The Guide to Mycoplasma Contamination Treatment

Contamination is a silent threat in cell culture, quietly undermining experimental accuracy and consistency. Among the top culprits—bacteria, fungi, and mycoplasma—mycoplasma is by far the most insidious, with contamination rates as high as 30%-60%. In this issue of Cell Culture Academy, we spotlight essential strategies for mycoplasma prevention and control to help safeguard your cultures and your results.

Ⅰ. What Is Mycoplasma?

Mycoplasma, also called pleuropneumonia-like organisms (PPLO), belongs to the class Mollicutes and is among the smallest known prokaryotes. It consists of a lipoprotein membrane, ribosomes, and circular double-stranded DNA (Figure 1), with a genome size of 580-2,200 kb.

While capable of growing in cell-free media, Mycoplasma has limited biosynthesis capacity and cannot produce essential molecules like cholesterol, relying on external nutrients. Cell culture systems provide ideal conditions for its growth. It adheres to host cell surfaces for long-term survival and replication. Its resistance to many common antibiotics makes Mycoplasma a persistent, often hidden contaminant in cell cultures.

Figure 1. Schematic Diagram of Mycoplasma

Ⅱ. Mycoplasma Contamination Characteristics

There are more than 120 species of mycoplasma in nature, but the strains commonly contaminating cell cultures mainly include Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, and Mycoplasma bovis. When these strains contaminate cells, they cause a series of characteristic changes, including:

1. Abnormal cell growth rate, with slowed or halted proliferation.

2. Morphological changes, including poorer adhesion and easier detachment of adherent cells.

3. Chromosomal number or structural abnormalities, leading to karyotype instability.

4. Altered cell membrane antigenicity and abnormal cell adhesion ability.

5. Disrupted metabolic pathways, with changes in glycolysis and amino acid metabolism rates.

6. Reduced survival rates after cell recovery.

These changes are nonspecific but can disrupt normal cellular functions, leading to skewed or unreliable experimental results. Mycoplasma are extremely small—measuring just 0.1-0.3 μm in diameter, much smaller than typical cells—and lack a cell wall, making them poorly refractive and difficult to detect with standard light microscopy. Their resistance to common antibiotics like penicillin further complicates both detection and eradication. As a result, unusual changes in cell growth or morphology often serve as early warning signs. When such issues arise without a clear cause, mycoplasma contamination should be one of the first possibilities considered.

Ⅲ. Mycoplasma Contamination Detection Methods

Effective mycoplasma control starts with accurate detection. Common methods include microscopic observation, culture method, immunoassay, DNA fluorochrome staining, molecular methods, and ATP bioluminescence assays (Table 1). Of these, the Chinese Pharmacopoeia  recommends culture method and DNA fluorochrome staining. Culture method remains the gold standard for its reliability, though it’s time-consuming. qPCR is the most sensitive rapid method, making it ideal for fast, accurate screening.

Table 1. Comparison of Mycoplasma Detection Methods

IV. Mycoplasma Contamination Treatment Strategies

Once mycoplasma contamination is identified in cell cultures, prompt and effective treatment is essential. Successful elimination targets the disruption of the mycoplasma’s lipoprotein membrane and inhibition of key metabolic pathways, such as protein and DNA synthesis. Because mycoplasma shares similar culture characteristics with host cells, the main challenge lies in eradicating it without harming cell viability.

1. Heat Treatment

Mycoplasma typically survives within a narrow temperature range of 35-37℃. Raising the temperature to 41℃ for 5-10 h (but no more than 18 h) can kill the organism. However, since cells vary in their sensitivity to heat stress, this method may damage certain cell lines and should be applied cautiously.

2. Antibiotic Treatment

Some antibiotics—such as kanamycin, gentamicin, doxycycline, tetracycline, and ciprofloxacin—are effective against mycoplasma. Cells can be passaged multiple times in media containing these antibiotics, and successful clearance is confirmed when repeated mycoplasma tests return negative results. Still, prolonged or excessive antibiotic use may trigger resistance or toxicity, so care must be taken when using this approach.

3. Combination Reagents

For reliable and efficient removal, combination mycoplasma elimination reagents are highly recommended. These products typically include antibiotics along with membrane-disrupting agents that penetrate the mycoplasma cell membrane, interfere with its metabolism, and block DNA replication.

Recommended product: Pricella® Anti-Mycoplasma Treatment Reagent(Cat. No.: P-CMR-001). Add the appropriate amount of reagent to the culture medium as directed. This approach is user-friendly, effective, and generally less toxic to host cells, preserving cell viability throughout the process.

Following treatment, it’s critical to confirm successful mycoplasma clearance using validated methods such as qPCR or DNA fluorochrome staining. This step ensures the contamination is fully resolved and guards against false negatives caused by residual reagents or the re-emergence of the mycoplasma.

Despite their tiny size, mycoplasmas pose a serious threat to cell culture, often causing significant disruption without obvious signs. Protecting the reliability and consistency of cell experiments requires a clear understanding of their biology, early identification of contamination, and the use of effective detection and elimination strategies.