Chory Lab Science Guidance

Scope

This guide covers phage work and continuous evolution, mammalian cell and virus culture, media and agar preparation, and automated workflows.

It describes practice, not procedure. Formulations, strain names, disinfectant concentrations, contact times, deck layouts, and instrument steps live in the individual protocols, SOPs, and the robot manual.

Where this guide and a protocol disagree, follow the protocol and flag the conflict.

How contamination behaves

Contamination spreads through shared stocks, media, pipettes, equipment, incubators, and robots. One contaminated reagent can compromise many experiments before anyone notices, because material moves through the lab faster than results come back.

Phage is the hardest case. It is almost never observed when it happens, and it surfaces weeks later as bacteria that will not grow, cultures that lyse, controls that fail, or an established experiment that stops working for no clear reason.

Four habits prevent most of it.

Protect what cannot be remade. A working stock can be regrown in a day. An archival glycerol stock from Addgene, from Emma, or from an alumnus often cannot be replaced at all.

Keep reusable materials closed around phage. If something could be used again, it should not be open while phage is open. This single habit prevents most untraceable events.

Move in one direction. Clean to exposed, never back. Hands, tips, pipettes, and robot methods all follow the same rule.

Discard rather than deliberate. Replacing media, water, antibiotics, agar, or a working stock costs a day. Letting contamination run costs weeks, usually paid by someone who had nothing to do with it.

When contamination is suspected, do not try to prove which item was responsible. Proof is rarely available. Identify everything that could reasonably have been exposed, and eliminate all of it.

Glycerol stocks

What is hard to replace

The freezer holds a spectrum, and the handling follows from how hard something is to recover:

• Hard or impossible to replace. Addgene deposits, Emma's stocks, and stocks left by lab alumni. There may be no one left who can remake these.
• Slow to replace. Parent strains, and strains already transformed with a plasmid someone may want later. Remaking one means retransforming, re-picking, re-validating, and often re-sequencing.
• Easy to replace. Working stocks and aliquots made for a current experiment.

Handle your own stocks the same way you handle inherited ones. Today's routine working stock is often what a labmate or future student ends up depending on, and by then nobody remembers how it was handled.

Glycerol stocks stay in the -80 C

Scrape inside the freezer, with the tube still in its box. The tube does not come out.

Open the -80 C, open the box, scrape, close the box, close the freezer.

Restarting cultures from a glycerol stock is routine, and people return to the same tube constantly. That frequency is exactly why each access has to look the same.

The failure mode is thawing. Stocks tolerate cold scraping indefinitely. They do not tolerate repeated partial thawing, and a surface that thaws and refreezes locks in whatever landed on it.

Never:

• Carry a stock to the bench or into the main lab space
• Put one on ice, on dry ice, or in a bucket, including "just for a minute"
• Set one down while you label tubes, set up, or answer a question
• Take one near an active phage workspace or an open phage tube
• Let one thaw through and then refreeze it
• Scrape twice in a sitting, or scrape a tube that has already softened
• Touch one with anything that has contacted another tube, culture, reagent, or surface

Wanting to bring the tube out means the bench was not ready before you opened the freezer.

Accessing a stock

Everything happens before the freezer opens:

1. Prepare and label all receiving culture tubes.
2. Gather sterile, single-use cotton-tipped applicators or approved disposable scrapers.
3. Clear and disinfect the surrounding area.
4. Confirm no phage work is happening nearby.
5. Know which box and position you need.

Then go to the freezer. Scrape with one new sterile applicator per stock, recap, close the box, close the freezer, and bring the applicator to your prepared tube.

Never return an applicator, loop, or tip to a stock after it has touched anything else. There is no way to undo this. The contamination freezes in and gets handed forward to everyone who uses that tube afterward.

Nothing previously used, exposed to the bench, or handled near phage should ever enter a bacterial, phage, or virus stock.

Make your own stock the first time you use someone else's

If you scrape a stock from Addgene, from Emma, or from an alumnus, your first priority with that culture is your own glycerol stock in your own box.

Grow up the culture, confirm it is what you expect, freeze your own vials, and work exclusively from those afterward. The inherited tube gets touched once.

Two reasons:

• Every scrape is a chance to contaminate something that may be irreplaceable, so the number of accesses should be as close to zero as possible.
• A strain you use repeatedly should have a known freeze date and handling history, not an unknown number of scrapes by an unknown number of people over years.

Do this from the first culture, before the strain is entangled in an experiment and before there is pressure to skip it.

Freezing a stock others can use

Assume someone will need your stock after you have left.

• Label it so it can be identified without asking you: strain, plasmid or construct, relevant markers, your initials, and the date.
• Record it wherever the lab tracks strains, with enough detail to know what it is and how it was made.
• Freeze more than one vial for anything that took real effort to build, stored so one freezer failure does not take out both.
• Keep one vial archival and untouched by routine work.

Close everything before opening phage

Nothing reusable is open while phage is open.

This is the highest-value habit in phage work.

Close all of these before any phage tube is opened:

• Media bottles
• Water
• Antibiotic stocks
• Glycerol and bacterial stocks
• Agar
• Shared buffers
• Reagent reservoirs
• Other phage tubes

The order matters:

1. Dispense clean reagents.
2. Close every shared and reusable material.
3. Only then open the phage tube.

Open one phage tube at a time and close it as soon as the transfer is complete.

Work within the flame zone

For approved open-bench bacterial and phage procedures, stay inside the flame zone.

A lit burner creates a convective updraft that carries airborne particles away from open vessels instead of letting them settle in. Flaming the neck of a glass vessel kills organisms on the glass and drives a brief outward current as you open and close it.

Both effects are real and both are limited. The flame does not sterilize the air, it does not extend far from the burner, and it does not replace closing reusable containers.

Technique:

• Arrange the bench first, so hands and tubes do not repeatedly cross between clean and phage-exposed areas.
• Keep the work inside the zone. Reaching out to grab something and returning defeats it.
• Briefly flame the necks of compatible glass tubes, flasks, and bottles on opening and closing.
• Keep vessels open as briefly as possible, and angled rather than straight up where the protocol allows.
• Replace caps immediately.

Never flame plastic caps, plastic tubes, pipettes, cap liners, filter units, or any other heat-sensitive material.

Never use an open flame inside a biological safety cabinet, inside a robot enclosure, or near ethanol.

Ethanol is used constantly here for surface disinfection. Confirm sprayed surfaces are dry and no ethanol container is open before lighting a burner, and keep the burner away from any surface you are about to spray.

Pipettes and tips at the bench

Filtered tips for all hand pipetting in phage work, fresh between every well, tube, culture, and reagent.

Hand pipetting has no wash step and no way to verify decontamination, so there is no acceptable reuse pattern at the bench. Robot tip reuse is a separate case, covered in Section 3.

• Use pipettes designated for phage workflows where possible.
• Do not move a pipette directly from a phage workspace into media-making, cloning, stock-preparation, or cell-culture areas.
• Wipe pipette exteriors during long experiments and at the end of every session.
• Apply disinfectant to a wipe. Never spray into the pipette mechanism.

Do not assume ethanol alone eliminates phage. Follow the phage decontamination procedure for the right disinfectant, concentration, contact time, and equipment compatibility.

Recognizing phage contamination after the fact

Phage contamination is usually invisible when it happens. It is suspected later, when:

• Bacteria fail to grow, or grow poorly
• Cultures lyse unexpectedly
• Overnight cultures are unexpectedly clear
• Controls fail
• A reliable strain stops behaving normally
• Continuous evolution cultures collapse
• Several related experiments fail without explanation
• The same problem appears across multiple users or workflows

When this happens, assume contamination occurred earlier in the workflow and work backward through everything that contacted the affected bacteria, directly or indirectly:

• Bacterial working stocks and overnight cultures
• Media, water, antibiotics, agar, plates, buffers
• Reagent reservoirs
• Pipettes, tubes, tube racks
• Incubators and shakers
• Robot decks and carriers
• Shared benches and equipment

The exposure window usually extends well beyond the day the failure appeared.

Not every failure is contamination. Poor growth also comes from disinfectant carryover, exhausted media, or antibiotic errors. The signatures tend to be opposite:

• Contamination looks like cultures growing too well, growing when they should not, or lysing.
• Carryover looks like cultures that will not grow at all.

Use that to direct the investigation. Do not use it to delay discarding replaceable materials.

What to discard

Discard anything replaceable that could reasonably have been exposed.

• Open or recently used media
• Water
• Antibiotic stocks
• Agar and plates
• Buffers
• Old bacterial working stocks
• Overnight cultures
• Prepared reagent plates
• Open reservoirs
• Partially used tubes

Do not keep a reagent because contamination has not been proven. It rarely can be, and one preserved bottle carries the problem into many more experiments.

Archival stocks stay usable precisely because they never enter phage workspaces. Restart from one only after the workspace, pipettes, incubators, shakers, robots, and shared equipment have been decontaminated.

When bacteria unexpectedly stop working, assume phage until there is a convincing alternative.

Remove biohazard waste after every session

This applies to mammalian cell culture, virus-associated culture, bacterial culture, phage work, media preparation, agar preparation, and plate pouring.

At the end of every session:

• Close and remove the bench waste container or bag, and install a fresh one.
• Remove used serological pipettes, tips, tubes, plates, swabs, and culture waste.
• Clean and decontaminate the work surface.
• Remove waste from the biological safety cabinet.

Never leave open culture waste on the bench or in the BSC.

Liquid biological waste needs the required disinfectant concentration and full contact time before disposal. Do not empty it early.

Virus work

Filtered tips for all mammalian virus work and virus-associated cell culture.

• Use pipettes designated for viral workflows where possible.
• Do not move pipettes from viral work into routine culture or clean media preparation without completing decontamination.
• Perform mammalian cell and virus work in the appropriate BSC, per the applicable biosafety SOP.
• Never use an open flame inside a BSC.

Keeping cell-culture areas clean

At the start and end of each session:

• Disinfect the BSC or work surface.
• Bring in only what the procedure requires.
• Keep clean materials separated from waste and exposed cultures.
• Keep caps and lids closed whenever possible.
• Remove waste immediately when finished.
• Return shared reagents only if they stayed closed and uncontaminated.
• Report and document spills, splashes, or suspected contamination.

Do not preserve an inexpensive shared reagent when there is a reasonable chance it was exposed to virus or contaminated culture.

Agar and antibiotics

Prepare and store agar without antibiotics if it may need remelting.

After remelting:

1. Mix thoroughly.
2. Cool while keeping it molten.
3. Add fresh antibiotic at the temperature specified for that antibiotic.
4. Mix thoroughly before pouring.

Roughly 55 to 60 C is a common range when no more specific protocol exists, but check the antibiotic. Several common ones are heat-sensitive and lose activity if added hot.

Never repeatedly heat agar after antibiotics have been added.

The same applies to any heat-sensitive supplement added to autoclaved media: add it only after the base has cooled, and label the vessel with what was added and when.

Vessel size

Always leave substantial empty headspace when preparing, autoclaving, or microwaving media and agar.

Molten agar foams, boils over, and erupts. Do not remelt 1 L of agar in a 1 L flask.

• 1 L flask: no more than about 500 mL of molten agar
• About 1 L of agar: use a 2 L or larger vessel
• Follow the autoclave and microwave SOPs for cap position, secondary containment, and maximum fill

An oversized flask is also easier to mix and to handle aseptically.

Preparing 1 L of media

1. Add the powder required for 1 L.
2. Dissolve in approximately 800 mL of water.
3. Transfer to an appropriately oversized autoclave vessel.
4. Autoclave per the media-specific protocol.
5. Cool enough to reduce burn and thermal-shock hazards.
6. Aseptically bring to 1 L with sterile water.

Water added after autoclaving must itself be sterile. Do not assume freshly dispensed Milli-Q water is sterile. Use autoclaved or filter-sterilized water.

The Milli-Q system and its filters must be maintained on schedule and recorded in the lab maintenance log.

Avoid adding cold water to very hot glass, which can crack the vessel.

Label every prepared vessel with contents, date, and any post-autoclave supplements including antibiotics. An unlabeled bottle of media is not usable and should be discarded.

Flaming glass containers

When opening or closing a glass media flask at an open bench:

• Work near the flame.
• Keep the opening exposed as briefly as possible.
• Briefly flame the opening.
• Briefly flame the compatible glass or metal-facing portion of the lid where appropriate.
• Replace the lid immediately.

Never expose plastic caps, plastic liners, or heat-sensitive components to the flame.

Automation amplifies contamination. One bad well, reservoir, or tool can be distributed across an entire plate, an entire run, or a set of shared reagents before anyone sees a result.

This section covers practice only. Setup, cleaning, washing, error recovery, and troubleshooting live in the robot manual for each method. Where the manual is more specific, the manual wins.

Keep shared and irreplaceable materials off the deck

Aliquot what the run needs.

Never bring a shared antibiotic bottle, a shared media bottle, or a sole phage or virus stock to a robot because aliquoting felt like extra work. That bottle spends the run beside cultures and waste, then goes back on the shelf for everyone else.

Never return unused material from a reservoir, plate, or tube to a shared bottle.

Robot tip reuse is not bench tip reuse

Robot methods reuse tips by design, through dedicated dirty racks and an automated wash cycle. That is validated as part of the method and it is what makes long runs affordable.

It works only when the wash infrastructure works, so the job is to protect the wash rather than skip it.

• Never bypass or shorten wash steps to save time.
• After any error, tips that were on the deck are not clean.
• Do not change a method's tip-reuse pattern without explicit evaluation and approval. A pattern that looks harmless in a simple transfer can distribute phage, cells, DNA, or aerosols across an entire experiment.

None of this extends to hand pipetting. There is no wash step at the bench, so manual work uses fresh filtered tips every time.

Separate workflows by biological class

Keep separate channels, tools, accessories, and workflows where possible for clean media preparation, bacterial culture, phage work, mammalian cell culture, and mammalian virus work.

Do not assume a tool is clean because it holds no visible liquid. Contamination moves through pipetting channels, grippers, carrier surfaces, plate lids, tube racks, tip-ejection areas, droplets, aerosols, condensation, and the undersides of plates and reservoirs.

Never mix bleach and ethanol

Bleach and ethanol must never be mixed or allowed to contact each other while wet.

When a protocol calls for both, the bleach must be fully removed and the surface fully dry before ethanol goes on.

Never combine them in bottles, waste containers, aspirators, reservoirs, wet wipes, spill trays, or on a still-wet surface.

Stop rather than restart

Stop the run after any splash, spill, stray droplet, collision, displaced lid, spilled well, aspirated foam, tip pickup or ejection failure, unexpected tip contact, or suspected cross-contamination.

Restarting the protocol is not a response. Instead:

1. Quarantine the affected materials.
2. Record the run ID and the affected deck positions.
3. Tell the relevant project lead.
4. Decontaminate before the instrument is used again.

Discard before diagnosing

When a run fails or cultures behave unexpectedly, discard the replaceable materials that could connect that run to future experiments: reservoirs, working stocks, media, antibiotics, prepared plates, affected cultures.

The investigation and the cleanup happen in parallel, not in sequence.

Document every biological run

Record:

• The identity and source of the biological materials
• What went on the deck
• The deck layout
• The tip-use strategy
• Any deviations from the planned protocol
• Any spills, collisions, or handling failures
• Any unexpected culture behavior
• The decontamination performed afterward

This is what makes a retrospective investigation possible. A run that was not documented cannot be investigated, and the usual result is that weeks of unrelated work get discarded because nobody can rule it out.