LB and other media as carbon and salt

LB is a rich, undefined broth, and the decision it forces is which carbon-and-salt class you actually mean before you blame a cloning strain for growing oddly. The hero photograph on this page is an analytical instrument with sample vials. It is not a picture of powder, and it should not be read as one. How that broth is used to grow and transform non-pathogenic E. coli is set out in bacterial culture and transformation. The antibiotic you stir in after cooling is explained in antibiotic selection and blue-white screening. The plasmid that will live in the flask is plasmid cloning from insert to colony.

Powders and agar bases are specified from the reagents and chemicals catalogue. The method context is the molecular biology pathway. If the gene you will express has special medium needs, say so through the custom gene synthesis reference as an enquiry. The specification goes to the quote request. A method can be discussed.

What the three powders are doing

Classic LB, whatever expanded name a label prefers, is built from three classes of ingredient. Tryptone is an enzymatic digest of casein. It supplies peptides and amino acids, which is a carbon source and a nitrogen source at once, and it saves the cell from synthesising every amino acid. Yeast extract supplies more amino acids, vitamins, and some carbohydrate. Sodium chloride supplies salt. Together they make a medium in which a K-12 cloning strain can double quickly because the hard biosynthetic work is already done.

That speed is the point of a rich medium, and it is also the limit. The exact sugars and peptide lengths vary with the lot of hydrolysate. Two tins that both say LB can shift lag length, final density and even how a fussy plasmid behaves. You cannot write a carbon balance on an undefined broth. You can write which recipe class you poured, and you can stop changing tins in the middle of a comparison.

Salt is the ingredient people forget is a variable. A widely used Miller-style LB is the saltier end of the family. Lennox-style LB carries less sodium chloride. A low-salt version carries less again. The cells grow in all three. Some antibiotics do not work the same way in all three. Zeocin-type selection is the usual teaching example of a drug that wants the low-salt end. High salt can also follow cells into a wash if you are making electrocompetent cells: the broth is allowed to be salty, the cuvette is not. Rinsing has to remove what the recipe put in. Naming "LB" without the salt level hides that fork.

pH, water and the autoclave, briefly

Salt and pH are why a broth is a little like a buffer even when nobody calls it one. Preparing a buffer and checking pH is the companion if you are adjusting pH before you sterilise. Rich media are often close to neutral after dissolving. Do not titrate by habit with a strong acid you have not recorded. The water you dissolve the powder in is part of the salt you think you weighed. Laboratory water types and where they fail is the right note if the still or the polisher is the suspicious reagent. NIST sits behind the idea that a gram and a pH unit are measurements, not moods.

Sterilise the medium, cool it until you can hold the bottle, then add heat-labile antibiotics. Agar is the same liquid plus a gelling agent, poured after that cooling step. Adding the drug before the autoclave is how a selective label ends up on a rich plate. The Addgene protocol for inoculating a culture assumes the broth you start from is the broth you named. Keep that assumption true.

SOB, SOC and a defined alternative

SOB is a different rich class. It is still undefined, built for higher cell densities, and it changes the salt pattern: less sodium chloride than salty LB, plus potassium and magnesium salts. People use it when they want a dense culture or as the base of a recovery medium. It is not "LB with a nickname".

SOC is SOB plus a small amount of glucose, used for the recovery hour after transformation. The sugar gives wounded cells an easy carbon source while they express the resistance protein. It is a short-term medium. Leaving a culture in SOC overnight, still without antibiotic, is how you grow biomass that may already be losing the plasmid. Recover, then plate on selective agar, or dilute into selective broth if the method says so.

Defined media, M9 plus a stated carbon source and any supplements the genotype needs, are the opposite bargain. You know the carbon source. You can label it, or you can starve a pathway on purpose. Growth is slower. An auxotroph will not grow at all until you add what the genotype demands. That failure is informative in a defined medium and invisible in LB, because the yeast extract already fed it. Choose defined medium when the question is metabolic. Choose rich medium when the question is "please copy this plasmid".

ATCC culture guides list the medium a collection strain expects. Follow that note for a strain you received from a collection. Do not assume your house LB is what the strain sheet named.

What you gain and what you stop controlling

When the medium, not the clone, changed the result

A plasmid prep that suddenly yields less DNA after a new tin of broth is a medium story until proven otherwise. So is an antibiotic that used to select and now produces a lawn, if the new tin is a saltier LB and the drug was salt-sensitive. Write the salt version in the notebook the way you write the drug. A competent-cell prep that starts arcing after you "used LB as usual" may be salt left from a saltier recipe plus a shorter wash. The fix is the wash and the named recipe, not a new strain.

A defined-medium culture that does not grow at all is often a missing supplement, not a dead plasmid. Check the genotype for auxotrophies before you repeat the transformation. Rich medium would have hidden the same genotype inside yeast extract.

The Addgene molecular biology reference is a public place to see these recipe names used consistently. Use it to stabilise your own naming.

Powders are still chemicals

Tryptone and yeast extract are not kitchen ingredients in a laboratory sense. Weigh them as the method states, and keep them closed. Antibiotics added later have their own hazards. This explainer is about media for non-pathogenic cloning strains your institution has already accepted. It is not a recipe for growing an unapproved organism, and it is not food advice. Waste broth is inactivated by the existing route. The WHO Laboratory Biosafety Manual is a reference for the people who own that route.

Humidity, water and an autoclave that stopped

Open tubs of tryptone and yeast extract take up moisture in a humid season. A damp scoop does not weigh what a dry scoop weighs, and a wet powder can cake or grow mould. Close the tub. Do not leave a spoon in the tin. If the laboratory water supply changes, because a still failed or a tank was refilled from a different source, the salt and the pH of "the same" recipe move. Remeasure. A power cut during an autoclave cycle is an unfinished cycle. Do not add antibiotic to a bottle that never reached temperature and call it sterile medium. Start the cycle again from a bottle you trust, or discard it according to the autoclave rule your laboratory already uses.

What to put in the enquiry

Name the class: Miller-style LB, Lennox-style LB, low-salt LB, SOB, SOC, or a defined base with the carbon source written out. Say broth or agar. Say which antibiotic will be added after cooling, and the water grade. Ask for the written composition, including grams of sodium chloride, so two suppliers' "LB" can be compared as recipes. Use the quote request. A method can be discussed. The custom gene synthesis reference belongs beside that request only when the construct's medium needs are part of the design question. It does not mean a gene is already being made.