Sensitivity Matters: An Independent Evaluation of Flip Mycoplasma Test Sensitivity Using the Flip Nest

1 Oct

An independently performed^* limit-of-detection study of Flip Mycoplasma using a European Pharmacopoeia (EP) 2.6.7 (12.2) compliant quantified standard, with all tests incubated in the USB-C powered Flip Nest.

^*Testing performed as a service by Mycoplasma Experience Ltd.

Executive Summary

This whitepaper reports the outcome of a study demonstrating the sensitivity of Flip Mycoplasma using an EP 2.6.7 (12.2), USP <77> and JP 18 G3 compliant quantified standard in a dilution series experiment. All of the reported testing used the Flip Nest to perform the required incubation, and was undertaken independently by leading mycoplasma contamination experts.

Undetected mycoplasma contamination is one of the most common and costly problems in cell culture. Surveys have found 15–35% of continuous cell lines to be infected [Drexler & Uphoff, 2002], and mycoplasma sequences have been detected in around 11% of cell lines deposited in public RNA-sequencing archives, many of them used to generate published data [Olarerin-George & Hogenesch, 2015]. Because it typically produces no turbidity, no pH shift and nothing visible under standard microscopy, mycoplasma contamination can quietly distort experimental results for a long time before it is suspected. The value of a research screening test therefore depends on two things working together. It must detect very low levels of contamination, and it must be quick and easy enough to be run frequently.

Flip Mycoplasma is a versatile test aimed at research users, enabling fast, simple and accurate detection of mycoplasma contamination. The fundamental ease of use of the Flip test is supplemented by the low-cost dedicated Flip Nest, which gives users without a suitable PCR machine or dry bath a convenient alternative for incubation. The Flip Nest's USB-C power source means it is highly portable and can even be powered from a phone or battery pack, so testing can be performed anywhere.

The key results are summarised below and detailed in the sections that follow.

Key Findings

In this independent study, a dilution series of Mycoplasma orale (ATCC 23714) standard 100CFU® (EP-compliant product cat. 106-2001) was prepared in cell culture media and tested in triplicate according to the Flip Mycoplasma instructions for use. The results obtained are shown in Table 1 and reveal an indicative limit of detection below 10 CFU per mL.

This work used a new M. orale standard designed to comply with the recently updated EP chapter 2.6.7 (12.2), which mandates certain controls on the reference standard preparations used in mycoplasma test validation, in particular the requirement for the ratio of genome copies to CFU to be less than 10. It provides independent endorsement of the published limit of detection of Flip Mycoplasma, and also evidences for the first time the performance of the Flip Nest when used as the incubator in place of a PCR machine or heat block.

Table 1 — Flip Mycoplasma Results Across the M. orale Dilution Series

Target concentration	Result
90 CFU/mL	3/3 positive
30 CFU/mL	3/3 positive
10 CFU/mL	3/3 positive
3.3 CFU/mL	3/3 positive
1.1 CFU/mL	2/3 positive

Highlighted row: lowest concentration at which all three replicates were positive.

Bench setup for the study, with the Flip Nest, sample tubes in racks and pipettes — Study setup at the bench

Three Flip Mycoplasma tests incubating in the USB-C powered Flip Nest — Three tests incubating in the Flip Nest

Completed Flip Mycoplasma tests from the dilution series laid out on the bench in triplicate groups — Completed tests from the dilution series

Three Flip Mycoplasma cassettes labelled 3.3, each showing a positive test line — 3.3 CFU/mL — 3/3 positive

Method

Materials and Equipment

Item	Supplier and details
100CFU® M. orale standard	Minerva Biolabs cat. 106-2001, lot 106-590-2602, exp. 28.02.2027
Flip Mycoplasma	Fuse Diagnostics cat. FPR003-0013-10, lot 000002071, exp. 30.06.2027
Flip Nest	Fuse Diagnostics cat. FPR001-0029
Cell culture media	Gibco™ DMEM, high glucose, pyruvate. Thermo Fisher Scientific cat. 41966-029, lot 3425150, exp. 06-2027
Pipettes	Thermo Scientific Finnpipette Novus electronic single channel, 10–100 µL and 100–1000 µL, with corresponding tips
Consumables	1.5 mL tubes

Steps

A single vial of 100CFU® was centrifuged briefly for 5 s, then reconstituted with 1 mL DMEM and incubated for 10 minutes. After 10 minutes it was mixed thoroughly by pulse-vortexing (3 × 10 s), then centrifuged briefly again.
Dilutions were prepared according to Table 2. Each sample was mixed thoroughly after preparation.
The Flip Nest was plugged in and pre-heated until it reported that it was at temperature, via its LED indicators and sounder.
Three Flip Mycoplasma tests were performed for each diluted target preparation according to the kit instructions, as follows:
1. 50 µL of the diluted target preparation was added to 450 µL Flip Mycoplasma sample buffer and mixed.
2. Flip Mycoplasma assay tubes were placed in a rack and the foil removed.
3. 30 µL of the prepared sample from (a) was added to each assay tube.
4. Three readout cassettes were promptly clipped onto the assay tubes.
5. Within 2–3 minutes the three assembled assays were placed onto the Nest and the timer start button pressed immediately.
6. When the Nest alarm sounded, all three were removed together, and each test was flipped upside down and given a gentle shake before being laid on the bench.
When the previous test was half way through incubation, the next samples in the down-titration series were prepared. Three Flip tests were prepared according to the kit instructions in step 4 above, and placed in the Nest once the previous three had been laid on the bench.
Steps 4 and 5 were repeated for all dilutions.
Negative controls were performed before and after testing, using DMEM only as the test sample. All tests gave the expected result.

Table 2 — Preparation of the Dilution Series

Step	Concentration	Preparation
Stock	100 CFU/mL	Reconstituted vial
1	90 CFU/mL	900 µL stock + 100 µL DMEM
2	30 CFU/mL	333 µL of 90 CFU/mL + 667 µL DMEM
3	10 CFU/mL	333 µL of 30 CFU/mL + 667 µL DMEM
4	3.3 CFU/mL	333 µL of 10 CFU/mL + 667 µL DMEM
5	1.1 CFU/mL	333 µL of 3.3 CFU/mL + 667 µL DMEM

Why Sensitivity and Convenience Matter in Practice

Sensitivity is critical because it determines how readily a contamination can be caught. While certain cultures can accumulate mycoplasma to several thousand cells per millilitre with no visible effect on growth or morphology, others can persist at low level or take days to accumulate, influenced by the cell line, the growth conditions (media, antibiotics) and the contaminating species. As a result, low-level infections can persist and spread long before they are noticed. A test that only registers a positive at high titres leaves this window open. The contamination continues undetected, corrupting results and, in the worst case, spreading through a cell bank, where one infected culture commonly means others are already affected. Reliably detecting contamination at 1–10 CFU per mL closes much of that blind spot. It is also what gives confidence that a culture is genuinely clean after an attempted decontamination, rather than merely suppressed below the threshold of a less sensitive test.

Real-world protection depends on how little a test can detect, and on how often it can realistically be run. Slow-growing contaminations are caught only if testing happens close to the point of infection, which is unlikely under an infrequent regime, so even the most powerful assay offers little protection if it is run rarely. This is where Flip Mycoplasma's combination matters. Because it is both highly sensitive and fast and simple to run, it can be used frequently, on small samples, as an in-process check at the bench. Each test takes around 12 to 15 minutes and needs only the Flip Nest or a simple heat block, with a visual strip read-out and no thermocycling, gel or dedicated molecular facility. It therefore pairs excellent sensitivity with a format built for routine cell-culture screening in any location, so users no longer have to trade sensitivity for convenience.

Discussion and Conclusions

This study provides independent evidence of the excellent sensitivity of Flip Mycoplasma, and further demonstrates that this performance can be achieved using the low-cost, highly portable Flip Nest to incubate the tests, providing an easy and accessible solution for cell culture screening.

This testing supplements other published evidence of the analytical performance of Flip Mycoplasma, including the Flip Mycoplasma performance characteristics study and a comparative performance evaluation against four other commercially available mycoplasma tests. Both are also available via the Flip Mycoplasma product page.

Although Flip Mycoplasma detects M. orale and other mycoplasma species such as M. arginini below the 10 CFU per mL limit prescribed in the EP, it is designed for routine screening applications in the research laboratory. Within regulated settings, Flip Mycoplasma can be deployed alongside an EP-compliant release test, enabling rapid in-process checks that reduce contamination risk. If you would like to find out more about Flip Mycoplasma, or to discuss your mycoplasma testing requirement, please contact info@fuse-dx.com.

The testing reported here was performed independently as a professional service by Mycoplasma Experience Limited. Mycoplasma Experience are fully independent of Fuse Diagnostics and do not endorse specific mycoplasma tests, including Flip Mycoplasma.

Product Information

Flip Mycoplasma is available from Fuse Diagnostics and selected distributor partners in sizes of 10 tests, 50 tests and 100 tests. Contact Fuse on info@fuse-dx.com if you wish to request a free 10-test evaluation kit or a quotation.

Product	Code
Flip Mycoplasma test kit (10 tests)	FPR003-0013-10
Flip Mycoplasma test kit (50 tests, 5 kits)	FPR003-0013-50
Flip Mycoplasma test kit (100 tests, 10 kits)	FPR003-0013-100
Flip Mycoplasma external positive control	FPR003-0045
Flip Nest	FPR001-0029

Evaluate Flip Mycoplasma in your laboratory. Fuse Diagnostics supplies free evaluation kits to research groups. The full analytical performance summary is available on the Flip Mycoplasma product page; contact us at info@fuse-dx.com if you would like further information or to request a quote.

References

Drexler HG, Uphoff CC (2002). Mycoplasma contamination of cell cultures: incidence, sources, effects, detection, elimination, prevention. Cytotechnology 39(2):75–90.

Olarerin-George AO, Hogenesch JB (2015). Assessing the prevalence of mycoplasma contamination in cell culture via a survey of NCBI's RNA-seq archive. Nucleic Acids Research 43(5):2535–2542.

Appendix: Result Images

Flip Mycoplasma readout cassettes for each dilution, tested in triplicate. Cassettes are labelled with the target concentration in CFU per mL and the replicate number.