Cell culture contamination:

• It is possible to describe a cell culture contaminant as any elements in the culture system that are unacceptable because of their possible adverse effects on either the system or its usage.
• Contamination cannot be entirely removed, but both the level of occurrence and the severity of its effects can be controlled to decrease.

Consequences of contamination in cell culture lab:

• Loss of time, cash and commitment.
• Unfavorable impacts on cultures.
• Inaccurate or false scientific observations.
• Personal awkwardness.
• Loss of valuable items

Contamination issues in cell culture:

• The contamination issues in cell culture can be classified into three classes:

1. Minor annoyances: When several plates and flasks are lost to contamination.
2. Severe problems: When the frequency of contamination rises and whole experiments or cell cultures are lost.
3.  Major disasters: Contaminants that are usually other cell lines or mycoplasma that bring into question the validity of your past and present work

Types of Contamination:

1. Biological contamination
2. Chemical contamination

I. Chemical contamination:

Types and Sources of chemical contamination:

• The involvement of any nonliving material that results in adverse effects on the culture system is best explained as chemical contamination.
• Media: Most chemical contaminants are present in the media for cell culture and come either from the reagents and water used to make them, or from the additives used to supplement them, such as sera.
• Sera: Cause of both biological and chemical contaminants, variance of hormone and growth factors.
• Water: A common cause of chemical contamination is water used for the manufacture of media and cleaning glassware and needs careful precautions to ensure its quality.
• Endotoxins: lipopolysaccharide-containing gram-negative bacteria by-products typically present in water, serums, and some culture additives (especially those manufactured using microbial fermentation)
• Storage vessels: Another cause of contaminants may be media stored in glass or plastic bottles that have already contained heavy metal solutions or organic compounds, such as electron microscopy stains, solvents, and pesticides.
  • – During storage of the initial solution, the contaminants can be adsorbed on the surface of the container or its cap (or ingested into the bottle if it is plastic).
  • Fluorescent lights:
  • – the application of media containing HEPES (N-[2-hydroxyl ethyl] piperazine-N’-[2-ethanesulfonic acid])-an organic buffer widely used to supplement bicarbonate-based buffers), riboflavin or tryptophan to regular fluorescent lighting is a significant yet sometimes underestimated cause of chemical contamination.- These media components are likely to be photoactivated resulting hydrogen peroxides and free radicals that are harmful to cells, the longer the exposure, the more toxic it is.
  • Incubators:
  • – The incubator may also be a source of chemical contamination, often viewed as a significant source of biological contamination.
  • – Gas mixture perfused through certain incubators (usually including carbon dioxide to help control media H) which contain toxic contaminants, especially oils or other gases such as carbon monoxide that may have previously been used in the storage cylinder or tank.

II. Biological contamination:

• Centered on the difficulties of detecting them in cultures, biological contaminants can be subdivided into two groups:
• 1. Those that are easy to detect: Bacteria, molds and yeast.
• 2. Those which are more difficult to detect and cause more severe problems in culture: Viruses, protozoa, insects, mycoplasmas, and cross-contamination from other cell lines.

Sources of biological contamination:

• Accidents and failures.
• Contact with nonsterile supplies, media or solution.
• Particulate or aerosol fallout during manipulation, transportation, or incubation.
• Swimming, crawling, or growing into culture vessels

1. Bacteria, yeasts and molds:

• Found almost anywhere and able to colonize and grow rapidly in the environment provided by cell culture.
• In the absence of antibiotics, microbes can be identified either by direct microscopic analysis or by their effects on culture in a culture within a few days (pH shifts, turbidity and cell destruction).
• Resistant species may establish low-level infections that are very difficult to detect by direct visual examination when antibiotics are regularly used.
• a. Bacterial contaminants:
  • -common in nature
  • – maybe mistaken as cellular debris, usually at lower levels.
  • – Search for:
    • Signs of motility
    • acidic pH
    • Size and shape uniformity
• b. Fungal and yeast contaminants:
  • – also common\- formation of clumps or mats
  • – Easier identification because of large size .
  • – Fungi will form small colonies on medium initially.
  • – Yeast will normally show signs of budding.

2. Viruses:

• Viruses are the most challenging cell culture pathogens to detect in culture due to their small size.
• Their small size also makes it difficult to extract them from biological media, solutions, and other solutions.
• While viruses may be more widespread in cell cultures than many researchers know, unless they have cytopathic or other adverse effects on cultures, they are typically not considered a serious issue.
• Their effect on cultures is not a major concern for the use of virally infected cultures, but they pose possible health risks for laboratory staff.
• Viral contaminants:
  • – Not very common:
  • identified by their adverse affects on  cultures
  • Many unknown consequences are wrongly blamed on viruses
  • Fetal bovine sera consists of bovine viruses
  • – No reliable way to completely eliminate viral contamination
  • -Detection:
    • Immunostaining
    • ELISA
    • PCR

3. Protozoa:

• Single-celled protozoa, such as amoebas, have sometimes been classified as cell culture contaminants, both parasitic and free-living.
• Amebas, typically of soil origin, can form spores and are readily isolated from the air, sometimes from tissues, as well as from laboratory personnel’s throat and nose swabs.
• Cytopathic symptoms similar to viral damage can be induced and a culture can be completely killed within ten days.
• Amoebas are very hard to detect in culture due to their sluggish growth and morphological similarity to cultured cells.
• Fortunately, contaminants of this kind are uncommon, but it is important to be aware of the likelihood of their occurrence.

4. Invertebrates:

• Insects and arachnids are commonly found in laboratory environments, and both cultures and sterile supplies may be infected by flies, ants, cockroaches and mites.

5. Mycoplasmas:

• Mycoplasmas were first observed by Robinson and coworkers in 1956 in cell cultures.
• They attempted to research the effects of PPLO (pleuropneumonia-like species, the original name for mycoplasma) on HeLa cells, when they found that the control HeLa cultures were already contaminated by PPLO.
• The most popular form of contaminant in today’s cell culture!
• There are approximately 180 different species.
• The most popular transmission method is from other cultures that have been contaminated.
• The smallest (0.2-0.3μm) free living organisms.
• No cell wall; it cannot be seen under phase contrast microscopy.
• Effects of Mycoplasma:
  • Effects on almost every aspect of cell behavior & growth, including microarrays.
  • A medium of up to 108 mycoplasma/mL without turbidity
  • Intervention with screening assays.
  • This triggers chromosome breakage.
  • This leads to inaccurate or incorrect results.

How to reduce contamination of cultures ?

• The basic step is autoclaving, it reduces the contamination.
• Commercial cleanup kits: It can help with temporary reduction of contamination between detectable levels. It may alter characteristics or kill cells.
• In case of detection of mycoplasma:
  • – Tylosin
  • – BM Cycline 
  • MRA; mycoplasma removal  agent 
  • – Ciprofloxacin
  • Non- Antibiotic: Mynox Mycoplasma Elimination  Reagent