Coming into the lab and spotting an obvious fungal growth, or the tell-tale signs of cloudy media, can be a stressful experience for researchers. When carrying out cell culture in the lab, contamination from microbial agents such as bacteria and fungus can occur and have disastrous effects on ongoing experiments and cell expansion plans. With the ability to introduce unexpected, uncontrolled variables into experiments, they also present a threat to other cell lines and can result in the immediate disposal of valuable resources. Having access to tests that can detect these infections at early stages can help scientists improve the success of cell-based experiments as well as the reproducibility of results.
Ruth Peat, Head of Cell Services at the Crick Institute, London, UK, says her team regularly quarantines all cell lines that come into her Institute. “It is important that cell lines are deposited with our Cell Services team to screen for the other culture contaminants, such as bacteria, fungus as well as mycoplasma, which particularly cannot be detected by the naked eye. We always screen prior to a researcher receiving a cell line.”
In cell culture, two main types of fungi may be detected: yeast, which are round to ovular single-cell particles that may form chains; and hyphae, which form long multicellular filaments. Molds or floating fungal colonies form when contamination grows significantly. Here we talk about tests to help you detect fungal infections in human cell cultures.
Naked eye and microscopic observations
Both yeast and hyphae can be detected by the naked eye once they have grown to a sufficient size and/or density (1). With yeast, cell culture media will turn cloudy (although this can also be indicative of a bacterial infection). Peat adds: “There are occasions when fungus can be at a low level and does not appear for several sub-cultures. That is why we keep the cell line in our Scientific Tech Platform for at least 3-4 weeks, to discover low-level infections.”
Under the microscope, yeast can be seen as small (3-10 mm) bright particles that may form chains as the yeast “bud” to grow. In contrast hyphae-based molds often look white, green, or black, but can be other colors too. They can appear fuzzy on the surface and vary in shape. When detectable by the naked eye, infected cell culture dishes/vessels should not be opened, and kept closed and disposed of immediately so as not to risk exposing other cell lines or equipment to fungal spores. If the pH has increased in the cell culture media, then phenol red-containing media might also look pink.
Peat advises: “Cell lines that have mold/fungus or bacteria are discarded – in our experience it is not beneficial to keep these cell lines and they will not be cured. We always make a note of what infections have been found in a particular cell line – this is dated and if the origin is outside of the Crick, then we keep that noted too.”
Testing turbidity against microbial standards
This method should be carried out in a microbiology lab, far away from mammalian cell cultures. It is useful for confirming the absence of microbial infection in normally healthy cell lines that are passaged twice without antibiotic and antifungal agents. Bacillus subtilis (gram-positive aerobic bacteria), Candida albicans and Clostridium sporogenes (gram-positive anaerobic bacteria) can be used as positive controls and purchased from suppliers such as the American Type Culture Collection (ATCC) or the European Collection of Authenticated Cell Cultures (ECACC) (2).
Human cell lines can then be brought into suspension with a cell scraper and the suspension inoculated into aerobic (tryptone soy) and anaerobic (thioglycollate) nutrient broths. Inoculate both types of broths with sterile phosphate buffered serum (PBS) to serve as negative controls and incubate all samples for 14 days, checking test samples against controls at various timepoints (e.g., 3, 7, 14 days). Positive control broths become turbid showing evidence of bacteria and fungi at around 7 days of incubation while negative PBS control broths show no evidence of bacteria and fungi and stay clear.
Lactophenol cotton blue staining
Cultures can be scraped and dropped onto a slide, then stained with phenol which kills fungi, lactic acid to preserve fungal structures and cotton blue to stain chitin present in fungal cell walls (4). Hyaline fungi, which have colourless hyphae, will stain blue and dematiaceous fungi that synthesize melanin, will stain hyphae brown. If moulds are visible, then disposal without opening the cell culture vessel is the safest and most recommended approach.
Rapid fluorescent staining
The Calcofluor white stain is useful for confirming fungal infection. It binds to the cellulose and chitin in fungal cell walls (4). Stained fungi appear bright fluorescent green or blue. For this method Calcofluor white is applied to samples in combination with equal parts 10% potassium hydroxide, to clear the specimen and aid visualization of fungal elements. Samples can then be viewed after one minute under ultraviolet light for the presence of fluorescent staining. Samples can be counterstained with Evans blue to diminishes background fluorescence when using blue light excitation instead of ultraviolet light. Other biological materials fluoresce reddish-orange.
Routine environmental monitoring
The final type of testing is one that scientists should adopt as standard practice in cell culture facilities – routine environmental monitoring. For this, equipment such as Class 2 lamellar flow hoods and HEPA filters should be checked every six months to ensure proper functioning. It is also useful to regularly test for the existence of contaminants in hoods and other locations in the lab. In the hood, bacteriological culture plates (such as Tryptone Soya Bean Agar) can be placed on work surfaces for four hours. Plates can then be observed to see if they capture and grow any bacteria over seven days when stored in sealed boxes at 32°C. “Settle plates” can also be used to check for airborne microbes elsewhere in the lab by leaving each plate in a different location for four hours, then covering, storing in sealed boxes at 22°C for as long as seven days.2 Here, careful labelling of plates and regular recording and monitoring of results can help flag issues in advance of end-stage cell culture contamination.
Making infection prevention routine
When fungal infections strike, the best way to treat cells is, unfortunately, to discard them and start again with either fresh (frozen or live) stock culture (1). To prevent the development of severe infection it is therefore critical that cells are scanned regularly, macroscopically and microscopically, during routine media changes or passaging, for the potential presence of low-level infection. This, paired with good aseptic technique and careful handling of cells and reagents, can ensure that infections do not hinder lab work.
References
- Geraghty, R.J., et al., 2014. Guidelines for the use of cell lines in biomedical research. Br J Cancer 111:1021-1046.
- ECACC, 2018. Fundamental Techniques in Cell Culture Laboratory Handbook, 4th Edition. 48-9.
- Smith, A.C., Hussey, M.A., 2005. Gram Stain Protocols. American Society for Microbiology.
- Wanger, A., Chavez, V., Huang, R.S.P, et al., 2017. Biochemical Tests and Staining Techniques for Microbial Identification. Microbiology and Molecular Diagnosis in Pathology. Chapter 5: 61-73.
