Q1:How to determine if cells are contaminated?
A1:Step 1: Observe with naked eyes;
Is the medium turning yellow? Is the liquid becoming turbid?
Step 2: Observe under microscope (200X, 400X);
Are there rapidly moving particles in the medium showing non-Brownian motion? Is there sand-like material covering the gaps between cells and in the culture medium? Are there "little mushrooms" growing in the culture dish? Has the cell condition changed?
Q2:What are the types of cell contamination?
A2:(1) Bacterial contamination: For cells with bacterial contamination, the medium turns significantly yellow and turbid. Under the microscope, spherical or rod-shaped bacteria can be seen moving rapidly and irregularly in the medium. Bacteria have regular shapes and uniform distribution. After bacterial contamination, intracellular granules increase and coarsen, and cells eventually round up, detach, and die;
(2) Fungal/mold contamination: For cells with fungal contamination, the medium color usually does not change and is difficult to detect in the early stage of contamination. Fungal hyphae can be seen under the microscope. 2-3 days after fungal/mold contamination, visible white or yellow fungal colonies form in the medium, and colony structures can be observed under the microscope. After fungal contamination, cell growth slows, and eventually cells detach and die due to nutrient depletion and toxic effects;
(3) Mycoplasma contamination: Cannot be directly observed with naked eyes or microscope. Cells with mycoplasma infection show significantly deteriorated condition, slow growth, and increased cell debris. PCR detection, one-step colorimetric method, or fluorescent staining can be used for verification;
Q3:What to do if cell contamination is detected?
A3:(1) For bacterial contamination, if detected early and cell condition is not affected, try washing with 10% antibiotics, or use gentamicin 50U/mL, tetracycline 10μg/mL, or other antibiotics; if cell condition is already very poor, it is recommended to discard directly and revive new cells;
(2) For fungal contamination, it is difficult to remove, so it is recommended to discard directly;
(3) For mycoplasma contamination, add 50μg/mL mycoplasma removal reagent and treat for at least 2 weeks;
Q4:How to avoid cell contamination?
A4:I. Environmental requirements
(1) Maintain clean air in the cell culture room;
(2) Place laminar flow hoods and biosafety cabinets properly to avoid mutual interference;
(3) Regularly irradiate the cell culture room with UV light and fumigate with ozone. Clean the incubator regularly. Mop the floor of the cell culture room with 84 disinfectant;
(4) Irradiate the laminar flow hood with UV light for 30min before use, and wipe with 75% alcohol after use;
(5) Wear sterile lab coat dedicated to the cell culture room, put on gloves and mask, and change into dedicated shoes when entering the cell culture room;
II. Experimental operation requirements
(1) Clean hands with alcohol before taking cells from or placing cells into the incubator. Minimize door opening time and frequency;
(2) During operation, cover reagents promptly when not in use and move them to the periphery of the operation area. Try not to pass over open containers;
(3) When using a pipette, tilt the container for easier pipetting. Do not touch the bottle mouth or inner wall with the pipette tip. In case of aspiration, wipe immediately with alcohol cotton to prevent bacterial growth caused by residual liquid;
(4) When discarding waste into the waste container, do it gently to avoid splashing that may contaminate the pipette tip and body. After the experiment, promptly discard the waste, rinse the container, spray with alcohol, and place it in the laminar flow hood for UV sterilization;
Q5: How to handle bacterial contamination?
A5: Treatment methods
(1) For cells with bacterial contamination, the medium becomes obviously turbid and forms a layer of sand-like sediment at the bottom after standing. Under the microscope, cells are surrounded by bacteria.
(2) For bacterial contamination detection, take about 1ml of cell culture supernatant and inoculate it into 3ml LB medium, then incubate overnight with shaking. If the medium becomes significantly turbid, it confirms bacterial contamination; if there is no obvious color change, it indicates no contamination.
(3) Discard contaminated cells promptly after treatment to avoid affecting other cells.
(4) Cell debris should be distinguished from bacterial and fungal contamination. The main difference is that cell debris does not proliferate significantly, while bacteria and fungi can be clearly observed to proliferate after overnight incubation, even completely covering the cells.
(5) Bacterial contamination generally breaks out within 1-2 days after the contaminant enters, so you can trace back the source when contamination is detected.
Q6: How to handle fungal contamination?
A6: Treatment methods
(1) For cells with fungal contamination, branched mold hyphae or other forms of fungi can be clearly seen under the microscope. Obvious white spots or granular substances floating in the medium can be seen with the naked eye.
(2) Fungi do not need testing and can be distinguished by microscope and naked eye.
(3) Discard cells with fungal contamination promptly after treatment. Disinfect the incubator and workbench to avoid contaminating other cells.
(4) Fungal contamination generally breaks out within 2-4 days after the contaminant enters. Trace back the source when contamination is detected.
Q7: How to handle mycoplasma contamination?
A7: Treatment methods
(1) Mycoplasma contamination is a common problem in China. Most laboratories lack methods for detecting and controlling mycoplasma contamination, resulting in a large proportion of cells being contaminated with mycoplasma.
(2) There are various mycoplasma detection methods, including PCR, fluorescence method, culture method, and kits. Different methods have different sensitivities, specificities, and target mycoplasma types, so detection results may vary. That is, for the same sample, some methods may detect positive, some negative, some strong, some weak.
(3) For mycoplasma treatment, culture in 6-well plates, change medium daily, passage and discard part of the cells when confluent. This can basically ensure complete removal within 2-3 weeks.