Exercise 5 - ENDOPHYTIC MICROORGANISMS FROM LEAVES AND ROOT NODULES

roots

Endophytic bacteria are most abundant in — followed by stems and leaves.

vertically and horizontally

endophytic bacteria can be transferred by:

vertical transmission

via asexual reproduction, pollen, and seed

horizontal transmission

via airborne spores and soil

natural openings or wounds

endophytic bacteria enter the tissue via the —

Nutrient Agar (NA) Plates (supplemented with nystatin or any antifungal), Potato Dextrose Agar (PDA) Plates, Nutrient Agar (NA) Plates without antimicrobial supplements, Modified Yeast Extract Mannitol Agar (YEMA)

what agars were used in the exercise

Isolation and Characterization of Different Leaf Endophytes

Characterization of Root Nodule-associated bacteria

what are the two parts in the exercise

9 petri plates

A. Isolation and Characterization of Different Leaf Endophytes

how many petri plates should be prepared per leaf source?

30 mL

A. Isolation and Characterization of Different Leaf Endophytes

how many mL of the solutions should be filled in the petri plates?

Running sterile distilled water, 70% ethanol, 6% NaClO, 70% ethanol, sterile distilled water

A. Isolation and Characterization of Different Leaf Endophytes

what are the different solutions used in disinfecting the leaf samples

Until dust and dirt are dislodged on the leaf surface

A. Isolation and Characterization of Different Leaf Endophytes

what is the SOAKING/RINSING TIME of the Running Sterile Distilled Water (1st plate)?

3 minutes

A. Isolation and Characterization of Different Leaf Endophytes

what is the SOAKING/RINSING TIME of the 70% ethanol (2nd plate)?

6 minutes

A. Isolation and Characterization of Different Leaf Endophytes

what is the SOAKING/RINSING TIME of the 6% NaClO (3rd plate)?

30 seconds

A. Isolation and Characterization of Different Leaf Endophytes

what is the SOAKING/RINSING TIME of the 70% ethanol (4th plate)?

Enough to rinse off the disinfectants

A. Isolation and Characterization of Different Leaf Endophytes

what is the SOAKING/RINSING TIME of the Sterile Distilled Water (5th-9th plate)?

100 µl

A. Isolation and Characterization of Different Leaf Endophytes

how many microliters of the rinsate from the last plate should be blotted onto the duplicated nutrient agar w.o antimicrobial supplement?

duplicate nutrient agar (w/o antimicrobial supplement)

A. Isolation and Characterization of Different Leaf Endophytes

what agar will the 100 ul rinsate be blotted onto

30 °C for 5-7 days

A. Isolation and Characterization of Different Leaf Endophyte

what is the incubation period for the duplicate nutrient agar (w/o antimicrobial supplement)

sterile scalpel

A. Isolation and Characterization of Different Leaf Endophyte

what tool should be used to cut out two 1 cm2 squares from one disinfected leaf sample?

1 cm2

A. Isolation and Characterization of Different Leaf Endophyte

Using a sterile scalpel, cut out two — squares from one disinfected leaf sample

6 mm; sterile cork borer

A. Isolation and Characterization of Different Leaf Endophyte

You can also use — circles, cut out using a —

two

A. Isolation and Characterization of Different Leaf Endophyte

Aseptically place — cut-out leaf samples in PDA and NA, equidistant from each other.

PDA and NA

A. Isolation and Characterization of Different Leaf Endophyte

Aseptically place two cut-out leaf samples in —, equidistant from each other.

in the dark at 25 °C for 1-2 weeks

A. Isolation and Characterization of Different Leaf Endophyte

what is the incubation period for PDA

30 °C for 5-6 days

A. Isolation and Characterization of Different Leaf Endophyte

what is the incubation period for NA

simple staining

A. Isolation and Characterization of Different Leaf Endophyte

what staining should be sued on the isolates from PDA

lactophenol cotton blue; HPO

A. Isolation and Characterization of Different Leaf Endophyte

what reagent should be used in doing a simple stain from PDA isolates and what objective should it be viewed?

Gram staining

A. Isolation and Characterization of Different Leaf Endophyte

what staining should be used on the isolates from NA

OIO

A. Isolation and Characterization of Different Leaf Endophyte

what objective should the gram staining from the NA isolates be viewed?

microscopic examination and cultural characterization

what are the two parts in the Characterization of Root Nodule-associated bacteria

Disinfect root nodules according to Steps A 1-3.

B. Characterization of Root Nodule-associated bacteria

what is the first step in Microscopic Examination

1-2

B. Characterization of Root Nodule-associated bacteria

Microscopic Examination

Crush one root nodule in — drops of sterile water in a clean glass slide

clean glass slide

B. Characterization of Root Nodule-associated bacteria

Microscopic Examination

Crush one root nodule in 1-2 drops of sterile water in a —

red coloration inside the nodule

B. Characterization of Root Nodule-associated bacteria

Microscopic Examination

what should be noted when crushing the root nodule

loopful

B. Characterization of Root Nodule-associated bacteria

Microscopic Examination

Transfer a — into another glass slide and prepare a heat-fixed smear.

heat-fixed smear

B. Characterization of Root Nodule-associated bacteria

Microscopic Examination

Transfer a loopful into another glass slide and prepare a — .

Gram staining; OIO

B. Characterization of Root Nodule-associated bacteria

Microscopic Examination

Perform — and record microscopic features viewed using —-

sterile microcentrifuge tube; 1.5 ml

B. Characterization of Root Nodule-associated bacteria

Cultural Characterization

In a — (—capacity), aseptically crush one root nodule in 0.5 ml 0.85 % NaCl solution.

0.5 ml 0.85 % NaCl

B. Characterization of Root Nodule-associated bacteria

Cultural Characterization

In a sterile microcentrifuge tube (1.5 ml capacity), aseptically crush one root nodule in — solution.

0.1 ml

B. Characterization of Root Nodule-associated bacteria

Cultural Characterization

Take — of the resulting supernatant and spread plate in modified YEMA with bromothymol blue

modified YEMA; bromothymol blue

B. Characterization of Root Nodule-associated bacteria

Cultural Characterization

Take 0.1 ml of the resulting supernatant and spread plate in — with —

28-30 °C for 10 days with daily observation for color change

B. Characterization of Root Nodule-associated bacteria

Cultural Characterization

what is the incubation period for the modified YEMA

fast growers

B. Characterization of Root Nodule-associated bacteria

Cultural Characterization

Colonies that turn surrounding culture media yellow are considered —

slow growers.

B. Characterization of Root Nodule-associated bacteria

Cultural Characterization

colonies that turn surrounding culture media blue are considered —