Phage contamination as a concept

Several cloning strains that fail on the same afternoon, in the same shaker, are a different problem from one ligation that produced no colonies. Bacteriophage contamination is the concept this guide is for: a virus of bacteria, classically a T1-like phage in an E. coli cloning room, that lyses cultures and survives on surfaces after the person who spilled them has gone home. The decision is whether the pattern follows a single DNA tube or follows a shared tool. Culture and transformation of non-pathogenic cloning strains, when the bench is fit for them, are described in bacterial culture and transformation. The barrier habits around any spill sit with biosafety basics for research benches.

The hero photograph is amber broth in clamped flasks. Those flasks are exactly where a phage problem becomes obvious, because a culture that was cloudy can drop clear as cells lyse. They are also a good way to spread the problem if a wet cap is handled and then a pipette is shared. Reagent and plastic classes are in the reagents and chemicals catalogue. The cloning workflow is the molecular biology pathway. A DNA design question can be framed from custom gene synthesis as a reference only. Questions about strain class, including resistance to a phage receptor, go through the quote request.

What you are looking at

Bacteriophages are viruses that infect bacteria. In a cloning laboratory the one with the longest reputation is T1, because dried material stays infectious on surfaces and because it infects many standard E. coli strains through the FhuA outer-membrane protein, historically called TonA. Strains mutated in that receptor are sold as a T1-resistant class. That class is a genotype choice. It does not sterilise a pipette.

The visible events are ordinary and easy to mis-name. A flask becomes turbid on schedule and is clear the next morning, sometimes with debris. A streak that should have grown is empty, or shows glassy plaques, clear spots in a lawn of the host. Transformations that used to work fail across plasmids that share nothing but the room. People blame competent-cell lots one after another. The lot is sometimes the culprit. When the replacement lot fails in the same incubator and a different incubator is fine, the geography is the data.

This guide does not tell you how to grow phage, how to raise a titre, or how to enrich it from a lysed culture or from the environment. Those are propagation methods, and they are out of scope. The useful act is to notice the pattern, stop, and hand the cleaning decision to the institution.

Why it follows objects rather than plasmids

Phage particles adsorb to cells and are also carried on gloves, pipette shafts, flask mouths and aerosol from a sloshed culture. A single contaminated micropipette, used for every "quick" inoculation, will visit every strain on the bench. A shaker tray that caught a drip does the same for every flask clamped on it. A plasmid tube cannot do that. The plasmid tube only touches reactions you pipette it into.

That is the branch. List the failures by strain, by plasmid and by tool. One plasmid, one host, other work healthy: debug the DNA, the antibiotic and the cells, using the controls in the culture article. Many strains, many plasmids, one incubator or one pipette set: treat it as a surface pattern until the safety office says otherwise. Mixed patterns happen. A genuinely bad DNA prep can land in a room that also has a phage. The shared-tool list is still the first map.

Some cloning strains are marketed as T1 resistant. Use that as a purchasing class when the committee has accepted the genotype, and keep cleaning. A resistant host can hide a contamination that will destroy the sensitive strain next door. Sharing a pipette between a resistant room and a sensitive room is how the hide fails.

Ordinary inoculation technique, which you return to only after the area is cleared for use, is illustrated by the Addgene protocol for inoculating a bacterial culture. The broader set of cloning notes is the Addgene protocols index. Neither is a decontamination method.

What to stop touching

Stop opening new cultures in the suspect area. Do not "just streak one more plate" with the same loop handle. Do not carry the suspect flask to the clean incubator to see if it recovers. Recovery in a second room is how a local problem becomes a departmental one. Close the flasks you already have, leave them in a tray the safety office considers contained, and label the tray as a suspected phage event rather than as ordinary waste, until they tell you which stream it is.

Disposable plastic that cannot be cleaned is part of the event. Open tip boxes, wooden sticks, spreaders and flasks with porous closures that sat in the splash zone are cheaper than a month of failed transformations. Reusable glass is decontaminated only by the method the office names. Do not soak a chlorine bleach product into a mystery cleaner. Do not mix bleach with ammonia or with acid. Name the incompatibility to the office if you are unsure what is already on the cloth. The WHO Laboratory Biosafety Manual is a public reference for the conversation. Your spill card is the instruction.

An indicator test, if it is allowed at all, uses a phage-sensitive cloning strain the institution names, under the containment they name, to ask whether unexpected clearing still happens. This page does not write that test. Ask, and wait. Collection-strain paperwork, if you need a fresh host afterwards, is handled in the spirit of the ATCC culture guides, under your own assessment.

One bad plasmid versus a bench-wide failure

Patterns that feel similar and are not this

A wrong antibiotic makes every strain that lacks the marker refuse to grow. The cultures do not typically cloud and then clear, and a strain you know is resistant still grows on the same drug. Dead competent cells fail at the transformation step and spare the streaks you make from glycerol stocks with a sterile stick. A shaker left off by a power cut produces stalled cultures with a clock time in common, including flasks that were not handled by the suspect pipette. Write the clock time down. If only the handled flasks cleared, the clock is not the cause.

Medium poured too hot with a dead antibiotic grows lawns. Lawns are the opposite appearance of plaques. Do not fold those notes into one "contamination" word.

Institutional disinfection, and nothing improvised

The safety office chooses the agent, the contact time and the waste stream. Cloning strains remain inside the assessment you already have. A suspected phage does not authorise you to culture a new organism, to take swabs home, or to run an enrichment. Waste that may contain phage is still waste they classify. The manual linked above supports the conversation. Your card is the procedure. This page is not medical advice and not a permit to change containment.

Humidity, shared kit, and the cupboard everyone uses

Dried droplets persist. A humid bench that never quite dries is a poor reason to delay a spill cleanup, because the spill is the object being carried. Shared tip boxes left open beside a shaker are a common route. In a building where several groups use one orbital shaker, the enquiry and the local rule both need a named responsible person for a contamination hold. A power cut belongs in the incident note as a time stamp. It is not, by itself, a lysis pattern. When work resumes, start from stocks that never entered the segregated area, and keep the T1-resistance genotype, if you use it, written on the flask so a sensitive strain is not the first test of a bench that was only wiped quickly.

What to ask when you specify a host after an event

Name the strain class, whether a T1-resistance genotype is required, the antibiotic markers, and the institutional assignment so packaging can be discussed. Say that the bench is returning to cloning work only under the local clearance. A method for routine culture can be discussed once that clearance exists. Use the quote request. Keep custom gene synthesis as a design reference if the plasmids must be rebuilt, and do not describe a phage experiment as something a supplier should help you run. The request is for a cloning strain and clean consumables, specified in writing.