Bacteriophage: What is it, how do I prevent contamination, and how do I get rid of it?

What is bacteriophage? A Bacteriophage (known informally as phage), is a virus that infects and replicates within a bacterium. Bacteriophages are ubiquitous viruses, found wherever bacteria exist.

Phage contamination can lead to rapid lysis of bacterial cultures, resulting in a previously high OD culture becoming clear (a sign of lysis). In some cases, infection provokes only a weak lysis of bacterial culture; however, this affects the metabolism of the host cells, causing slow growth rates and low production. The continuous liberation of phages to the growth medium can be even more problematic than fast lysis Transmission electron micrograph of of whole cultures, as it is often not recognised. multiple bacteriophages attached to a

bacterial cell wall Some bacteriophages can survive even in dry places for many years, so careful microbiological work, especially during inoculation of cultures and sample withdrawal, is absolutely essential. Good lab hygiene is crucial, but even in very clean laboratories, where people work carefully, phage infections occur from time to time, as it is impossible to keep sterile conditions during all cultivation procedures, especially on a large scale.

Serious problems occur in laboratories when cultures, either in flasks or in large bioreactors, are contaminated by Phage contaminated culture bacteriophage, leading to a complete loss of the desired bio product and spread of bacteriophages throughout the laboratory

Bacterial cultivation is widely used within the MIB; bacterial cells are used as model organisms in biochemical, genetic, and molecular biology studies, and are used for the production of biotechnologically important substances (proteins, etc). Phage contamination can result in a significant cost implication, both in terms of time and resources, so it is important that all lab workers use good microbiological technique at all times, and infections are identified and dealt with promptly.

Recognition of phage infection or prophage induction

Common signs of phage infection include:

• Lysis of cultures (unexpected decrease in optical density, low density after overnight cultivation) • Visible clumps and threads in the culture • Thick white foam on the culture (even when the culture is dense), which does not disappear if the flask is left for some time on the table • Observation by light microscopy: clumps, fatty drops, dirty-like threads, no rod-shaped cells

Common sources of phage in laboratories

• Dirty pipettes • Open bacterial plates (always put waste plates into a biohazard bag inside one of the blue bins) • Contaminated incubators and centrifuges (eg. Following a spill which hasn’t been cleaned up properly) • Strain and plasmid collections (test all incoming strains!) • Waterbaths - Clean out water baths regularly. If necessary add a “germicide” to keep water baths free of bacterial growth. • Any buffers/solutions that have been in contact with phage-infected cultures (eg. Stocks of antibiotics, IPTG, etc)

Prevention – best practice

• Clean/disinfect your lab bench before and after working with micro-organisms. Use a 1% solution of Virkon, followed by 70% ethanol if desired. A fresh solution of 1% Virkon should be prepared weekly (in the fume cupboard). • Employ meticulous aseptic techniques while working, making sure to always flame your flasks when handling your cultures. • When possible, avoid draughts and areas of air turbulence when working with micro- organisms. Expose cultures to the air as little as possible. A communal Class II microbiological safety cabinet is available on the 2nd floor (outside 2.062) for GM Class 1 and Hazard Group 1 microbial work. All users must clean/disinfect the safety cabinet thoroughly before and after use. • The communal laminar flow cabinet on the 2nd floor (outside 2.042) can be used for preparing sterile solutions and pouring/drying agar plates. This laminar flow cabinet must NOT be used for handling cultures. • Do not open flasks (i.e. remove bungs) in shaking incubators. Transfer flasks to your bench before taking samples or adding components. Where possible, inoculate everything in the communal class II microbiological safety cabinet on the 2nd floor until the problem goes away, and flame flash sterilise EVERYTHING just before placing in the hood/cabinet. Please note that the class II microbiological safety cabinets available in the MIB tissue culture facility are not available for use for microbiological work. • If you suspect a culture is infected with phage DO NOT open the flask or plate in the lab – send it to be autoclaved (either take directly down to the MIB autoclave facility, or place in a blue bin). • Avoid generating aerosols. Take extra care when vortexing cultures, flaming inoculating loops, pipetting and using centrifuges. • Transfer samples into centrifuge bottles/tubes, balance and seal them on your bench (or in a class II microbiological safety cabinet), not in the equipment parks. Decant supernatants on your bench (or in a class II microbiological safety cabinet). • Clean up and disinfect all spills promptly and thoroughly, especially in communal equipment (e.g. orbital incubators and centrifuges) and areas with 1% Virkon or 70% ethanol. NB. Virkon should not be used on metal! • Dispose of all microbiologically contaminated materials correctly. If in doubt about waste routes, please ask a senior member of your group, your floor coordinator or Dr Tanya Aspinall. • Place used agar plates in biohazard bags in a lidded blue bin, not in open bags in stands. • Discard microbiologically contaminated tips, tubes etc. into Virkon solution. Soak, decant, and place the tips/tubes in a biohazard bag. Virkon solution needs to be changed weekly or more frequently if the colour has changed. Make up Virkon solution in a fume cupboard. • Place microbiologically contaminated glassware in the appropriate blue bin or if you are disinfecting flasks/centrifuge bottles ensure that the Virkon is the correct strength (its activity is impaired by the presence of organic matter), all internal surfaces are covered and the vessels are left for > 1 hour. • Use autoclaved water to prepare solutions which will be filter sterilised (e.g. ampicillin, IPTG) as filter membranes will not exclude phages. Aliquot the solutions. • Re-autoclave all sterile water containers, and any other sterile liquid, and leave autoclave tape on. Only allow one use of the liquid (say a set of flasks), followed by immediate re- autoclaving of it and leave the tape on again. Never use an aliquot twice, and never use one that another person has used. • Use filtered tips (or sterile syringes) where possible. • Do not keep old plates of bacteria. Restreak strains to single colonies. • Consider growing your culture more slowly (eg. at 20°C) – slow bacterial metabolism means slow phage growth. Moreover, spontaneous prophage induction is less frequent in slowly growing bacteria. This may not be possible, as the growth rate obviously must be sufficiently high to synthesise a desired product in cultivated cells effectively. • Maintain a high standard of “housekeeping” in your lab area and communal areas. • Co-operate with your floor coordinator’s arrangements for cleaning communal orbital incubators, cold rooms etc. • Ensure lab coats are laundered regularly. All lab coats are autoclaved before being sent for cleaning. Ensure that you change gloves regularly and follow good microbiological practice to the letter! • Wash your hands at the hand-washing basin before leaving the lab area.

Elimination of phage

• If you suspect that a specific incubator is the source of infection, strip it down and autoclave as many of the components as possible; non-autoclavable parts should be cleaned thoroughly with 70% ethanol. MIB technical staff can provide advice on how to do this correctly. • Discard all of the solutions you used to prepare the culture (including antibiotic stocks, IPTG, etc), and start making fresh for each set of flasks/containers. This is expensive, but is sometimes the only way to ensure that you are not re-contaminating your cultures/lab area • Clean automatic pipettors regularly. Some pipettors, or parts of pipettors, can be autoclaved if necessary. Consider this when you buy new pipettors. Another suggestion would be to use sterile 1ml/2ml syringes (these are relatively cheap, come as single packs and are sterile. Use a different one for every addition if necessary). • Put all your flasks (and all of your glassware, ideally) through the oven at 180°C. An oven is available for use in the MIB autoclave facility – please speak to technical staff for help/advice. • You can make agar plates with bacterial lawns and spread them around to see where the phage is. Inside the laminar flow hood should give zero plaques after a few hours of being left open in there. If you puff air from pipettes onto plates, you will probably find the current source of the phage – these can then be dismantled and cleaned/sterilised.

Alternative experimental design

• You could consider using E. coli strains which are resistant to certain phages. A number of such strains are available from New England Biolabs, Invitrogen etc. including derivatives of BL21. • You could consider auto induction, which will minimise opening of flasks. • You could consider other hosts (yeast, other species of bacteria).

Further reading: Bacteriophage contamination: is there a simple method to reduce its deleterious effects in laboratory cultures and biotechnological factories? Los M, Czyz A, Sell E, Wegrzyn A, Neubauer P, Wegrzyn G