Inter-laboratory and inter-operator differences can yield highly inconsistent—even opposite—results across the three methods. The principal cause is human operation. SOPs should be detailed yet not overcomplicated. Focus on critical checkpoints and avoid nonessential items (e.g., routine growth curves). A simple, structured approach can reduce operator burden and errors: always include a negative control; check every 12 hours; if slight color change appears within 12 h, extend culture by 12 h; if around 2 days, extend by 24 h. This standardization improves reproducibility.

Yes, these limitations can lead to both false negatives and false positives. The emphasis should be on SOP execution and primer design. With time, more suitable commercial kits are expected to appear.

No single method detects all Mycoplasma species; medium formulations are continuously being developed and updated. Culturing feline mycoplasmas remains notably unsatisfactory. For each material, first identify likely contaminating species, then decide whether culture alone is sufficient.

Cost ranking is roughly: culture < cell-based assays < PCR (which varies greatly by instrument and kit brand). Under regulatory requirements, validate multiple methods and vendors before finalizing a routine approach.

Antibiotic resistance is severe worldwide; quinolone-resistant, uncommon field mycoplasmas (e.g., wild boar, some plant mycoplasmas) have been reported. Removal strategies depend on sample type: serum → filtration; reagents → high-temperature/pressure sterilization; cells → consider antimicrobial peptides. Prevention hinges on testing incoming materials, lab disinfection, and, most importantly, personnel training.

At low magnification, Mycoplasma colony morphology is consistent; to confirm species, increase magnification to 100×. A positive should be based on agar colonies; liquid color change is only supportive and error-prone. Growth curves are of limited qualitative value because color-change timing varies widely:
— ATCC 15531: red → yellow in ~3–5 days
— ATCC 23714: red → dark red/orange, then deeper red in ~2–4 days
— M. synoviae: orange to yellow within ~1 day.
Observe at least daily and subculture promptly to plates to avoid missing transiently viable cells.

Preservation is paramount: use CDC-recommended viral transport tubes or compatible buffers for Mycoplasma, keep samples cold, and test within 3 days to avoid death-related false negatives. In fermenters, dead zones and poor mixing make distribution uneven; therefore, multi-site sampling (e.g., ≥10 mL as many regulations require) is necessary. Begin Mycoplasma testing only after sterility has been confirmed. If antibiotics or inhibitors are suspected, filter to collect Mycoplasma and reculture rather than testing the raw liquid directly.

Each of the three regulatory methods (culture, cell culture, PCR) has gaps for comprehensive Mycoplasma detection. Culture media are often outdated and inadequate for many fastidious species, hence the use of cell culture. Cell-based methods are difficult to read and some species cannot be grown in eukaryotic cells, making medium-based culture necessary. PCR suffers from false negatives/positives due to insufficient primer coverage. Regulations define a baseline, but in practice one must optimize media and, especially, SOP.

Growth rates vary greatly among species. “Autotrophic” strains that are relatively easy to culture, such as ATCC 15531 and ATCC 23714, typically require ~3–5 days for a color change in liquid media and 7–10 days on agar. In contrast, fastidious “heterotrophic” strains (e.g., porcine nasal species) can show color changes within 1–2 days on suitable media. For faster microscopic assessment, raise CFU in liquid culture to shorten time to visibility. Optimizing the medium formulation is the key to speed.

Given the global prevalence of antibiotics, do not assume samples—especially those containing animal- or plant-derived materials—are free of inhibitory substances. Highly pathogenic Mycoplasma can expand rapidly in the human body once such inhibitors are diluted in vivo, posing significant risk.

Historically, penicillin was added to media to suppress contaminants (β-lactams do not affect Mycoplasma), but widespread antibiotic resistance has made this far less meaningful. Medium design should prioritize meeting Mycoplasma growth requirements rather than antibacterial activity. Species with missing metabolic pathways may grow poorly in artificial media yet proliferate rapidly in hosts. Common inhibitors include coexisting bacteria and their secreted antibiotics, enzymes, antimicrobial peptides, and even proteases that can disrupt Mycoplasma.

In principle, even a single CFU of Mycoplasma can be detected by culture. However, certain highly fastidious species from cats, dogs, or pigs often require cell culture because many of their metabolic pathways are missing and standard media are nutritionally inadequate. The Friis formulation in regulations is relatively old and can be underpowered for these strains; therefore, using an optimized Friis medium is crucial. In addition, SOPs for Mycoplasma detection differ from bacterial testing: proper sampling, testing within the sample’s stability window, and excluding growth-inhibitory substances are all essential.

Mycoplasma typically takes ≥3 days to show visible changes (e.g., color shift), and the broth remains clear.
Bacteria cause rapid changes—color within 24 hours, along with turbidity and often a smell.
In summary:

Fast color shift → likely bacteria

Turbidity or odor → bacteria

Slow red shift + clear medium → likely mycoplasma

Many users focus on whether antibiotics are included. However, usable antibiotics for mycoplasma are very limited, and combining several is not feasible. Thus, antibiotics in media are only marginally useful and not a foolproof solution.

Commercial media are quality-controlled, but lab contamination is still possible—especially from prior mycoplasma contamination that wasn’t properly eliminated.
Other issues include poor aseptic technique: leaving bottles open too long or accidentally introducing contamination with pipette tips.
Routine lab disinfection and good aseptic practices are critical.

Set up a negative control.
In positive cultures, color change (yellow or red) occurs progressively and becomes faster. In sterile controls, slight yellowing may occur but is slow and stable for at least 2 weeks.
For agar media, this is easier to judge by microscopy.

The smaller the dilution, the faster the growth—but also the faster pH may reach critical levels, leading to mycoplasma death. The 1:10 ratio is a lab-standard SOP for easy dilution and safe buffering.
However, it can be adjusted based on lab needs. Be cautious: lower dilution (higher concentration) can be risky. If the sample contains inhibitory agents (e.g., antibiotics or EDTA), a larger volume of medium should be used to dilute out these factors.

Serum not only contains BSA (which provides protection and buffering) but also lipids, growth factors, and trace enzymes that benefit mycoplasma growth.
Experiments show that using BSA or DMEM instead of serum leads to ≥60% lower CFU counts in even M. pneumoniae. DMEM may even cause growth arrest. Therefore, if using BSA as a substitute, you must also supplement with the missing nutrients.

Unlike bacteria, mycoplasma lacks motility, so it tends to cluster in some areas while being sparse in others. For small-volume samples, thorough mixing (pipetting/blowing) is essential. For larger sample sources, take multiple site samples to ensure representation.

Excessive antibiotic use, bacterial metabolites (e.g., proteases, nucleases), and serum enzymes can all inhibit mycoplasma growth—causing false negatives in testing. This may compromise pharmaceutical quality and scientific results. Inhibition factor testing helps detect such issues.

CO₂ does stimulate growth for some strains, but not all. For example, M. pneumoniae grows well with or without CO₂, while oral strains show stronger dependency. The need can often be predicted based on the natural environment of the strain. However, using CO₂ incubation universally is a safe standard.

Microscopy is the gold standard. At low magnification (4× objective, 10× eyepiece), all mycoplasmas look similar. At higher magnification (~100× objective), morphological differences emerge:

M. pneumoniae appears as tight clusters,

M. hyorhinis has dispersed, filamentous cytoplasm,

M. orale looks like rounded clusters of small cells.

Hayflick medium, developed in 1965, was the first to culture mycoplasmas without using chicken embryos. However, it struggled to support avian mycoplasmas.
In 1975, Frey medium was developed in the U.S. as an improvement, especially for more types of mycoplasmas.
Friis medium came later, further optimized for difficult strains like Mycoplasma hyorhinis (M.h.).

Slight yellowing within the first 24 hours is mainly due to changes in ionization from temperature increase, which lowers pH. As incubation continues, sugar degradation further drops the pH, reaching around 7.4, which activates oral mycoplasma growth. Once activated, they produce alkali, shifting the color toward red or reddish-purple.
If the strain is highly active or the inoculum is large, the shift to red may be fast, masking the initial drop in pH.
If the medium turns bright yellow directly, it may indicate weak strain viability or low inoculum size, suggesting SOP optimization is needed—especially if yellowing occurs within a week, which likely indicates a flaw in the liquid medium formulation.

Precipitates may appear in cold-stored media due to high-lipid serum content. This is normal. When the medium returns to room temperature or is gently warmed (e.g., water bath), the precipitate dissolves and does not impact usability.

Except for Mycoplasma synoviae-specific media, most mycoplasma media (including for M. hyorhinis) can be stored at 2–4°C for up to six months. Liquid media can be stored at -20°C or -80°C for up to one year (longer storage unverified). Kusada media stability claims are based on experimental data.

Mycoplasma synoviae is arguably the most difficult strain to culture, with strict dependence on NAD (nicotinamide adenine dinucleotide) for initiating growth pathways. NAD is highly unstable, leading to declining medium efficacy after 3–4 days.
Solutions include periodic NAD supplementation or maintaining NAD stability in both liquid and agar formulations.

Mycoplasma grows slowly, especially on agar. When the initial CFU is low, liquid culture is necessary for enrichment. Relying solely on agar plates may result in false negatives due to low visibility.
On the other hand, liquid cultures may change color due to many unrelated factors. Thus, using only liquid or only agar is insufficient—both are required for accurate detection.

Microscopic observation is the global gold standard for microbial detection. Although PCR is widely used, a lack of comprehensive mycoplasma research means many commercial kits suffer from false positives and negatives. Thus, agar plate microscopy is still crucial.
At 4x objective and 10x eyepiece magnification, all mycoplasma colonies exhibit a characteristic “fried egg” morphology. At 100x magnification, species-level identification is often possible.
If it's difficult to distinguish mycoplasma from cell debris, use Kusada ready-to-use mycoplasma stain. After staining, rinsing, and resting for 30–60 minutes, debris will fade, while mycoplasma retains a blue color with clearer “fried egg” morphology.

Mycoplasma is more fragile than bacteria and must be preserved and transported in media with protective agents. The CDC recommends viral transport medium (VTM) formulations or cold-stored culture media (with glycerol if frozen). Transport should be completed within 72 hours (CDC recommendation), and in any case, within 5 days, as longer durations result in significant viability loss or complete cell death.

Like bacteria, mycoplasma can be stored long-term via lyophilization or short-term by adding 20% sterile glycerol to cultured media for cold storage. Unlike bacteria, it's critical that the pH of the medium does not approach the mycoplasma’s survival limit before freezing. Also, avoid -40°C; use -20°C or -80°C instead, as -40°C may weaken or kill the cells.
ATCC lyophilized strains often contain preservatives that inhibit growth, so they should be diluted before culturing. However, passaged or glycerol-preserved strains can be cultured directly.

Mycoplasma hyorhinis is one of the least-studied mycoplasmas and has highly demanding nutritional requirements, making it among the most difficult to culture. Method II is often used for its detection. Porcine serum is commonly added to culture media to meet its high lipid demands. However, antibiotics present in porcine serum can hinder growth. This strain is also sensitive to many common components in traditional mycoplasma media and is highly susceptible to most veterinary antibiotics (except β-lactams). Thus, extreme caution must be taken with media formulation and raw materials.

Oral mycoplasmas are generally one of the most difficult types to culture. They require an environment where they can adhere to surfaces and have higher nutritional requirements, particularly for lipids and phospholipids. Additionally, the strain or pH of the medium can cause cell death during culture. For example, if the CFU in the sample is very low or the mycoplasma is poorly viable, the pH of the medium may continuously drop due to nutrient breakdown in the incubator, eventually falling outside the optimal range for oral mycoplasma growth, resulting in complete death. Therefore, selecting proper storage conditions and an appropriate culture medium formulation is essential.