Isolation of protease producing microorganism from the rhizosphere of Zingiber officinalis.

2. INTRODUCTION
Bacteria are large prokaryotic microorganisms that are widely spread on most habitats
of earth. The study of bacteria is called bacteriology, a branch of microbiology.
Rhizosphere is a zone of increased microbial activity in the immediate vicinity of
plant root as compared to distant part of soil .It is due to the exudation of plant roots.
Enzymes are proteins that catalyze chemical reaction .the study of enzymatic
activities in environmental samples is a useful tool accessing the functional diversity
of soil microbial community of soil organic mass turnover ,measuring enzyme
activities in soil has a long tradition in connection with evaluating soil fertility and
quantifying process in natural and semi natural ecosystems with a high turn of organic
compounds .
A protease is and enzyme that conduct proteolysis, that is,
protein catabolism by hydrolysis of the peptide bonds that line amino acids together in
the polypeptide chain forming the protein proteases occurs naturally in organisms.
Bacteria also secrete proteases to hydrolyses the peptide bonds in proteins and
therefore break the proteins down into their constituent monomers. Some viruses with
HIV/AIDS among them , dependon proteases in their reproductive cycle. Thus
proteases inhibitors are developed as antiviral means . Application of proteases-
Rennet, Papain ,detergents, Aging , Tanning.
The present study aimed to isolate soil bacteria from the rhizosphere zingiber
officinalis to demonstrate the antimicrobial activity of soil bacteria isolated from
rhizosphere .

3. OBJECTIVES
Objectives of the study includes :
1.Sampling of rhizosphere soil.
2.Isolation of bacteria from the soil sample.
3.Screening of protease producing in microorganism.
4.Determination of the staining characteristics of the selected microorganism.

4. MATERIALS AND
METHODS
Materials required for this project are petriplates , test tubes , autoclave , beaker ,conical flasks ,
balance , soil from medicinal garden , nutrient agar , skimmed milk power ,wire loop , distilled
water , cotton , pipette , glass rod and spirit lamp
1. sampling
soil was taken from the medicinal garden. 1gm of soil was taken using a weight balance.
100ml water was taken in a flask and plugged using cotton. 1gm soil was suspended in
100ml water and shaken well and allowed to settle .
Isolation of bacteria from the soil sample
a. serial dilution.
b. preparation of nutrient Agar.
c. inoculation
.

5. ISOLATION OF BACTERIA
1. 5ml of nutrient agar was poured into 6 test tubes and were
autoclaved. Then they were kept in a slanting position and
allowed to solidify. Bacterial colony from the petriplates
transferred very carefully to the agar slant tubes in zig-zag
manner using previously incinerated wire loop and then
incubated.
2. Isolation of bacteria from the soil.
3. Screening and selection of protease producing bacteria.
4. Determination of staining characteristics of the selected
micro-organism

6. . preparation of nutrient agar
Mediacomposition
•1The chemical composition of the nutrient a agarbroth was weighed accurately and transferred them into
beaker containing 500ml distilled water.
•The contents were gently heated with slightly agitated to dissolve the ingredients
•Distilled water was added to make the volume to 1 litre.
•pH of the broth was measured by using a ph meter and adjusted the ph to-7 by adding drops of either HCL or
NAOH solution.
•10ml broth was dispensed to each culture tubes.
•cotton plugs was prepared and applied into the mouth of broth tubes.
•The mouth of cotton plugs was tightly covered withaluminiumfoil or a paper and tied with a rubber band or
thread.
•All the broth tubes were transferred into a test tube stand or iron basket.
•The plates were placed inside the autoclave/pressure cooker and sterilize at 121degree Celsius for 30 minutes.
•When temperature cools down,the broth tubes were taken.
•The broth tubes are stored at room temperature for further use.

7. NUTRIENT AGAR MEDIUM
BROTH TUBES

8. STREAKED CULTURE PLATE

9. •14g of nutrient agar was prepared in 100ml
water and autoclaved.
•10ml water was taken in a beaker and boiled
it.
•5gm skimmed milk powder was added to 10ml
boiled water as the source of casein and mixed
well.
•The 10 ml solution was added to the agar
solution and mixed well and poured into 6
petriplates are allowed to set.
•Isolated bacteria were inoculated in the
petriplates in zig-zag manner and allowed to
grow.
•The plates were observed for bacteria to
identify the clear zone surrounding to it.
The clear zone indicated the protease producing
abilityof bacteria
.Screening and selection of protease
producing bacteria

10. 4.Determination of the staining
charecteristics of the selected
microorganism.
1. Bacterial smear was prepared on a
glass slide heat fixing was using a spirit
lamp.
2. The smear was covered with crystal
violet and kept for 2-3’.
3. The stain was briefly washed off using
a wash bottle of distilled water.
4. The excess water was cleared off.The
smear was covered with grams iodine solution
and kept for 1-2’.
5. The grams iodine was poured off and
the smear was washed with ethyl alcohol for
10-20 sec.
6. The action of the alcohol was stopped
by rinsingthe slide with wash bottle for a few
sec.
7. Apply safrannin for 20 sec.
8. The smear was washed gently for a few
sec blotted with bibulose paper and dried at
room temperature.

11. 9. The slide was examined under the
microscope.Purplecoloured cells indicate Gram
+ve bacteriawhere as pinkcoloured cells
indicated Gram-ve bacteria.
The gram reaction is determined by the
interaction of crystal violet by the integrity and
the structure of the cellwall (peptidoglycan ).

12. RESULTS & DISCUSSION
1. Sampling
1 g of soil was taken from the medicinal garden aseptically.
2. Isolation of bacteria from the soil sample.
3. Screening of protease producing micro-organisms
4. Staining

13. STAINING RESULTS ARE SHOWN BELOW
No. Name Gram +ve / -ve Shape of bacteria
1 SZ1 Gram+ve Rod
2 SZ2 Gram+ve Coccus
3 SZ3 Gram+ve staphylococci
4 SZ4 Gram+ve Staphylococci
5 SZ5 Gram+ve coccus
6 SZ6 Gram-ve Rod

14. ROD NEGATIVE BACTERIA POSITIVE COCCUS BACTERIA
POSITIVE COCCUS BACTERIA ROD POSITIVE BACTERIA
POSITIVE COCCUS BACTERIA ROD POSITIVE BACTERIA

15. POSITIVE STAPHYLOCOCCUS
BACTERIA

16. CONCLUSION
Soil sample were taken from the rhizosphere of zingiber officinalis.Medium was prepared in petriplate
for the isolation of bacteria.Bacterial growth was observed on the plate.Subculturing was clone to agar
slant in tubes inorder to isolate them.They were named as SZ1-SZ6.By gram staining 2 +ve
coccus,2+ve staphylococcus,1 rod –ve and 1rod+ve were obtained.Out of the six bacteria one were
able to produce protease as they show hallow zone around the colony which grow on casein
containing agar medium.

18. THANK YOU…