Contamination control
PCR of culture supernatant with primers to a conserved region of the mycoplasma 16S rRNA gene, read on a gel or by qPCR, with positive, water and internal amplification controls. Fast and sensitive, though it detects DNA rather than live organisms.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Mycoplasma PCR
PCR of culture supernatant with primers to a conserved region of the mycoplasma 16S rRNA gene, read on a gel or by qPCR, with positive, water and internal amplification controls. Fast and sensitive, though it detects DNA rather than live organisms.
How it differs from the standard method
- Sample 3+ days after feeding, near confluence, from cultures without anti-mycoplasma agents for two passages.
- Heat-treat the supernatant; use the volume the kit specifies.
- Set up in a pre-PCR area; add the positive control last.
- Alternatives: luminescent enzyme assays, DNA staining (Hoechst) of indicator cells, and culture on mycoplasma agar.
1Three ways a culture stops being what you think it is
A cell culture can be wrong in three different ways, and each needs its own test. Bacteria, yeasts and moulds usually announce themselves within days as cloudy, yellowed medium, unless antibiotics are holding them down; a formal sterility test of a cell bank looks for the few that do not.
Mycoplasma
Amplifies a conserved 16S rRNA region from culture supernatant; an internal control shows each reaction worked.
Identity
Lengths of short tandem repeats at eight or more loci give a profile matched against the line's reference.
Sterility
Sample inoculated into thioglycollate and soybean-casein digest broth and watched for turbidity.
2What each test actually measures
Mycoplasma PCR detects genomes shed into the supernatant, so the sample matters as much as the reaction: cultures left three or more days without a medium change, near confluence, and grown without anti-mycoplasma agents give the titre a chance to build up. Supernatant is heat-treated to release DNA and limit inhibitors, and every run needs a positive control (the PCR works), a water control (the reagents are clean) and an internal amplification control in each tube (that particular reaction was not inhibited). Amplicon carry-over from previous PCRs is the commonest cause of false positives.
3How the tests go wrong
- Sampling straight after feeding, or from cultures in anti-mycoplasma reagents: false negatives.
- Too few PCR cycles for a low-level infection, or raw supernatant inhibiting the reaction.
- Setting up PCR where gels are run: carry-over gives a band in every lane, including the water.
- Comparing an STR profile with the lab's own earlier profile, which may already have been of the wrong line.
- Profiling the clean bank vial instead of the working culture that was contaminated.
- A calling threshold below the stutter level (false mixtures) or so high that a minor contaminant is missed.
- Reading a sterility test after a few days, in one medium, or with antibiotic carried over from the culture.
Key terms
Practise these as flashcards →Wall-less bacteria (Mollicutes) that contaminate cell cultures without visible signs; unaffected by penicillin.
A template amplified in every tube to show that a negative is a real negative and not an inhibited reaction.
Contamination of new reactions with products of earlier PCRs, a common cause of false positives.
A locus of 2–6 bp repeats whose length varies between people; the basis of human cell line authentication.
Percentage match between two STR profiles: 2 × shared alleles ÷ (alleles in query + reference).
A minor PCR artefact one repeat shorter than the true allele, typically a few to ~10% of its height.
Inoculating each medium batch with a small number of reference organisms to show it supports growth.
Showing that the sample does not inhibit growth of spiked organisms (for example through carried-over antibiotic).
When to use it
- Every new line, in quarantine, before it shares an incubator with others.
- Before banking: mycoplasma, sterility and STR testing of the culture the bank is made from.
- Routinely every 1–3 months for mycoplasma, and at intervals and before publication for identity.
- Whenever cells behave unexpectedly or results stop reproducing.
Compared with alternatives
Prevention: the habits that keep contaminants out. Testing tells you whether they worked.
Mycoplasma detection is an endpoint PCR with a conserved target; the same rules on controls and carry-over apply.
Routine antibiotics in the medium hide contamination rather than prevent it.
Testing before a bank is frozen means every vial thawed from it starts clean and authentic.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
PCR of culture supernatant with primers to a conserved region of the mycoplasma 16S rRNA gene, read on a gel or by qPCR, with positive, water and internal amplification controls. Fast and sensitive, though it detects DNA rather than live organisms.
- Sample 3+ days after feeding, near confluence, from cultures without anti-mycoplasma agents for two passages.
- Heat-treat the supernatant; use the volume the kit specifies.
- Set up in a pre-PCR area; add the positive control last.
- Alternatives: luminescent enzyme assays, DNA staining (Hoechst) of indicator cells, and culture on mycoplasma agar.