An optimized and High Yielding Protocol for Isolation and Amplification of Bacteriophages Against Methicillin-resistant Staphylococcus aureus (MRSA)

Collect untreated sewage water samples from the domestic sewage treatment plant and hospital wastewater treatment plant.

Collect solid fecal samples from cages of chicken poultry farm and store them at 4°C until further processing.

4°C

Measure 40 mL of each sewage sample and centrifuge them in 50 mL centrifuge tubes at 7000 rpm for 15 min.

40mL 7000rpm,25°C,0h 0m 0s

Equipment

Value	Label
Eppendorf™ 5810R Centrifuge	NAME
Centrifuge	TYPE
Eppendorf	BRAND
02-262-8187	SKU

Filter the supernatant obtained through a 0.2 µm filter, and store the filtrate at 4 ⁰C.

4°C

Weigh 10 g of dry poultry farm fecal sample and mix it with 100 mL of sterile saline in a sterile 250 mL conical flask.

10g 100mL

Keep the suspension at 30 ⁰C for 5 – 6 h at 120 rpm to detach the phages from solid particles.

30°C

Collect 40 mL of this sample and centrifuge it at 7000 rpm for 15 min .

40mL 7000rpm,25°C,0h 0m 0s

Filter the supernatant obtained through a 0.2 µm filter, and store the filtrate at 4 ⁰C.

4°C

Mix all three filtrates (Domestic sewage, hospital wastewater, and poultry farm) in a sterile environment to make a single filtrate suspension (pooled sample) for isolation of MRSA-specific bacteriophages.

Thaw the glycerol stock of MRSA stored at -80 ^°C.

Inoculate 5 µL of the thawed culture in 20 mL of TSB in a 50 ml sterile conical flask and incubate for 24 h at 37 ⁰C and 120 rpm.

5µL 10mL 37°C

The grown culture was centrifuged at 10,000 rpm for 10 min and resuspended the pellet in 10 mL of fresh TSB. Use this suspension for the isolation and enrichment of MRSA-specific bacteriophages.

10mL 10000rpm,25°C,0h 0m 0s

Mix 10 mL of pooled sample filtrate (obtained Step 1) and 10 mL of sterile 2X TSB in a 50 mL sterile glass conical flask.

10mL

Add 1 mL of an overnight grown pure culture of MRSA (obtained from Step-2) to the above suspension and mix gently. Incubate the suspension at 30 ⁰C for 24 h at 85 rpm.

1mL 30°C

Following incubation at 30 ⁰C, Incubate the suspension at 4 ⁰C for 24-48 hr.

4°C

After 24-48 hr, centrifuge the suspension at 14,000 rpm for 15 min and filter the supernatant through a sterile 0.2 µm syringe filter.

14000rpm,25°C,0h 0m 0s

Quantify the titer of bacteriophages in the filtrate using the drop cast method (as described in step 4).

Quantification of bacteriophages in the filtrate obtained in any step of the experiment can be done using the drop cast method.

Take 100 µL of overnight grown pure culture of MRSA (from Step-2) and mix with 3 mL of molten soft agar (TSB + 0.6% Agar).

100µL 3mL

Pour the suspension on prepared hard agar (TSB + 2% Agar) and allow it to solidify.

To quantify bacteriophages in the given filtrate, serially dilute the filtrate in SM buffer and spot 5 µL of each dilution on the bacterial lawn. Allow the plates to solidify completely.

5µL

Incubate the solidified plates overnight at 37 ⁰C for the development of clear visible plaques.

37°C

Calculate the phage titer (PFU/mL) by using the following formula:

Number of PFU/mL= Number of plaques in a drop X Dilution factor

                                                          Volume of drop spotted