The short answer
A selective medium relies on inhibitors (bile salts, dyes, antibiotics, high salt, tellurite) to hold back unwanted organisms and on indicators to make the target visible. Failure comes in two forms. "The wrong organisms grew" usually means the inhibitor has lost strength: over-sterilisation, plates stored too long, or a post-sterilisation supplement that was never added. "The right organism did not grow" usually means inhibition is too strong or nutrition too weak: supplement overdosed, plates too dry, or target cells that were injured to begin with. In both cases a parallel test with reference strains settles within a day whether the medium or the sample is at fault.
Wrong organisms growing: the inhibitor has failed
- Over-sterilisation. Bile salts, crystal violet, brilliant green and neutral red are heat-sensitive; 121 °C for 15 minutes is the maximum, and re-autoclaving or long heating of large bottles weakens them. Media whose instructions say "boil to dissolve, do not autoclave" (m-Endo, some chromogenic media) must never go in the autoclave.
- Supplement missing or wrong. Baird-Parker needs egg yolk tellurite, MYP needs polymyxin, Oxford needs its antibiotic supplement. The base medium alone has no selectivity; leave the supplement out and everything grows.
- Stored too long after preparation. Plates containing dyes and antibiotics lose inhibition after one to two weeks refrigerated; once an antibiotic supplement is added the shelf life is shorter still. Follow the instructions.
- pH off. At low pH the bile salts in MacConkey inhibit less; if pH falls too far after sterilisation, revisit the preparation.
- Inoculum too heavy. With a very high load, inhibitors reduce rather than eliminate; background flora still comes through. Dilute before plating.
Right organism not growing: too much inhibition or too little nutrition
- Supplement overdosed. Double the antibiotic or tellurite and the target is suppressed too. Dose by measurement, not "about a few drops".
- Plates too dry or too old. Injured target cells cannot establish on a dehydrated surface, which gives background flora its chance.
- Injured target cells. Samples that have been heated, frozen or acidified contain cells that must first recover in a non-selective medium or enrichment broth; plated straight onto a selective medium they are killed by the inhibitor. This is why test methods include pre-enrichment.
- Incubation conditions. Wrong temperature, time or atmosphere (vibrios and campylobacters each have their requirements) and the target does not grow through no fault of the medium.
Deciding with reference strains
Every selective medium has defined performance-test strains: targets that should grow with the correct reaction and non-targets that should be inhibited. For example:
| Medium | Should grow | Should be inhibited |
|---|---|---|
| MacConkey Agar | E. coli (red), Salmonella (colourless) | S. aureus, Enterococcus |
| Baird-Parker | S. aureus (black with clear halo) | E. coli, Bacillus |
| XLD | Salmonella (red with black centre) | E. coli (yellow), S. aureus |
| TCBS | Vibrio parahaemolyticus (green), V. cholerae (yellow) | E. coli |
| Cetrimide | Pseudomonas aeruginosa (fluorescent) | E. coli, S. aureus |
Method: inoculate freshly prepared medium and the suspect medium with both target and non-target strains, incubated alongside the sample. If non-targets grow on the suspect plates, the medium has failed; if the reference strains behave normally and only the sample is odd, the problem is the sample. ISO 11133 sets out this test in full, and laboratory quality systems usually require it for every lot of medium.
When to remake
- Reference-strain test fails: discard the batch, remake, and find the cause (sterilisation record, supplement lot and dose, water).
- Plates past their storage limit, cracked or dehydrated surfaces, unusual colour: do not use.
- Powder caked or discoloured after opening: see Can caked or discoloured dehydrated medium still be used.
FAQ
What is the difference between a base and a complete medium? A "base" still needs a supplement (egg yolk, blood, antibiotic, tellurite) added after sterilisation because those components do not survive heat. Any product with "Base" in its name is this type, and the instructions state what to add, how much and at what temperature.
The colours on my chromogenic medium are faint. Is the medium at fault? First check incubation time (most colour reactions need a full 24 hours) and whether the plates were stored in the dark (chromogenic substrates break down in light). If both are fine, test with reference strains.
Can I add antibiotics myself to make a selective medium? Yes, but the type, concentration and addition temperature must follow the method, and the shelf life afterwards is short. Where the method specifies a supplement, a commercial supplement is more consistent.
Do STBIO selective media come with performance data? Every lot ships with a COA including cultural response against reference strains; a full performance-test report is available on request. Each product page also states which official method the medium belongs to and in which step; see find media by standard method.