Basic techniques in Isolation of Soil Bacteria

CHARACTERIZATION OF ISOLATED BENEFICIAL SOIL MICROORGANISMS

MOHAMMED TAYYABBAGF10M036

SOIL BIOLOGY & BIOCHEMISTRY SECTIONLAND RESOURCE RESEARCH INSTITUTE

NATIONAL AGRICULTURE RESEARCH CENTERISLAMABAD

3RD MARCH TO 26TH OF MAY, 2014

STRUCTURE OF PRESENTATION

• BRIEF INTRODUCTION TO LRRI,NARC ISLAMABAD• INTRODUCTION TO SOIL BIOLOGY AND BIOCHEMISTRY SECTION• WHAT IS NEED OF CHARACTERIZATION OF SOIL MICROORGANISM• WORK DONE AT SOIL BIOLOGY AND BIOCHEMISTRY SECTION• SAMPLING METHODS/TECHNIQUES• SERIAL DILUTION• ISOLATION• BIOCHEMICAL EXAMINATION OF NEWLY ISOLATED MICROBES• SUMMARY

INTRODUCTION TO LRRI,NARC ISLAMABAD

LAND RESOURCE RESEARCH INSTITUTE• ESTABLISHED IN 1982 • FOCUSES ON PRODUCING MORE FOOD AND FIBER• USING FEWER INPUTS WHILE PROTECTING THE ENVIRONMENT

SECTIONS

Soil Fertility and Plant Nutrition

Soil Salinity Soil Biology and Biochemistry

Soil Physics and Mineralogy Soil

Environment

SOIL BIOLOGY & BIOCHEMISTRY SECTION

• AREAS OF RESEARCH UNDERTAKEN, ARE :• BIOLOGICAL NITROGEN FIXATION (BNF)• PLANT GROWTH PROMOTION BY PGPR (PLANT GROWTH PROMOTING

RHIZOBACTERIA)• PHOSPHORUS SOLUBILIZATION BY PSM (PHOSPHORUS SOLUBILIZING

MICROBES)• INOCULANTS PRODUCTION AND SUPPLY.• COMPOSTING TECHNOLOGIES.

WORK DONE AT SOIL BIOLOGY & BIOCHEMISTRY

WHY NEED OF BIOFERTILIZER???

• GREEN REVOLUTION BROUGHT IMPRESSIVE GAINS IN FOOD PRODUCTION BUT WITH INSUFFICIENT CONCERN FOR SUSTAINABILITY(NILABJA GHOSH, 1991)

• THIRD WORLD COUNTRIES, NOT SELF-RELYING FOR FERTILIZER PRODUCTION THAT ARE PURELY FOSSIL FUEL BASED

• CHEMICAL FERTILIZERS: A POTENTIAL SOURCE IN REDUCING SOIL QUALITY, WATER CONTAMINATION AND UNSUSTAINABLE BURDEN ON ECONOMY

• WHILE THE ECO-FRIENDLY BIO FERTILIZERS ARE QUITE OF PRIME IMPORTANCE‘OBJECTIVE’

CHARACTERIZE ISOLATED STRAINS OF BENEFICIAL MICROBES FOR THEIR POSSIBLE USE AS BIOFERTILZER

MATERIAL & METHODS

1-SAMPLING

• THE PLANT IS NATURAL HABITAT FORMICROBIAL LIFE: RHIZOSPHERE ENDOSPHERE PHYLLOSPHERE

SAMPLE PREPARATION

• 1G ADHERING SOIL WAS SUSPENDED IN 10ML STERILE DISTILLED WATER• DILUTION WAS MADE UP TO 10-6

FURTHER STEPS !!

• LURIA-BERTANIA (LB) MEDIUM WAS PREPARED (MANIATIS ET AL.1982) • AUTOCLAVING AT 121OC AND 17 PSI PRESSURE FOR 40 MIN• POURING IN PRE-AUTOCLAVED PLATES • WAIT FOR SOLIDIFICATION(SAMASEGARAN ET. AL, 1982)

SPREADING

• 0.1ML FROM DESIRED SERIAL DILUTIONS 6, 5 AND 4 TAKEN• DROPPED WITH TIP • SPREAD ON THE MEDIA PLATE WITH THE HELP OF SPREADER AND MARKED

THEM ACCORDINGLY

BIOCHEMICAL EXAMINTAION OF ISOLATED STRAINS

• VARIOUS BIOCHEMICAL TESTS WERE CONDUCTED:• CATALASE TEST• AMYLASE PRODUCTION TEST• PECTINASE TEST• PROTEASE TEST• PHOSPHATE SOLUBILIZING TEST• INDOLE TEST

CATALASE TEST

• FACILITATES THE DETECTION OF THE ENZYME CATALASE IN BACTERIA• CATALASE ENZYME SERVES TO NEUTRALIZE THE BACTERICIDAL EFFECTS OF

HYDROGEN PEROXIDE(MACFADDIN 2000)• SINGLE COLONY OF EACH ISOLATE WAS PICKED AND PUT ON A GLASS SLIDE• FORMATION OF BUBBLES WITHIN 10 SECONDS WAS OBSERVED

AMYLASE PRODUCTION TEST

• PURPOSE IS TO SEE IF THE MICROBE CAN USE STARCH AS A SOURCE OF CARBON

• THESE ISOLATES WERE STREAKED ON FRESHLY PREPARED PLATES• INCUBATED AT 35°C• CLEAR ZONES WERE CHECKED AFTER 24 HRS. OF INCUBATION (ASHWINI ET

AL., 2011).

PECTINASE PRODUCTION TEST

• ISOLATES WERE STREAKED ON SPECIFIC MEDIUM• INCUBATION AT 35°C• ZONES WERE CHECKED AFTER 24 HRS

PHOSPHATE SOLUBILIZING TEST

• PREPARATION OF PIKOVASKAYA’S MEDIUM(PIKOVASKAYA, 1948)• AUTOCLAVED FOR 40 MINUTES AT 1210C TEMPERATURE & 15PSI PRESSURE• POURING WAS DONE• LEFT FOF SOLIDIFICATION• INOCULATION OF BACTERIAL STRAINS• KEPT THE PLATES FRO 48 HOURS • LOOKING FOR CLEAR ZONES

PRESERVATION OF STRAINS

RESULTS1-CATALASE PRODUCTION TEST

Catalase Test

Sr. No. Strain Name Results

1 KTRS 2 Positive

2 KTRS 3 Positive

3 KTRS 4 Positive

4 KTRP 2 Negative

5 KTRP 3 Positive

6 KTRP 4 Positive

7 KTES 2 Positive

8 KTES 3 Negative

9 KTES 4 Positive

AMYLASE PRODUCTION TESTSr.No. Strain name Results

1 KTRS 3 -ve

2 KTRS 4 +ve

3 KTRP 2 +ve

4 KTRP 3 +ve

5 KTES 3 +ve

6 KTES 4 +ve

PECTINASE PRODUCTION TEST Pectinase Test

Sr. No. Strain Name Results

1 KTRS 2 +ve

2 KTRS 3 +ve

3 KTRS 4 +ve

4 KTRP 2 +ve

5 KTRP 3 +ve

6 KTRP 4 +ve

7 KTES 2 +ve

8 KTES 3 +ve

9 KTES 4 +ve

PHOSPHORUS SOLUBILIZATION TEST

Phosphorus Solubilization Test

Strain No. Strain Name Results

1 KTRS 2 +ve

2 KTRS 3 +ve

3 KTRS 4 +ve

4 KTRP 2 -ve

5 KTRP 3 +ve

6 KTRP 4 +ve

7 KTES 2 +ve

8 KTES 3 -ve

9 KTES 4 -ve

CELL MORPHOLOGY OF BACTERIAL STRAINS

Cell Morphology of PGPR bacterial strains

Sr. No. Strain no. Shape Group Colour Gram Reaction

1 KTRS 2 Coccus Streptococcus Purple Positive

2 KTRS 3 Bacillus Bacillus Purple Positive

3 KTRS 4 Spirochete Bacillus Purple Positive

4 KTRP 2 Coccus staphylococcus Purple Positive

5 KTRP 3 Coccus Sarcina Purple Positive

6 KTRP 4 Coccus Micrococcus Pink Negative

7 KTES 2 Coccus Streptococcus Purple Positive

8 KTES 3 Coccus Micrococcus Purple Positive

9 KTES 4 Coccus Micrococcus Purple Positive

SUMMARY

• 7 WERE TENTATIVELY IDENTIFIED AS COCCUS 1 AS BACILLUS. AND 1 ISOLATES AS SPIROCHETE

• . IN CASE OF P.SOLUBILIZATION 6 STRAIN PRODUCED CLEAR ZONE THIS IS THE INDICATION OF POSITIVE RESULT AND 3 ISOLATE SHOWED NEGATIVE RESULT.

• MOST OF THE STRAINS SHOWED POSITIVE RESULTS FOR CATALASE PRODUCTION• AMONG ALL STRAINS SHOWED CLEAR ZONE AND HAVE POSITIVE PECTINASE

PRODUCTION ABILITY

QUESTIONS??