What is Dr. Zubair Khalid's research focus?

Dr. Zubair Khalid specializes in molecular virology, mRNA vaccine development, and computational biology, with a focus on avian pathogens like IBDV and Avian Reovirus.

Where is Dr. Zubair Khalid currently working?

Dr. Zubair Khalid is a Postdoctoral Research Associate at the University of Maryland (UMD), specifically within the Department of Animal and Avian Sciences.

Mycoplasma Testing in Cell Culture: Methods and Best Practices

By Dr. Zubair Khalid, DVM, MS, PhD · 2026-08-11

Introduction to Mycoplasma Testing

What Are Mycoplasmas?

Mycoplasmas are the smallest self-replicating prokaryotes known to science, belonging to the class Mollicutes. Unlike typical bacteria, they lack a rigid cell wall, which renders them resistant to many common antibiotics such as penicillin, streptomycin, and other β-lactams that target peptidoglycan synthesis. Their genome is drastically reduced—ranging from 580 to 1,400 kilobase pairs—and they depend on a parasitic lifestyle, scavenging precursors such as amino acids, nucleotides, and lipids from their host environment. In cell culture, mycoplasmas grow attached to the exterior of the plasma membrane or in the extracellular space, where they compete with the cultured cells for nutrients and release metabolic byproducts, including ammonia and hydrogen peroxide.

Eight species account for the vast majority of cell culture contaminants: Mycoplasma arginini, M. hyorhinis, M. orale, M. fermentans, M. hominis, M. pirum, M. salivarium, and Acholeplasma laidlawii. These species are fastidious, requiring sterols (typically supplied as serum) for growth, and they replicate slowly, with doubling times of 1 to 6 hours. Because they do not produce the turbidity or pH shifts that characterize bacterial or fungal contamination, mycoplasma infections can persist silently for weeks or months, spreading throughout a laboratory before being detected.

Why Testing Is Essential

Mycoplasma contamination is a pervasive problem in cell culture laboratories, with historical surveys reporting infection rates of 15% to 35% in continuous cell lines. The consequences are not merely cosmetic. Mycoplasmas alter nearly every measurable aspect of cell biology: they deplete arginine and other amino acids, induce chromosomal aberrations, modulate signal transduction pathways, and change the expression of hundreds of genes. For a graduate student or postdoc, an undetected infection can invalidate months of experiments, produce irreproducible data, and compromise the integrity of cell lines shared with collaborators. Testing is therefore not a one-time validation but a continuous quality-control measure that underpins the reliability of all downstream work. The Mycoplasma Contamination PCR method, described later, is a cornerstone of modern detection strategies.

Sources and Routes of Mycoplasma Contamination

Common Contamination Sources

Mycoplasmas enter cell culture through several well-characterized routes. The most frequent source is contaminated cell lines themselves. When a new line arrives from another laboratory, a repository, or a collaborator, it may already harbor mycoplasmas without showing any visible signs. This is particularly dangerous because the contamination is silently propagated during every passage.

A second major source is animal-derived reagents. Fetal bovine serum (FBS) is a classic vehicle; although commercial suppliers filter serum through 100-nm pore membranes, some mycoplasma species can survive or be introduced post-filtration. Trypsin solutions prepared from porcine pancreas have also been implicated, as have other biological additives such as growth factors, cytokines, and conditioned media. Water baths are another notorious reservoir: mycoplasmas thrive in the warm, humid environment, and droplets from a water bath can land on flasks, pipettes, or gloves, transferring organisms into cultures.

Laboratory personnel are a third source. Humans naturally harbor M. orale, M. salivarium, and M. fermentans in the respiratory and urogenital tracts. Talking, coughing, or breathing over an open flask can aerosolize these organisms. Poor aseptic technique—such as touching the inside of a flask neck, using shared media bottles without proper sterilization, or failing to change gloves after handling contaminated materials—provides a direct route of entry.

Cross-Contamination Risks

Once a single culture is infected, mycoplasmas spread rapidly within a laboratory. The primary mechanism is aerosolization during pipetting, centrifugation, or disposal of spent media. Mycoplasmas are small enough (0.2 to 0.3 µm in diameter) to pass through standard 0.22-µm filters used for sterilizing media, meaning that a contaminated solution can contaminate a fresh batch of media even if filtered. Shared equipment—laminar flow hoods, incubators, centrifuges, and even the same bottle of medium used for multiple cell lines—serves as a vector for cross-contamination.

The risk is amplified by the fact that mycoplasmas can survive for extended periods on surfaces, including stainless steel, plastic, and glass, particularly in the presence of organic material such as serum. They are resistant to drying and can persist in biofilms. Consequently, a single contaminated culture in a busy laboratory can compromise every other line within weeks. This is why routine testing of all cultures, not just newly acquired ones, is essential for containment.

Biological Impact of Mycoplasma Infection

Effects on Cell Metabolism

Mycoplasmas exert their effects through multiple concurrent mechanisms. The most well-characterized is arginine depletion. Several species, including M. arginini and M. hominis, express the enzyme arginine deiminase, which converts arginine to citrulline and ammonia. Since arginine is an essential amino acid for many cultured cells, its depletion arrests cell growth in the G1 phase of the cell cycle and induces apoptosis in sensitive lines. This is particularly problematic for experiments involving cell proliferation, differentiation, or metabolic flux.

In addition to arginine catabolism, mycoplasmas consume other nutrients—glucose, glutamine, uridine, and thymidine—competing directly with the host cells. They also produce hydrogen peroxide and superoxide radicals as byproducts of their own metabolism, creating oxidative stress that damages host DNA, proteins, and lipids. The accumulation of ammonia in the culture medium further shifts the pH and osmolarity, creating a suboptimal environment that can subtly alter gene expression even before visible effects on growth are apparent.

Mycoplasmas also modulate host cell signaling. For example, mycoplasmal lipoproteins and membrane components can activate Toll-like receptors (TLRs), particularly TLR2 and TLR4, triggering NF-κB signaling and the production of pro-inflammatory cytokines such as IL-6 and TNF-α. In immune cells, this can skew differentiation and activation states. In epithelial or fibroblast lines, it can activate stress-response pathways, including the unfolded protein response and the p38 MAPK cascade, leading to changes in the expression of genes involved in apoptosis, proliferation, and adhesion.

Impact on Experimental Reproducibility

The biochemical changes induced by mycoplasma infection translate directly into experimental irreproducibility. Consider a typical experiment measuring the transcriptional response to a drug. In an infected culture, the baseline expression of hundreds of genes is already altered—some upregulated as part of the stress response, others downregulated due to nutrient depletion. The drug's effect is then superimposed on this distorted baseline, producing results that differ from those obtained in a clean culture. The magnitude of the effect can be large enough to reverse the direction of a response, turning an apparent induction into a repression.

Functional assays are equally affected. Mycoplasma contamination alters cell proliferation rates, migration, invasion, and apoptosis. It can change the surface expression of receptors, including integrins and growth factor receptors, confounding flow cytometry and binding studies. In transfection experiments, infected cells often show reduced transfection efficiency and altered reporter gene expression. Even protein purification and biochemical assays are compromised, as mycoplasmal proteases can degrade target proteins, and mycoplasmal nucleic acids can contaminate DNA or RNA preparations, leading to spurious results in sequencing or qPCR.

The insidious nature of mycoplasma infection is that these effects are often quantitative rather than qualitative. The cells may look healthy under the microscope, grow at a normal rate, and pass routine viability checks. Only when the data are compared across experiments—or between laboratories—does the inconsistency become apparent. This is why testing is not merely a matter of quality assurance but a fundamental requirement for scientific validity.

Mycoplasma Testing Methods

Culture-Based Detection

Culture-based detection is the oldest and most definitive method for mycoplasma testing. It relies on the ability of mycoplasmas to form visible colonies on agar medium. The standard protocol uses a complex medium, such as Hayflick's medium, which contains beef heart infusion, peptone, yeast extract, and 20% horse serum, supplemented with penicillin (1000 U/mL) to suppress bacterial growth and amphotericin B (2.5 µg/mL) to suppress fungi. The medium is inoculated with a sample of cell culture supernatant or cells, then incubated at 37°C in an atmosphere of 5% CO₂ and 95% nitrogen for 14 to 28 days.

Mycoplasma colonies are small (10 to 100 µm in diameter) and have a characteristic "fried egg" appearance—a dense central zone of growth penetrating the agar, surrounded by a thinner peripheral zone spreading across the surface. This morphology is due to the organism's preference for growing into the agar rather than on top of it. The sensitivity of culture-based detection is relatively low, typically 10 to 100 colony-forming units (CFU) per milliliter, and the method is slow, requiring weeks before a definitive result is available. Moreover, some strains of M. hyorhinis are difficult to cultivate in vitro, leading to false negatives.

Despite these limitations, culture-based detection remains the gold standard for confirming the presence of viable mycoplasmas and for validating other detection methods. It is also the only method that can provide a live isolate for further characterization or for testing antibiotic sensitivity.

PCR-Based Detection

PCR-based detection has become the method of choice for routine mycoplasma testing due to its speed, sensitivity, and specificity. The approach targets conserved regions of the mycoplasmal genome, most commonly the 16S rRNA gene, which contains both highly conserved and variable regions. Primers are designed to anneal to conserved sequences flanking a variable region, allowing the amplification of a broad range of mycoplasma species while excluding other bacteria.

A typical PCR assay uses a primer set that amplifies a 280- to 500-base-pair fragment of the 16S rRNA gene. The reaction mixture contains 1× PCR buffer, 1.5 to 2.5 mM MgCl₂, 200 µM each dNTP, 0.2 to 0.5 µM each primer, 1 to 2.5 units of Taq DNA polymerase, and 1 to 5 µL of template DNA in a total volume of 25 to 50 µL. The thermal cycling protocol typically includes an initial denaturation at 95°C for 5 minutes, followed by 35 to 40 cycles of denaturation at 95°C for 30 seconds, annealing at 55 to 60°C for 30 seconds, and extension at 72°C for 30 to 60 seconds, with a final extension at 72°C for 5 to 10 minutes. The amplified products are then visualized by agarose gel electrophoresis or, more sensitively, by real-time PCR using SYBR Green or TaqMan probes.

Real-time PCR offers several advantages over conventional PCR. It provides quantitative data, allowing the estimation of the mycoplasma load in the sample. It is also more sensitive, with detection limits as low as 1 to 10 genome copies per reaction, and it eliminates the need for post-PCR processing, reducing the risk of contamination. The PCR Testing page provides a broader overview of PCR principles, while the Mycoplasma Contamination PCR page details the specific application to cell culture.

ELISA and Immunodetection

Enzyme-linked immunosorbent assay (ELISA) and other immunodetection methods rely on antibodies that recognize mycoplasmal antigens. These assays detect the presence of mycoplasma proteins or lipoglycans in cell culture supernatant, cell lysates, or conditioned medium. The most common format is a sandwich ELISA, in which a capture antibody is immobilized on a microtiter plate, the sample is added, and a detection antibody conjugated to an enzyme (such as horseradish peroxidase) is then applied. A chromogenic substrate produces a color change proportional to the amount of mycoplasma antigen present.

ELISA kits are commercially available and are designed to detect the most common cell culture contaminants, including M. arginini, M. hyorhinis, M. orale, and A. laidlawii. The sensitivity of ELISA is generally lower than that of PCR, with detection limits around 10⁴ to 10⁵ CFU/mL. However, ELISA is simple, rapid (results in 2 to 4 hours), and does not require specialized equipment beyond a plate reader. It is well suited for high-throughput screening of many samples, but its limited sensitivity means that early-stage infections may be missed.

A related approach is immunoblotting or dot blot, in which cell lysates are spotted onto a membrane and probed with anti-mycoplasma antibodies. This method is less quantitative than ELISA but can be useful for confirming positive results or for detecting mycoplasmas in samples that are difficult to analyze by other means.

Fluorescent Staining

Fluorescent staining is a direct visualization method that detects mycoplasmas in cell cultures using DNA-binding fluorochromes. The most commonly used dye is Hoechst 33258, which binds to the A-T-rich regions of DNA. When a cell culture is stained with Hoechst 33258 and examined under a fluorescence microscope, the nuclei of the cultured cells appear as large, brightly stained structures. In an infected culture, mycoplasmas appear as small, punctate, extranuclear fluorescent bodies—either attached to the cell surface or dispersed in the extracellular space.

The staining protocol is straightforward. Cells are grown on coverslips or in chamber slides, fixed with a 3:1 methanol-acetic acid mixture for 10 minutes at room temperature, and then stained with Hoechst 33258 at a concentration of 0.05 to 0.1 µg/mL in phosphate-buffered saline (PBS) for 10 to 15 minutes. After washing, the coverslips are mounted and examined under a fluorescence microscope with a UV excitation filter (340 to 380 nm) and an emission filter (430 nm or above).

Fluorescent staining is rapid and inexpensive, but it has several limitations. It requires experience to distinguish mycoplasmas from cellular debris, mitochondrial DNA, or other artifacts. The sensitivity is moderate, typically detecting infections at a load of 10⁵ to 10⁶ CFU/mL. Moreover, the method is subjective and can produce false negatives if the infection is low-grade or if the mycoplasmas are not uniformly distributed. It is best used as a screening tool or as a complement to more definitive methods such as PCR or culture.

Choosing the Right Testing Method

Sensitivity and Specificity

The choice of mycoplasma testing method depends on the specific application, the required sensitivity, and the available resources. The table below summarizes the key characteristics of the main methods.

Method	Sensitivity (CFU/mL)	Time to Result	Specificity	Cost per Sample	Best Use
Culture-based	10–100	14–28 days	High (species-level)	Moderate	Gold standard, confirmatory testing
PCR (conventional)	10–100	4–6 hours	High (genus-level)	Low–Moderate	Routine screening, validation
PCR (real-time)	1–10	2–4 hours	High (genus-level)	Moderate	Quantitative screening, high sensitivity
ELISA	10⁴–10⁵	2–4 hours	Moderate (species-specific)	Low	High-throughput screening
Fluorescent staining	10⁵–10⁶	1–2 hours	Low (non-specific)	Very low	Quick screening, educational use

For routine monitoring of cell lines, real-time PCR is generally recommended because it offers the best combination of sensitivity, speed, and quantitative information. It can detect a single genome copy per reaction, which corresponds to a contamination level that is clinically and experimentally significant. Conventional PCR is a reasonable alternative if real-time instrumentation is unavailable, but it is slightly less sensitive and requires post-PCR processing.

Culture-based detection, while slow, remains the definitive method for confirming a positive PCR result and for isolating the organism for further study. It is also the only method that can distinguish viable from non-viable mycoplasmas, which is important when evaluating the effectiveness of eradication treatments.

Throughput and Cost Considerations

Throughput and cost are practical considerations that often dictate the testing strategy. For a laboratory with a small number of cell lines (fewer than 20), conventional PCR or real-time PCR is cost-effective, as the reagents are relatively inexpensive and the labor is minimal. For larger laboratories or core facilities that screen hundreds of samples per month, ELISA offers a lower per-sample cost and can be automated for high-throughput screening. However, the lower sensitivity of ELISA means that it should be used in conjunction with PCR for confirmatory testing.

The cost of commercial mycoplasma detection kits varies widely. PCR kits typically cost $5 to $15 per sample, while ELISA kits cost $3 to $8 per sample. Culture-based detection is the most expensive when labor and incubation time are factored in, often exceeding $50 per sample. For most laboratories, a tiered approach is optimal: use real-time PCR for routine screening and validation, and reserve culture-based detection for confirming positive results or for troubleshooting persistent contamination.

Sampling and Sample Preparation

When to Test

The frequency of mycoplasma testing depends on the risk profile of the laboratory and the criticality of the experiments. As a general rule, all new cell lines should be tested immediately upon arrival, before they are introduced into the main culture room. This includes lines from commercial repositories, collaborators, and other laboratories. A negative test at this stage is not sufficient to declare the line clean, as the infection may be below the detection limit; therefore, the line should be quarantined and tested again after at least two passages.

For established cell lines, testing should be performed at least once a month, and more frequently (weekly or biweekly) if the laboratory handles many lines, uses shared equipment, or has experienced a contamination event. Critical experiments—such as those intended for publication, drug screening, or clinical translation—should be preceded by a mycoplasma test on the exact batch of cells to be used. Additionally, any cell line that shows unexplained changes in growth rate, morphology, or experimental results should be tested immediately.

Sample Handling and Storage

Proper sample collection is essential for reliable results. For PCR-based testing, the sample can be either cell culture supernatant or a cell pellet. Supernatant is easier to collect and is sufficient for detecting mycoplasmas that are released into the medium. However, some mycoplasmas adhere tightly to the cell surface, so a cell pellet provides a more representative sample. The recommended approach is to collect both: centrifuge 1 to 2 mL of culture medium at 200 × g for 5 minutes to pellet the cells, then transfer the supernatant to a fresh tube and retain the cell pellet.

For DNA extraction, commercial kits based on silica membrane binding or magnetic bead technology are recommended, as they provide consistent yields and remove PCR inhibitors. The extraction should include a proteinase K digestion step (typically 20 mg/mL proteinase K in a lysis buffer containing 10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 25 mM EDTA, and 0.5% SDS) at 56°C for 30 minutes, followed by heat inactivation at 95°C for 10 minutes. The final DNA should be eluted in 30 to 50 µL of elution buffer or nuclease-free water.

Samples that cannot be processed immediately should be stored at −20°C for short-term storage (up to 1 week) or at −80°C for long-term storage. Repeated freeze-thaw cycles should be avoided, as they can degrade DNA and reduce sensitivity. For culture-based detection, the sample must be processed fresh, as freezing kills the mycoplasmas and prevents colony formation.

Interpreting Mycoplasma Test Results

Understanding Positive and Negative Results

A positive result in any mycoplasma test indicates the presence of mycoplasma DNA, antigen, or viable organisms in the sample. In PCR-based testing, a positive result is typically defined by the appearance of an amplification product of the expected size on a gel, or by a threshold cycle (Ct) value below a cutoff (e.g., Ct < 35) in real-time PCR. The strength of the signal correlates with the mycoplasma load, but it does not distinguish between viable and non-viable organisms. A positive PCR result should therefore be confirmed by a second method, such as culture-based detection or a different PCR primer set, to rule out contamination of the reagents or the sample.

A negative result indicates that no mycoplasmas were detected under the conditions of the assay. However, a negative result is not an absolute guarantee of cleanliness. The sensitivity of the method, the quality of the sample, and the presence of PCR inhibitors can all contribute to false negatives. For example, if the sample contains high levels of cellular DNA, it can inhibit the PCR reaction, leading to a false negative. To control for this, a spike-in control—adding a known amount of mycoplasma DNA to a parallel reaction—should be used to verify that the PCR is not inhibited.

Confirmatory Approaches

When a positive result is obtained, it is essential to confirm the finding before taking drastic action such as discarding the cell line. The first step is to repeat the test on a fresh sample, ideally using a different method. If the initial test was a PCR, confirm with a culture-based assay or an ELISA. If the initial test was an ELISA, confirm with PCR. This dual-method approach reduces the risk of false positives due to reagent contamination or cross-reactivity.

If the positive result is confirmed, the next step is to identify the species of mycoplasma. This can be done by sequencing the PCR product or by using species-specific PCR primers. Knowing the species is important for selecting an appropriate eradication strategy, as some species are more susceptible to certain antibiotics than others. It also helps trace the source of contamination, as certain species are associated with specific sources (e.g., M. orale from human oral flora, M. hyorhinis from porcine trypsin).

Prevention and Eradication Strategies

Preventive Measures

Prevention is the most effective strategy for managing mycoplasma contamination. The cornerstone of prevention is a strict aseptic technique. This includes working in a laminar flow hood that is certified and regularly maintained, using sterile pipettes and tips, and never touching the inside of flasks or bottle caps. All media and reagents should be purchased from reputable suppliers and tested for mycoplasma before use, particularly serum and trypsin. Antibiotics should not be used as a substitute for good technique, as they can mask low-level contamination and select for resistant strains.

A quarantine protocol for new cell lines is essential. New lines should be maintained in a separate incubator or a dedicated area of the laboratory until they have tested negative for mycoplasma on at least two consecutive occasions, separated by at least two passages. During quarantine, the lines should be handled with dedicated media and equipment, and the operator should use gloves and a lab coat that are not used for other cultures.

Regular testing is a preventive measure in itself. By detecting contamination early, before it spreads, the laboratory can isolate and eliminate the source. Testing should be documented, and the results should be reviewed periodically to identify trends or recurring issues.

Quarantine and Treatment Options

If a cell line is found to be contaminated, the safest course of action is to discard it and replace it with a clean stock from a repository. This is particularly recommended for valuable or irreplaceable lines, as treatment is not always successful and can introduce additional stress to the cells.

For lines that must be saved, antibiotic treatment is the most common approach. The most widely used regimen involves a combination of a macrolide (e.g., clarithromycin at 10 µg/mL), a quinolone (e.g., ciprofloxacin at 10 µg/mL), and a tetracycline (e.g., doxycycline at 10 µg/mL). These antibiotics are added to the culture medium and the cells are treated for 14 to 21 days. The treatment should be performed in a quarantine area, and the cells should be tested weekly during and after treatment to monitor the effectiveness.

It is important to note that antibiotic treatment does not guarantee eradication. Mycoplasmas can develop resistance, and some organisms may survive in a non-replicating state. Moreover, the antibiotics themselves can have toxic effects on the cells, altering their phenotype and gene expression. After treatment, the cells should be cultured for at least two weeks in antibiotic-free medium and tested again to confirm that the contamination has been eliminated. If the treatment fails, the line should be discarded.

Common Pitfalls in Mycoplasma Testing

Infrequent Testing

The most common mistake in mycoplasma testing is not testing often enough. Many laboratories test only when a problem is suspected, by which time the contamination may have spread to multiple lines and compromised months of experiments. Routine testing—at least monthly for established lines and upon arrival for new lines—is the only way to catch contamination early. Infrequent testing is particularly dangerous in shared laboratories, where a single contaminated line can silently infect others.

Method Limitations

Each testing method has its limitations, and relying on a single method can lead to false confidence. For example, fluorescent staining is quick and inexpensive, but its low sensitivity means that early-stage infections are easily missed. ELISA is suitable for high-throughput screening but may not detect low-level contamination. Even PCR, the most sensitive method, can produce false negatives if the sample is poorly prepared or if PCR inhibitors are present. The best practice is to use a combination of methods, with PCR as the primary screen and culture-based detection as the confirmatory test.

Misinterpretation of Results

Misinterpreting test results is another common pitfall. A negative result is often taken as proof that the culture is clean, but it only indicates that no mycoplasmas were detected under the specific conditions of the assay. A positive result, on the other hand, is sometimes dismissed as a false positive without confirmatory testing. Both responses are problematic. A negative result should be interpreted in the context of the method's sensitivity and the quality of the sample, while a positive result should always be confirmed before taking action. Additionally, borderline results—such as a faint band on a gel or a Ct value near the cutoff—should be repeated and investigated, not ignored.

Frequently Asked Questions

What is mycoplasma testing?

Mycoplasma testing is the process of detecting the presence of mycoplasmas in cell cultures, reagents, or biological samples. It is a critical quality-control measure in cell culture laboratories, as mycoplasma contamination can alter experimental results without producing visible signs.

Why is mycoplasma testing important?

Mycoplasma contamination is common and often silent. Infected cultures can produce irreproducible data, altered gene expression, and compromised experimental outcomes. Testing is essential for ensuring the validity of research and for preventing the spread of contamination within a laboratory.

What are the common mycoplasma testing methods?

The main methods are culture-based detection, PCR (conventional and real-time), ELISA, and fluorescent staining. Each method has its own sensitivity, specificity, cost, and time requirements. PCR is the most sensitive and widely used method for routine testing.

How often should I test for mycoplasma?

New cell lines should be tested immediately upon arrival and again after at least two passages. Established lines should be tested at least once a month, and more frequently in high-risk environments. Critical experiments should be preceded by a test on the exact batch of cells to be used.

Can mycoplasma be eliminated from cell cultures?

Yes, but eradication is not guaranteed. Antibiotic treatment with a combination of macrolides, quinolones, and tetracyclines can be effective, but it may fail due to resistance or non-replicating organisms. The safest approach is to discard contaminated lines and replace them with clean stocks.

What is the most sensitive mycoplasma detection method?

Real-time PCR is the most sensitive method, with detection limits as low as 1 to 10 genome copies per reaction. It is also quantitative, allowing the estimation of the mycoplasma load in the sample.

How do I collect a sample for mycoplasma testing?

For PCR-based testing, collect 1 to 2 mL of cell culture supernatant and a cell pellet. Centrifuge the sample at 200 × g for 5 minutes, transfer the supernatant to a fresh tube, and retain the cell pellet. Extract DNA using a commercial kit and store the sample at −20°C or −80°C if not processed immediately.

Key Takeaways

Mycoplasma contamination is a common, silent problem in cell culture that can invalidate experimental results and compromise reproducibility.
The main sources of contamination are contaminated cell lines, animal-derived reagents, and laboratory personnel; cross-contamination spreads rapidly through aerosols and shared equipment.
Mycoplasmas alter cell metabolism, gene expression, and signaling pathways, leading to quantitative changes in experimental outcomes that are often invisible under the microscope.
Real-time PCR is the most sensitive and recommended method for routine mycoplasma testing, with culture-based detection serving as the gold standard for confirmation.
Test new cell lines upon arrival and established lines at least monthly; always test the exact batch of cells before critical experiments.
Proper sample collection, including both supernatant and cell pellet, and the use of spike-in controls are essential for reliable results.
Prevention through strict aseptic technique, quarantine of new lines, and regular testing is the most effective strategy; antibiotic treatment is a last resort and does not guarantee eradication.