Assessing the Coexistence of T4 Bacteriophage and E. coli in Murine Fecal Samples

Samenvatting

Source: Pett, N., et al. T4 Bacteriophage and E. coli Interaction in the Murine Intestine: A Prototypical Model for Studying Host-Bacteriophage Dynamics In Vivo. J. Vis. Exp. (2024).

This video demonstrates the in vivo interaction between T4 bacteriophage and E. coli in the murine intestine, and the fecal spot plating method to quantify their coexistence by measuring bacterial colonies and phage plaques.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Spot plating T4 phage to determine concentration in fecal pellets

1. Collect fecal pellets from each mouse into sterile, pre-weighed, microcentrifuge tubes to measure T4 phage and E. coli levels. Store tubes on ice until plating.
   NOTE: Place samples on ice in the interim between collection and plating to slow the growth of aerotolerant bacteria. Over several hours, there may still be growth of aerobic bacteria, or some death of anaerobic bacteria due to oxygen exposure which could lead to skewed bacteria counts. Therefore, bacteria and phage plating should be performed as soon as possible after sample collection. Obligate anaerobic bacteria will not tolerate oxygen exposure. To preserve the sample, collect samples into sealed tubes and transfer tubes to an anaerobic chamber as soon as possible after collection. If no growth is detected in fecal samples, consider alternatives such as 16S rRNA quantitative polymerase chain reaction (qPCR) for bacterial quantification.
2. Record final weights of each tube and calculate the sample weight by subtracting the initial tube weight. This will be used for normalizing T4 phage and E. coli concentration to sample weight (palque forming units - pfu/g or colony forming units - cfu/g, respectively).
3. Add 1 mL of sterile saline magnesium (SM) buffer to each tube and vortex thoroughly at maximum speed (>1 min) to homogenize fecal pellets. If the sample is less than 15 mg, a smaller volume of SM buffer may be added. Record the volume of SM buffer added to each sample for calculations of cfu/g or pfu/g of sample.
4. Prepare a series of 8 (or more depending on the expected phage concentration) serial dilutions of 20 µL of each sample in 180 µL SM buffer, in factors of 10. Vortex each homogenized sample briefly to mix before adding to the first tube/well. Pipette to mix in between each addition and change tips between each dilution to prevent inflating phage or bacteria counts via sample carryover.
   NOTE: If fibers and debris present in the sample hinder pipetting, add additional buffer to the fecal slurry to further dilute the stock sample.
5. Spot 5 µL of each dilution onto Lennox broth (LB) soft agar plates containing E. coli (for phage plaque assays) or LB agar plates (1.5% agar, for bacterial colony assays) to determine T4 phage and E. coli concentration in each sample. For accuracy, spot each sample in triplicate.
   NOTE: If preparing serial dilutions in a 96-well plate, an 8-channel P20 multichannel pipette may be used to pipette each column of serially diluted sample onto plates. Change tips between each dilution, even if moving from most dilute to most concentrated, as phage may adhere to the walls of the pipette tip and alter the amount of phage added to each new well.
6. Allow each spot to dry before inverting the plate and placing it in the incubator. Incubate overnight at 37 °C.
7. For each sample, select the dilution at which there are 3-30 countable plaques per spot. Count and record the number of plaques in the spot and the dilution used.
8. Calculate pfu/g of sample by dividing the number of plaques by the volume plated in each spot to give pfu/µL. Multiply this by the dilution factor and by the volume of SM buffer added to each sample to give pfu/sample. Lastly, divide by the sample weight to give pfu/g.

 

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Fisher BioReagents™ Agar, Powder / Flakes, Fisher BioReagents™	Fisher Bioreagents	BP1423-500
Fisher BioReagents™ Microbiology Media: Lennox broth (Powder)	Fisher Bioreagents	BP1427-500