EXERCISE 6 - ANTIMICROBIAL ACTIVITY AND PHOSPHATE SOLUBILIZATION OF SOIL MICROORGANISM

mineral phosphate

bioavailable form of phosphorus which is a key factor restricting plant growth

rhizospheric phosphate-solubilizing bacteria (PSB)

• These microorganisms solubilize mineral phosphate by secreting organic acids to lower pH (mineral PSB) or release catalytic enzymes such as phosphatases (organic PSB).

• These microbial activities were proven to reduce fertilizer use

Pantoea, Kosakonia, and Bacillus

In the study of Chakdar et al. (2018), five isolates of the genera — from drought-prone areas were found to have high mineral phosphate solubilization potential.

Bacillus isolate

This isolate was found to have the highest solubilization index (SI) and solubilization efficiency (SE) in solid culture media

Pantoea sp.

isolate that showed the highest mineral solubilization in liquid medium

Phyllobacterium, Rhizobium, and Sinorhizobium.

• 21 mineral PSB strains and 14 organic PSB strains isolated from roadside poplar rhizosphere in China, with seven strains having high phosphate solubilization activity towards tricalcium phosphate

• strains were found to have weak lecithinase activity and belong to 12 distinct genera

soil microorganisms

• also exhibit antimicrobial properties, contributing further to their ecological significance

• One of the potential antimicrobial sources

antimicrobial agents

In the environment, microorganisms use — to control the population of competing and beneficial species

Avena fatua and Brachiaria reptans

reported bacterial strains from — and —rhizosphere and phyllosphere that exhibited heavy metal-resistance and showed antimicrobial activity against a range of pathogens

two Streptomyces isolates from Origanum majorana rhizosphere

produced metabolites with antibacterial activity and inhibitory properties against biofilms of Staphylococcus aureus.

Mueller Hinton Agar (MHA)

What is the growth medium used for the test microorganism in antibiotic activity screening?

Luria Bertani (LB) broth

What medium is used to prepare overnight cultures of the isolates?

A line 7 cm long and 0.5 cm wide should be streaked at the center of the plate.

How should the test microorganism be streaked onto the MHA plate?

Three time points: 1 day, 3 days, and 7 days

How many incubation time points are tested in the MHA culture setup? What are they?

Sterile distilled water and sterile ciprofloxacin solution (100 mg/100 ml)

What are used as controls in this experiment?

• Grown on Nutrient Agar (NA) and incubated at 30°C for 18–24 hours.

• Diluted to 0.5 McFarland standard using 5 ml of 0.85% saline solution.

How are the coliform pathogens prepared before being streaked on the test plates?

A perpendicular line (3 cm long, 0.5 cm wide) is streaked from the edge of the plate toward the central streak

Describe how the pathogen is streaked in relation to the central test microorganism streak.

Incubated at 30°C for 24 hours.

At what temperature and for how long should the final plate be incubated after pathogen streaking?

The zone of inhibition, specifically the distance between the central streak and the pathogen streak.

What is measured to assess antimicrobial activity?

• Streaking with a loopful for isolation

• Spotting 10 µl of culture onto the agar

What are the two methods of inoculating Pikovskaya Agar with the isolates?

37°C for 48–72 hours

At what temperature and for how long should the Pikovskaya Agar plates be incubated?

Colonies with a zone of clearing around them.

What should be observed to indicate phosphate solubilization on Pikovskaya Agar?

• Colony diameter

• Halo diameter (zone of clearing)

Which parameters are measured on the Pikovskaya Agar plate for calculating SI and SE?

SI = (Colony diameter + Halo diameter) / Colony diameter

What is the formula for Solubilization Index (SI)?

SE = (Halo diameter / Colony diameter) × 100

What is the formula for Solubilization Efficiency (SE)?

100 µl

How much culture volume is transferred to the NBRIP-BPB broth?

Incubation at 37°C with shaking for up to 72 hours.

What incubation conditions are required for the NBRIP-BPB broth test?

A rapid color change indicates good mineral phosphate solubilization activity

What is the significance of color change in NBRIP-BPB broth after 24 hours?

The pH of the culture.

What additional parameter is measured from the NBRIP-BPB culture?

Bromophenol blue (0.025 g/L)

Which indicator dye is added to the NBRIP-BPB broth?

• NBRIP-BPB: Glucose (10.0 g/L)

• Pikovskaya Agar: Dextrose (10.0 g/L)

What is the carbon source in both NBRIP-BPB and Pikovskaya Agar?

Tricalcium phosphate (5 g/L)

Which phosphate compound is used in NBRIP-BPB broth?

• Tryptone (lacks vitamins)

• Peptone (lacks essential amino acids and vitamins)

Which alternative to yeast extract can be used in Pikovskaya Agar, and what are its limitations?

Ammonium sulfate

Which compound is common to both media as a nitrogen source?