Live/Dead Staining for Quantifying Viable but Not Culturable Cells in Manuka Honey-Treated Wound-Causing Bacteria

This research project aims to investigate the impact of Manuka honey on bacterial physiology. Recent studies have provided further insights into the anti-microbial effects of Manuka honey on bacterial physiology. Our preliminary findings indicate that Manuka honey bacteria enter the antibiotic resistant, viable but not culturable dormancy state, but to a lesser extent than those treated with conventional antibiotics.

Based on our experiments, it would be interesting to compare the expression profiles of Manuka honey and conventional antibiotic induced VBNCs. To begin, take the vials containing frozen bacterial strains. Open each one sequentially and use a sterile stick to scrape a small amount of frozen bacteria onto the appropriately labeled Luria-Bertani or LB agar plate.

Replace the vial cap and quickly place it in the freezer. Incubate the bacterial cultures for 18 to 24 hours at 37 degrees Celsius. The following day, use a serological pipette to transfer two milliliters of LB broth into two sterile test tubes.

Sterilize the inoculating loop in the Bunsen burner flame. Then transfer a quarter-loopful of bacteria from the LB agar plate to the appropriately labeled test tube containing LB broth. Incubate the test tube under aerobic conditions in a shaking incubator at approximately 250 RPM at 37 degrees Celsius for 18 to 24 hours.

For the setup of Manuka honey and antibiotic treatments of bacteria, add 10 microliters of bacterial culture to 10 milliliters of Mueller Hinton broth to obtain the 1:1, 000 dilution of the bacterial culture, which has approximately 10 to the sixth colony forming units per milliliter. Use the diluted cultures to prepare three samples for each bacterial species, an untreated control, a Manuka honey treated sample, and an antibiotic treated sample. Add the Manuka honey and antibiotic to the appropriate tubes.

Transfer 0.1 milliliter and 0.5 milliliters of the untreated sample to sterile micro centrifuge tubes for the viable plate count and live/dead staining for the T equals zero samples. Then incubate the untreated control, Manuka honey treated, and antibiotic treated samples under aerobic conditions at 37 degrees Celsius in a shaking incubator at 250 RPM for 24 hours. Prepare a tenfold serial dilution of the T equals zero untreated sample in LB broth to obtain a countable number of cells for the viable plate count.

Spread 50 microliters of each diluted sample onto one half of an LB agar plate. Incubate the agar plates under aerobic conditions at 37 degrees Celsius for 24 hours. The following day, remove the LB agar plates from the incubator.

Identify the dilution plate containing approximately 25 to 150 colonies and count the exact number of colonies. Use the colony forming unit formula to determine the number of culturable cells per milliliter. On day three, add 0.5 milliliters of 0.85%sodium chloride to 0.5 milliliters of T equals zero untreated live/dead staining control sample.

Combine 1.5 microliters of SYTO 9 and 1.5 microliters of propidium iodide per sample. Then add three microliters of the mixture to each sample. Incubate the samples at 21 degrees Celsius in the dark for 15 minutes.

Transfer six microliters of the stained, gently mixed sample to a disposable hemocytometer and view under a fluorescent microscope using a fluorescein isothiocyanate or FITC filter and a 40X objective lens. Capture an image showing both the hemocytometer grid lines and the green cells. Count the green cells in six fields per sample, noting the grid dimensions to calculate the number of green cells per volume.

Finally, calculate the number of viable but not culturable cells by subtracting the number of culturable cells per milliliter obtained using the viable plate count from the number of viable cells per milliliter obtained using live/dead staining. On day four, after removing the untreated, Manuka honey treated, and antibiotic treated bacterial cultures from the incubator, collect 0.1 milliliters and 0.5 milliliters of each sample into micro centrifuge tubes for the viable plate count and live/dead staining. This figure shows the number of culturable and viable cells determined using the viable plate count and live/dead staining for S.aureus and P.aeruginosa cultures.

The results show that the number of viable and culturable cells detected in the untreated and Manuka honey treated cultures were not significantly different. S.aureus cultures treated with tobramycin had fewer culturable cells than viable cells, but this difference was not significant. Only meropenem treated P.aeruginosa showed a statistically significant reduction in culturable cells compared to viable cells.