A faster route to a mycoplasma growth curve
Mycoplasma cultures have always been hard to measure. The organisms are small, the media are complex, the growth is slow, and the standard quantification methods microbiologists use for mycoplasma — color-changing units (CCU/mL) from microbroth dilution, colony-forming units (CFU/mL) from agar plating, qPCR-based contamination tests — share one property in common: they take days, or longer, to read out. A growth curve sampled every few hours can take more than a week to assemble into a curve that can actually be interpreted.
BactoBox® is a benchtop instrument that produces a direct count of structurally intact bacterial cells in roughly two minutes per sample, by impedance flow cytometry. Applied to mycoplasma cultures, it gives the same kind of cells/mL number it produces for any other bacterial culture, with the same speed. In our work with the HUN-REN Veterinary Medical Research Institute, BactoBox® cell counts were measured in parallel with CCU/mL across full growth curves for Mycoplasma anserisalpingitidis and Mycoplasma gallisepticum, and the two methods tracked each other strongly across the curve.
The rest of this article covers what the established mycoplasma quantification methods measure, what BactoBox® adds to that set, how to read a mycoplasma growth curve from direct cell counts, where the method belongs in a research and process-development workflow, and where it does not.
Why counting mycoplasma is hard
Several things conspire. Mycoplasma cells are wall-less, fragile, and small — typically 0.3–0.8 µm in their dividing form, smaller than most bacteria and irregular in shape, with pleomorphic morphologies that resist clean optical resolution[1]. They grow slowly, in complex undefined media that often include serum and sterol supplements[1]. Doubling times are measured in hours — on the order of 6–8 hours for M. pneumoniae in rich broth — and visible growth on agar typically takes a week or more[2]. Cell densities at plateau sit close to the OD600 detection limit, and pigments in the medium dominate the optical signal at those densities anyway. The methods that work in mycoplasma cultivation labs work around these properties, but they all pay for the workaround in time.
How mycoplasma is counted today
Five methods are commonly used to quantify mycoplasma cultures. Each addresses a different question, and each pays for it differently.
| Method | What it measures | Time to readout | Where it fits |
|---|---|---|---|
| CCU/mL (color-changing units) | Lowest serial dilution that produces a metabolic color change in the medium's pH indicator[3] | 24–48 h for fast growers; up to a week or more for slow species[3] | The most widely used quantification approach in mycoplasma research |
| CFU/mL (colony-forming units) | Culturable cells, counted from the characteristic "fried-egg" colonies on agar[1] | Days to a week | Labor-intensive; undercounts aggregated samples; misses cells that have lost culturability without losing structural integrity |
| qPCR and other NAT methods | mycoplasma DNA copies (genome equivalents) | Hours, at the assay step | Widely used for contamination detection in cell culture[4] and for compendial release testing of biologics and cell-therapy products[5][6]; cannot distinguish viable from non-viable[4] |
| OD600 | Optical scattering of the culture | Minutes | Signal too low at typical mycoplasma densities; pH-indicator dyes in many mycoplasma media interfere. See Understanding OD600 |
| Direct microscopy | Cells visualized in a counting chamber | Minutes per sample | Small size, low contrast, and pleomorphic morphology make routine quantitative microscopy operationally hard; used for confirmation rather than growth-curve work |
The common thread is time. CCU and CFU read out in days. qPCR is fast at the assay step but answers a different question — is mycoplasma DNA present? — not how is the population changing?. OD600 and direct microscopy are fast enough but poorly matched to mycoplasma biology. A multi-point growth curve sampled every few hours can take a week or more to assemble into a form that can actually be interpreted.
What BactoBox® counts in a mycoplasma culture
BactoBox® uses impedance flow cytometry: each particle that crosses a pair of microelectrodes in a microfluidic channel is recorded as a single event, with an electrical signature determined by its envelope and cytoplasm[7]. A cell with an intact lipid membrane and a conductive cytoplasm is classified as a bacterial cell. A compromised cell, or a non-cell particle such as a salt crystal or debris fragment, is classified separately. The result is a count of structurally intact total cells per mL, available in roughly two minutes per sample. Understanding BactoBox® cell counts covers the principle, the classification logic, and the comparison with other methods in detail.
The classification is calibrated for particles in the 0.5–5 µm range. Mycoplasma cells sit at the small end of that range and, in some preparations, slightly below it. The 0.5–5 µm specification is best understood as a rule of thumb. BactoBox® cell counts have been benchmarked against colony-forming-unit plating across multiple bacterial species in fermentation processes, with near-perfect correlation through exponential, deceleration, and stationary phases[8]. The method has been verified across multiple mycoplasma species in our work with HUN-REN on M. anserisalpingitidis and M. gallisepticum, where BactoBox® cell counts tracked CCU/mL across the full growth curve. At the small end of the size range, the J. Craig Venter Institute uses BactoBox® to acquire growth data on JCVI-Syn3.0, the synthetic minimal-genome organism derived from Mycoplasma mycoides whose cells sit close to or below the nominal lower bound of the detection range. For a new species or a new medium, a brief check against a reference method remains good practice — and is something we are happy to help with.
A worked example: Mycoplasma gallisepticum
The figure below shows a full growth curve of M. gallisepticum measured in parallel by BactoBox® and the microbroth dilution method, from inoculation through 48 hours. In the animated build-up, BactoBox® cell counts appear as the measurements would become available in a real run, with each reading produced within minutes of sampling. A clock then ticks across days to convey the wait that follows every sample drawn for CCU/mL. Only at the end of that wait do the CCU/mL values appear — together, all at once, because in practice the dilution panel must finish changing color before the readout can be assigned.
The curve has the shape a mycoplasmologist would expect. A short lag through the first ~12 hours, with cell concentration drifting around 10⁶ cells/mL. A transition into exponential growth from roughly 16 to 20 hours. Rapid exponential expansion from 20 to 32 hours, where the population climbs three orders of magnitude. An early plateau by 48 hours, with cells/mL stabilizing in the 10⁹ range. BactoBox® and CCU/mL trajectories follow each other through these phases.
The difference between the two methods is when the curve is readable. The BactoBox® trajectory exists from T0 onward and is available in real time. The first CCU/mL value cannot be assigned until its corresponding microbroth-dilution panel has incubated long enough to change color, and the value associated with the sample drawn at, say, T8 is not available for at least 24 to 48 hours — and for slower-growing species, often a week or more. For a workflow that depends on knowing what the culture is doing right now — for harvest decisions, sampling-point selection, anti-mycoplasma assay readouts, or media-comparison work — direct cell counts produce a curve that does not yet exist by any other method.
What direct counts unlock
For decades, mycoplasma growth experiments have had to be designed around a measurement that is only readable days after the sample is taken. Curves have been sparse. Phase transitions have been inferred rather than observed. Comparisons between media or strains have rested on endpoint counts rather than full trajectory shapes. None of this is a fault of the methods — CCU and CFU work as designed — but the time scale of the readout has shaped the shape of the experiments.
A direct cell count in minutes does not replace those methods. It sits alongside them. CCU and qPCR remain the right tools for culturability and contamination detection respectively. What changes is the resolution available for studying how mycoplasma cultures actually behave: phase boundaries become observable as they happen, media comparisons become full curves rather than endpoint pairs, and the kind of process-development work that has long been routine for E. coli or Bacillus subtilis becomes feasible for organisms that have, until now, been too slow to measure in real time.
The HUN-REN team's continuing use of BactoBox® across multiple mycoplasma species is one example of what this looks like in practice. For specific organisms, media, or workflows, the right next step is usually a short conversation about fit.
What BactoBox® is not, for mycoplasma
BactoBox® counts structurally intact mycoplasma cells. It is not a measure of culturability, it does not detect mycoplasma DNA, and it is not a compendial method — it is not a substitute for USP <63>[5], Ph. Eur. 2.6.7[6], or any equivalent test used for biologics or cell-therapy release, and it is not a contamination test for routine cell-culture monitoring[4]. The question BactoBox® answers is the growth-curve question: how many structurally intact mycoplasma cells are in this culture, right now. That makes it useful for research and process-development work — studying growth itself, comparing strains, developing or optimizing media, evaluating anti-mycoplasma compounds, characterizing contamination kinetics in research settings — not for regulated release or routine screening.
Mycoplasma media: serum, background, and dilutions
Mycoplasma cultivation runs on a small family of complex undefined media[1]. Frey medium is the standard for avian mycoplasmas such as M. gallisepticum and M. synoviae[9]. Friis medium and its modified variants are used for M. hyopneumoniae and related swine species[10]. SP4 medium[11] and Hayflick medium[12] are general formulations used for M. pneumoniae and as starting points for many other species. Older formulations such as PPLO broth, and commercial preparations such as Mycoplasma Experience and MolliScience medium, sit alongside these. All share a common architecture: a rich peptone or beef-heart infusion base, a yeast-extract supplement, animal serum, and a pH indicator that shifts color as mycoplasma metabolism acidifies the medium[1].
The serum and undefined components in these media can contribute a small background signal to BactoBox® measurements at low dilution. Two approaches handle this cleanly.
The first is to measure a sterile-medium negative control on BactoBox® and subtract the average background from each sample reading. This is the approach used in our work with HUN-REN and it produces fully background-corrected counts.
The second, simpler, approach is to avoid 1:100 dilutions of mycoplasma culture for BactoBox® measurement, and to work instead with 1:1,000 dilutions or higher. At 1:1,000 the medium background contribution falls below the BactoBox® detection threshold, and the count can be read directly as a mycoplasma cell count with no correction step. For routine growth-curve work this is the simpler workflow. For samples at the low end of the curve where further dilution would push the count below the BactoBox® working range, the background-subtraction route remains available.
References
- Razin, S., Yogev, D., & Naot, Y. (1998). Molecular biology and pathogenicity of mycoplasmas. Microbiology and Molecular Biology Reviews, 62(4), 1094–1156. https://doi.org/10.1128/MMBR.62.4.1094-1156.1998
- Waites, K. B., & Talkington, D. F. (2004). Mycoplasma pneumoniae and its role as a human pathogen. Clinical Microbiology Reviews, 17(4), 697–728. https://doi.org/10.1128/CMR.17.4.697-728.2004
- Hannan, P. C. T. (2000). Guidelines and recommendations for antimicrobial minimum inhibitory concentration (MIC) testing against veterinary mycoplasma species. Veterinary Research, 31(4), 373–395. https://doi.org/10.1051/vetres:2000100
- Nikfarjam, L., & Farzaneh, P. (2012). Prevention and detection of mycoplasma contamination in cell culture. Cell Journal (Yakhteh), 13(4), 203–212. https://pmc.ncbi.nlm.nih.gov/articles/PMC3584481/
- United States Pharmacopeial Convention. USP General Chapter <63> Mycoplasma Tests. United States Pharmacopeia and National Formulary (USP–NF), Rockville, MD. https://www.usp.org/microbiology
- European Directorate for the Quality of Medicines & HealthCare. European Pharmacopoeia General Chapter 2.6.7: Mycoplasmas. Ph. Eur. 12.2, in force 1 April 2026. EDQM, Strasbourg. https://www.edqm.eu/en/-/epc-adopts-mycoplasmas-general-chapter-and-monographs-updated-to-incorporate-latest-analytical-developments
- Bertelsen, C. V., Skands, G. E., González Díaz, M., Dimaki, M., & Svendsen, W. E. (2023). Using impedance flow cytometry for rapid viability classification of heat-treated bacteria. ACS Omega, 8(8), 7714–7721. https://doi.org/10.1021/acsomega.2c07357
- Jordal, P. L., González Díaz, M., Aalund, F., & Skands, G. (2025). Performance qualification of impedance flow cytometry as a rapid in-process control proxy for colony-forming units in bacterial fermentation processes. Journal of Microbiological Methods, 238, 107284. https://doi.org/10.1016/j.mimet.2025.107284
- Frey, M. L., Hanson, R. P., & Anderson, D. P. (1968). A medium for the isolation of avian mycoplasmas. American Journal of Veterinary Research, 29(11), 2163–2171. https://pubmed.ncbi.nlm.nih.gov/5693465/
- Friis, N. F. (1975). Some recommendations concerning primary isolation of Mycoplasma suipneumoniae and Mycoplasma flocculare. Nordisk Veterinærmedicin, 27(6), 337–339. https://pubmed.ncbi.nlm.nih.gov/1098011/
- Tully, J. G., Rose, D. L., Whitcomb, R. F., & Wenzel, R. P. (1979). Enhanced isolation of Mycoplasma pneumoniae from throat washings with a newly modified culture medium. Journal of Infectious Diseases, 139(4), 478–482. https://doi.org/10.1093/infdis/139.4.478
- Hayflick, L. (1965). Tissue cultures and mycoplasmas. Texas Reports on Biology and Medicine, 23(Suppl 1), 285–303. https://pubmed.ncbi.nlm.nih.gov/5833547/