Autoclaving media and antibiotic timing

The words on a media bench get swapped until a plate looks selective and is not. This glossary fixes the sequence for non-pathogenic E. coli cloning strains: steam-sterilise the base medium, cool it, then add the antibiotics that heat destroys. The decision it supports is whether a bottle or a plate ever had a chance to be both sterile and selective. Growing the strain and reading a colony sit in bacterial culture and transformation. What a steam cycle does not prove is the subject of autoclaves and what sterilisation does not do.

The hero image on this page is a mass spectrometer and sample vials. It is not a picture of an autoclave. The work described here is the medium that later becomes a culture, not an instrument method. Medium components and antibiotic classes are in the reagents and chemicals catalogue. The cloning context is the molecular biology pathway. Design questions about the plasmid can be framed from the custom gene synthesis reference without implying that synthesis is already running. A specification for medium or a cycle goes through the quote request.

Terms worth pinning down

An autoclave is a pressure vessel that heats its load with saturated steam. Pressure is what lets water sit at sterilising temperature instead of boiling away. A condition often described for microbiological media is 121 Celsius for a hold long enough for the centre of that particular vessel. A large bottle needs longer than a small one. The hold starts when the load is at temperature, not when the panel first looks ready. Follow the machine, the load type and the medium method. This glossary is not a cycle you punch in.

A dry oven is hot air. It does not deliver the same moist heat, and it is the wrong machine to point at a bottle of broth because the two boxes both feel hot. Indicator tape changes colour on the outside of a pack when it has seen heat. It does not measure the middle of a two-litre bottle, and it says nothing about whether an antibiotic added later is still active.

Thermal death means organisms in the load are killed by the time and temperature the cycle was built to deliver, assuming steam actually reached them. It does not describe a bottle that was sterile at the end of the cycle and was contaminated when someone opened it over a dusty bench, poured with a non-sterile cylinder, or left the cap loose in a humid room. Those are handling events. They need aseptic habit, not a longer autoclave.

Caramelisation is the browning of sugars under hard heat, worse when peptides are present. The broth can look tea-coloured and can grow a cloning strain poorly. Glucose is the usual example. Many methods keep it out of the hard cycle and sterilise it separately, often through a filter of a sterilising pore class, then add it to cooled medium. Follow the recipe class you are actually making.

Heat-labile, for this page, means the antibiotic does not survive the autoclave. Ampicillin is the example every cloning lab learns. Other selective drugs are treated as heat-labile unless the sheet in your hand says that particular molecule tolerates the cycle. "It is an antibiotic" is not a stability class.

What meets the steam, and what waits

The base of a rich medium is a peptide source, yeast extract, salt, and, for plates, a gelling agent. Those go into the vessel in water of a grade your method accepts, with the cap loose enough for steam to move as the autoclave procedure requires. Sealed full bottles are a pressure hazard and a failed sterilisation. Liquids generally want a slow exhaust so they do not boil over. That setting belongs to the machine training, and the limits are expanded in the autoclave article linked above.

After the cycle, cool the agar or the broth. For plates, the practical window is while the agar is still a uniform liquid and the bottle is warm but touchable, a range often described as around 50 Celsius. Then add the antibiotic, swirl so the drug is not a single streak in the pour, and pour. If you are dosing broth, cool it below the temperature the sheet allows before the drug goes in. Swirl. Label the base of each plate with the drug, the date and the person. Lids are not a record.

A branch belongs here. Before you trust a new pour with a precious ligation, streak the empty cloning host, with no plasmid, onto one plate. It should not form colonies. Streak a strain you already know carries the marker. It should grow. If the empty host grows, the drug was omitted, cooked, or dead in the stock. Do not transform onto that stack. If the resistant strain fails too, you may have poured a different drug, overheated a drug that should have worked, or used a concentration class far above the one the marker can handle. The plasmid map and the antibiotic sheet set the working concentration. This page will not paste a house recipe.

Public method notes for starting a culture from a colony, which assume the medium was selective on purpose, include the Addgene protocol for inoculating a bacterial culture. Reagent classes used around that culture appear among Promega protocols. Neither replaces the sheet for the drug in your hand.

Add before, add after, keep out

Filter sterilisation is the partner technique for liquids that must not be steamed. A filter is a method class with a pore size the protocol names. It does not sterilise a flask you forgot to autoclave. It does not remove every toxin or every antibiotic. It is how a heat-sensitive solution becomes sterile enough for the use the protocol states, if the filter and the receiving bottle were themselves fit for that job.

Failures that wear the right label

Plates that grow the empty host were usually dosed too hot, dosed not at all, or poured from a stock that had already died in a warm freezer door. Broth that looks brown and grows slowly may have had its sugar in the autoclave. A bottle with a tight cap that "went through" the machine may never have reached temperature in the liquid. A film of growth in a bottle that was sterile last week is a handling story: the cap, the pour, the pipette that touched the bench and then the mouth.

Tape that turned black is not an antibiotic assay. A cycle printout is not an antibiotic assay. The empty-host streak is the assay that matches this glossary.

Safety stays on the institutional card

Steam, hot bottles and molten agar injure people without any bacterium involved. Training on the machine is a local requirement. Waste plates and leftover culture are inactivated the way the safety office requires. The WHO Laboratory Biosafety Manual is a public reference for that conversation. It does not set your cycle. This page is research-use explanation for cloning-strain media. It is not medical advice and not a permit to autoclave an organism the committee has not already assigned to that machine.

Power cuts, slow cooling, and a hot afternoon

A cycle that loses power before the hold is finished is an unfinished load. Do not add antibiotic and do not pour it as sterile. Run a cycle the procedure considers complete, or discard the load as the laboratory rule says. In a hot room, agar stays liquid for less long once you take it out, and people rush the drug in while the bottle is still too hot. Give the cooling its own line in the notebook. In a humid cold room, bottles sweat and labels slide off. Write the drug on the glass as well as on a tape that can fall. A delayed afternoon is not a reason to leave a half-dosed bottle "until tomorrow" without a label. Tomorrow's pour will be blamed on the strain.

What to put in a medium enquiry

Name the recipe class, whether you need agar or broth, the antibiotic and the concentration class, the volume class, and whether you will sterilise it yourself or you need it already sterile. Ask which components are expected to see the autoclave and which are added after cooling. The timing can be discussed against the antibiotic sheet and the machine you have. Send that note through the quote request. If the plasmid marker is still being chosen, the custom gene synthesis reference is a place to frame the DNA question, with the drug decision written beside it.