Bacteriophages
So You Think You have Phage
Step 1: Confirm that its actually phage
Phage contamination is somethng that can happen to anyone working with a phage susceptible strain (MG1655 derivatives). However, its more likely to occur over long culture times (MAGE) or creep into a lab with unsupervised waste (sitting on the bench for extended periods)
The tell tale symptoms of phage are as follows:
Plaques on plates:
The iconic symptom of a bacteriophage is the appearance of plaques (circular regions of low cellular density, "anticolonies") on a region of lawn growth on a plate.
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Seal any plates with this symptom and continue incubating. More plaques/larger plaques upon incubation confirms the presence of an active, lytic phage
Plaques/OD Crashes in liquid cultures:
If a culture you are working with grows successfully then suddenly arrests growth/ loses OD overnight/ over the course of an experiment, that might be phage. Plate to confirm, sanitizing the surrounding area after exposing the suspect culture.
Additionally, phage conatminated overnights will often form stringy plaques of cellular debris in a shaking culture. This can and has been easily mistaken for other sorts of contamination (fungi, other bacteria)
Step 2: Dont Panic
Our primary cloning strain, DH10B (NEB10Beta), is phage resistant as is DH5alpha. As a result most of our labs cloning work will be minimally affected by a phage outbreak.
However, if you are working with a phage susceptible strain and see the tell tale signs of an outbreak, there are immediate steps you can take to counteract the threat.
Step 3: How to fight Phage
1: SEAL ALL CONTAMINATED PLATES/MEDIA.
2: Bleach all surfaces that might have been exposed to Phage contaminated cultures or plates. This includes shared equipment (centrifuges, shakers, spectrophotometers). 3: Autoclave all autoclavable materials from a contaminated area. Dispose of any disposables from the contaminated area. 4: UV Lamps overnight (several days). UV radiation will kill any airborne phage particles and sanitize surfaces of phage. 5: Honeypots to track exposure/treatment. Make plates with lawn growth of a phage suceptible strain and leave the plates exposed to ambient air at various points in the lab space during a workday. Incubate these plates overnight and any phage contamination will indicate regions of the lab that are still contaminated 6: Purge contaminated cryostocks. If a particular strain regularly gets contaminated, the parent strain could be contaminated already. Destroy/replace any confirmed contaminated cell lines to prevent rexposure/recontamination
Step 4: Preventing Phage outbreaks
1: Dispose of laboratory waste media/cultures the same day as they are created. Waste materials exposed to the ambient air could become a vector for phage contamination 2: Clean glassware within 24 hrs of its use. Media and cells from the previous use of that glass could provide an entryway for phage. 3: Regularly clean/sanitize your lab bench. A clean bench minimizes the risk of initial contaminiation, and regular sanitizing reduces the likelihood of recontamination 4: Use aseptic technique (flame + sterile tips/media) when working with live cells. 5: Recognize the signs of a phage contaminated culture and seal/dispose of suspicious cultures/plates.
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