylase =0.212, protease = 0.471 according to the OD values

protease is having protein is more

ENZYME PLATE ASSAY :

Again we have prepare the both protease and amylase enzyme assay

PROTEASE ENZYME ACTIVITY :

Skimmed milk of 1g and 3g of agar is dissolved in 100ml of distill water and it
should maintain the PH of 7

AMYLASE ENZYME ACTIVITY :

1g of starch and 3g of agar in 100ml of distill water and it should maintain the
PH of 7

Place the both P.E.A & A.E.A in the autoclave at 121c for 15 to 20 mins and by using
the laminar air flow and pour the protease and amylase in petri-dish plates and allow it to
be solidified and invert the petri-dish plate and the do the gel punch on both protease and
amylase and then pour the broth culture of amylase in A.E.A &protease broth in P.E.A
Place it in incubator for 24 hrs

RESULT : we have observed amylase producing bacteria in soil and then and the iodine
is add to A.E.A and after 10 mins washed with hot water ( amylase producing bacteria is
present then it turns to blue ) we have noticed A.E.A has turned to blue so that we have
identified that amylase producing bacteria is present in the taken soil sample
ENZYME TUBE ASSAY

Add the 1N NAOH in 1g of starch in 100ml of distill water

And add the 2 ml of amylase broth in 100ml starch solution and leave it for 10 mins
then the sugar molecules is going to form and then to stop the reaction
Adding the 2 ml of TCA to the starch solution then the reaction will be stop
This method is called as DNS method
Take the DNS solution in two different ratio 0.5ml and 1ml and add the 1
ml of distill water to both the 0.5ml and 1ml and also add the 1ml of amylase broth
supernatant to the 0.5 and 1ml of the solution and allow it in incubator for 15 min and
then to stop the reaction place the test tueb in hot water bath for 5 mins then the reaction
is going to be stop cool the test tube and take the OD values at the 540nm

RESULT :
OD values of the amylase 0.5ml is 0.168 and amylase 1ml is 0.257 we have
observed that starch solution increases the range of the amylase is also increases

AMYLASE CARBON SOURCE CULTURE :

Prepare the maltose and fructose solution
MALTOSE SOLUTION
Adding 1g of starch and 1 g of maltose in 100 ml of distill water .maintain the PH
autoclave and add 2 ml of amylase to the maltose solution and incubate for 24 hrs
FRUCTOSE SOLUTION
Adding 1g of starch and 1 of fructose in 100ml of distill water .maintain PH .
autoclave and add 2 ml of amylase to the fructose solution and incubate for 24 hrs

Preparing starch solution add 1N NAOH in 1 g of starch in 100ml of distill water in two
different flask . add the 2ml maltose culture in 1 flask and add 2ml of fructose in another
flask and leave it for 10 mins and to stop the reaction add the TCA to the both maltose
and fructose
Centrifuge the 5ml amylase broth at 5000rpm and collect the supernatant fluid
Take two test tube add starch solution 0.5 ml in one test tube and add 1 ml in another test
tube and add 1ml of distill water I both 0.5 and 1ml test tube and add the 1 ml of
supernatant fluid to both test tubes place it in incubator for 15 mins and place it in hot
water bath and allow it to cool and take the OD values at 540nm
RESULT :
Fructose
0.5ml of starch = 0.024
1ml of starch = 0.005
Maltose
0.5ml of starch = 0.125
1 ml of starch = 0.188

Maltose is having the amylase

AMYLASE NITROGEN SOURCE CULTURE

Prepare the peptone and ammonium sulphate solution

PEPTONE SOLUTION
Adding 1g of maltose and 0.5 g of peptone and 0.3g of nacl in 100 ml of distill water
.maintain the PH autoclave and add 2 ml of amylase to the peptone solution and
incubate for 24 hrs
AMMONIUM SULPHATE SOLUTION
Adding 1g of maltose and 0.3g of nacl and 0.5g ammonium sulphate in 100ml of
distill water .maintain PH . autoclave and add 2 ml of amylase to the ammonium sulphate
solution and incubate for 24 hrs

Preparing starch solution add 1N NAOH in 1 g of starch in 100ml of distill water in two
different flask . add the 2ml peptone culture in 1 flask and add 2ml of ammonium
sulphate in another flask and leave it for 10 mins and to stop the reaction add the TCA to
the both peptone and ammonium sulphate
Centrifuge the 5ml amylase broth at 5000rpm and collect the supernatant fluid
Take two test tube add starch solution 0.5 ml in one test tube and add 1 ml in another test
tube and add 1ml of distill water I both 0.5 and 1ml test tube and add the 1 ml of
supernatant fluid to both test tubes place it in incubator for 15 mins and place it in hot
water bath and allow it to cool and take the OD values at 540nm

RESULT : PEPTONE
0.5 ml of starch = 0.891
1 ml of starch = 1.337
AMMONIUM SULPHATE
0.5 ml of starch = 0.755
1ml of starch = 1.379