Biological contamination is the dread of every person working with cell culture. When cultures become infected with microorganisms, or cross-contaminated by foreign cells, these cultures usually must be destroyed. Since the sources of culture contamination are ubiquitous as well as difficult to identify and eliminate, no cell culture laboratory remains unaffected by this concern. With the continuing increase in the use of cell culture for biological research, vaccine production, and production of therapeutic proteins for personalized medicine and emerging regenerative medicine applications, culture contamination remains a highly important issue. Cell line cross-contamination can be a problem for scientists working with cultured cells. Studies suggest anywhere from 15–20% of the time, cells used in experiments have been misidentified or contaminated with another cell line. Problems with cell line cross-contamination have even been detected in lines from the NCI-60 panel, which are used routinely for drug-screening studies. Major cell line repositories, including the American Type Culture Collection (ATCC), the European Collection of Cell Cultures (ECACC) and the German Collection of Microorganisms and Cell Cultures (DSMZ), have received cell line submissions from researchers that were misidentified by them. Such contamination poses a problem for the quality of research produced using cell culture lines, and the major repositories are now authenticating all cell line submissions. ATCC uses short tandem repeat (STR) DNA fingerprinting to authenticate its cell lines.

1. A Literature Review on
Mycoplasma contamination of cell cultures: A
major problem in cell lines
-The Ugly Truth about Biological Contamination
CALCUTTA INSTITUTE OF PHARMACEUTICAL TECHNOLOGY AND ALLIED HEALTH SCIENCES, ULUBERIA, HOWRAH
(WEST BENGAL)
P r e p a r e d b y :
(M.Pharm in Pharmaceutical Biotechnology: 1st Year / 1st semester)
University Roll No.15920917004 & Registration No.171592310022

2. Contents Overview
 Introduction
 Literature Reviews
 Biological Contaminants
 Sources of biological contaminants in the laboratory
 Mycoplasma Contamination
 Effects of mycoplasma contamination on cell cultures
 Consequences of mycoplasma contamination
 Detection of mycoplasma contamination
 Microbiological colony assay
 Mycoplasma elimination methods
 Prevention of mycoplasma contamination
 Conclusion
 References
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3. Introduction
Cell culture is continuing a 60-year trend of increasing use and importance in academic research,
therapeutic medicine, and drug discovery, accompanied by an amplified economic impact.
Biological contamination is when cultures become infected with microorganisms. Since the sources of
culture contamination are ubiquitous as well as difficult to identify and eliminate, no cell culture
laboratory remains unaffected by this concern. Cell line cross-contamination can be a problem for
scientists working with cultured cells. Studies suggest anywhere from 15–20% of the time, cells used in
experiments have been misidentified or contaminated with another cell line. Authentication should be
repeated before freezing cell line stocks, every two months during active culturing and before any
publication of research; data generated using the cell lines.
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4. Literature Reviews
Mycoplasmas in Cell Culture, Rapid Diagnosis of Mycoplasmas.
Michael F. Barile 1, Shlomo Rottem 2
1Laboratory of Mycoplasma Office of Biologics Research, FDA, Bethesda, USA.
2Hebrew University-Hadassah Medical School, Jerusalem, Israel.
(1984) Plenum Press, New York.
This report will briefly review our current knowledge of: 1) the incidence, prevalence and sources of
mycoplasma contamination in cell cultures; 2) the procedures for isolation, detection and identification of
mycoplasmas, including procedures recommended for testing biological products produced for human
use; 3) the effects of mycoplasma contamination and /or infection on the function and activities of various
infected cell cultures; and 4) the recommended procedures for prevention and elimination of mycoplasma
contamination. Extensive reviews on mycoplasma contamination in cell culture have been reported earlier
[1].
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5. Literature Reviews
Mycoplasma Infection of Cell Culture: Effects, Incidence, and Detection, Uses and Standardization of Vertebrate Cell Cultures – In Vitro
Monograph.
Del Guidice RA and Gardella RS.
Department of Human and Animal Cell Cultures, DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig,
Germany.
(1984) Tissue Culture Association, Gaithersburg, Germany.
The contamination of cell cultures by mycoplasmas remains a major problem in cell culture. Mycoplasmas can produce a virtually
unlimited variety of effects in the cultures they infect. These organisms are resistant to most antibiotics commonly employed in cell
cultures. Here they provide a concise overview of the current knowledge on: (1) the incidence and sources of mycoplasma
contamination in cell cultures, the mycoplasma species most commonly detected in cell cultures, and the effects of mycoplasmas on the
function and activities of infected cell cultures; (2) the various techniques available for the detection of mycoplasmas with particular
emphasis on the most reliable detection methods; (3) the various methods available for the elimination of mycoplasmas highlighting
antibiotic treatment; and (4) the recommended procedures and working protocols for the detection, elimination and prevention of
mycoplasma contamination. The availability of accurate, sensitive and reliable detection methods and the application of robust and
successful elimination methods provide powerful means for overcoming the problem of mycoplasma contamination in cell cultures [2].
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6. Literature Reviews
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Assessing the prevalence of mycoplasma contamination in cell culture via a survey of NCBI's RNA-seq archive.
Anthony O. Olarerin-George and John B. Hogenesch.
Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine at the University of
Pennsylvania, Philadelphia, PA 19104, USA
(2015) Nucleic Acids Research.
Mycoplasmas are notorious contaminants of cell culture and can have profound effects on host cell biology by depriving cells of
nutrients and inducing global changes in gene expression. Over the last two decades, sentinel testing has revealed wide-ranging
contamination rates in mammalian culture. To obtain an unbiased assessment from hundreds of labs, we analyzed sequence data
from 9395 rodent and primate samples from 884 series in the NCBI Sequence Read Archive. We found 11% of these series were
contaminated (defined as ≥100 reads/million mapping to mycoplasma in one or more samples). Ninety percent of mycoplasma-
mapped reads aligned to ribosomal RNA. This was unexpected given 37% of contaminated series used poly(A)-selection for mRNA
enrichment. Lastly, we examined the relationship between mycoplasma contamination and host gene expression in a single cell
RNA-seq dataset and found 61 host genes (P < 0.001) were significantly associated with mycoplasma-mapped read counts. In all,
this study suggests mycoplasma contamination is still prevalent today and poses substantial risk to research quality [3].

7. Literature Reviews
7
Mycoplasma infection significantly alters microarray gene expression profiles.
Miller CJ, Kassem HS, Pepper SD, Hey Y, Ward TH, Margison GP.
Bioinformatics Group, Paterson Institute for Cancer Research, Christie Hospital Trust, Manchester, M20, 4BX, UK
(2003) Biotechniques.
Mycoplasmas are the smallest and simplest self-replicating prokaryotes. Most are parasites, and their contamination of primary and
continuous eukaryotic cell lines represents a significant problem in research, diagnosis, and biotechnological production. Unlike bacteria
and fungi, they rarely produce turbid growth or cellular damage, and most are resistant to the antibiotics commonly used in long-term
cell cultures. These factors combine to make them a stubborn contaminant, and recent surveys have shown that they affect up to 87%
of cell lines. Mycoplasmas have also been known to infect the respiratory, gastrointestinal, and urogenital tracts of many patients, often
without any apparent illness. Their ability to cause chromosomal re-arrangements has led some to postulate them as a cause of cancer.
Others have argued that they are cofactors for a va¬riety of conditions including arthritis, Crohn’s disease, and acquired immuno-
deficiency syndrome (AIDS) [4].

8. Biological Contaminants
 Bacteria and fungi Contamination
Bacteria and fungi, including molds and yeasts, are ubiquitous in the environment and are able to quickly colonize and
flourish in the rich cell culture medium.
 Mycoplasmas Contamination
Mycoplasmas are certainly the most serious and widespread of all the biological contaminants, due to their low detection
rates and their effect on mammalian cells.
 Viruses Contamination
Viruses do not provide visual cues to their presence; they do not change the pH of the culture medium or result in turbidity.
 Yeast Contamination
Yeasts are unicellular eukaryotic microorganisms in the kingdom of Fungi, ranging in size from a few micrometers (typically)
up to 40 micrometers (rarely).
 Mould Contamination
Moulds are eukaryotic microorganisms in the kingdom of Fungi that grow as multicellular filaments called hyphae.
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9. Sources of biological contaminants in the
laboratory
 Media, sera or reagents contaminated with mycoplasma.
 Nonsterile supplies, media and solutions.
 Incubators.
 Liquid Nitrogen.
 Airborne particles and aerosols.
 Overuse of antibiotics.
 Improper sealing of culture dishes.
 Other mycoplasma contaminated cell cultures.
 Discard or treat mycoplasma contaminated cells.
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10. Mycoplasma Contamination
Microorganism Profile: Mycoplasma are simple and smallest free-living organisms that lack a cell wall, and they are considered
the self-replicating Bacteria. Because of their extremely small size (typically less than one micrometer), mycoplasma are very
difficult to detect until they achieve extremely high densities and cause the cell culture to deteriorate; until then, there are often
no visible signs of infection. They belong to the bacterial class Mollicutes, whose members are distinguished by their lack of a
cell wall and their plasma-like form. Given their tiny size (~100 nm), mycoplasmas are undetectable by the naked eye or even by
optical microscopy; thus, they typically go undetected for extended periods.
Most common contamination mycoplasma species
Species Frequency - Natural host
 M. orale 20–40% Human
 M. hyorhinis 10–40% Swine
 M. arginini 20–30% Bovine
 M. fermentans 10–20% Human
 M. hominis 10–20% Human
 A. laidlawii 05–20% Bovine
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11. History: Mycoplasmas were first isolated from a contaminated cell culture in 1956. One mycoplasma cell can
grow to 106 colony-forming units per ml within three to five days in an infected cell culture. Eukaryotic cell
cultures contaminated with mycoplasma have titers in the range of 106 to 108 organisms per ml. Frequently,
there are from 100 to 1000 mycoplasmas attached to each infected cell.
Mycoplasma Contamination
Figure 1. Photomicrographs of mycoplasma-free cultured cells (panel A) and cells infected with mycoplasma (panels
B and C).
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12. Effects of mycoplasma contamination on cell cultures
General effects on eukaryotic cells
– Altered levels of protein, RNA and DNA synthesis
– Alteration of cellular metabolism
– Induction of chromosomal aberrations (numerical and structural alterations)
– Change in cell membrane composition (surface antigen and receptor expression)
– Alteration of cellular morphology
– Induction (or inhibition) of lymphocyte activation
– Induction (or suppression) of cytokine expression
– Increase (or decrease) of virus propagation
– Interference with various biochemical and biological assays
– Influence on signal transduction
– Promotion of cellular transformation
– Alteration of proliferation characteristics (growth, viability)
– Total culture degeneration and loss
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13. Consequences of mycoplasma contamination
Because of their typically high concentrations in cell cultures, mycoplasmas can often out compete with
the host cells for essential nutrients resulting in altered growth and production of proteins. In essence, the
cells no longer behave the way they should. Some of the effects are due to the removal of key medium
components by the mycoplasmas leading to diminished ATP levels, cytotoxicity and culture starvation.
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14. Detection of mycoplasma contamination
 Histological staining
Histochemical stains and light microscopy
 Electron microscopy
Transmission electron microscopy Scanning electron microscopy
 Biochemical methods
Enzyme assays, Gradient/electrophoresis separation
of labeled RNA, Protein analysis.
 Immunological procedures
Fluorescence/enzymatic staining with antibodies
ELISA
Autoradiography
 DNA fluorochrome staining
DAPI stain
Hoechst 33258 stain
 Microbiological culture
Colony formation on agar
 RNA hybridization
Filter hybridization
Liquid hybridization
 Polymerase chain reaction:
Species-/genus-specific PCR primers
Universal PCR primers
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15. Microbiological colony assay
Broths are transferred to agar plates after 4–7 days of incubation. Most mycoplasmas produce microscopic
colonies (100–400 μm in diameter) with a ‘fried egg’ For decades the mainstay of mycoplasma detection was
based on standard microbiological culture procedures. Specimens are inoculated into mycoplasma broth and
onto agar. Anaerobic incubation is recommended as aerobic incubation yields a lower detection rate.
appearance growing embedded beneath the surface of the agar (Figure 2). This procedure has the advantage
of ease of manipulation and visual recognition of colonies.
Figure 2. Detection of Mycoplasma Contamination by Colony Growth on
Agar. Mycoplasmas (M. arginini) from the human suspension cell line U-937.
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16. Mycoplasma elimination methods
 Physical procedures:
– Heat treatment
– Filtration through microfilters
– Induction of chromosomal or cell membrane damage with
photosensitizing
 Chemical procedures:
– Exposure to detergents
– Washings with ether-chloroform
– Treatment with methyl glycine buffer
– Incubation with sodium polyanethol sulfonate
– Culture in 6-methylpurine deoxyriboside
 Immunological procedures:
– Co-cultivation with macrophages
– In vivo passage through nude mice
– Culture with specific anti-mycoplasma antisera
– Exposure to complement
– Cell cloning
 Chemotherapeutic procedures:
– Antibiotic treatment in standard culture
– Antibiotic treatment plus hyper immune sera or
cocultivation with macrophages
– Soft agar cultivation with antibiotics
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17. Antibiotic treatment for elimination of mycoplasma
Mycoplasmas, which lack a cell wall and are incapable of peptidoglycan synthesis, are theoretically not
susceptible to antibiotics such as penicillin and its analogues, which are effective against most bacterial
contaminants of cell cultures.
Effective anti-mycoplasma antibiotics Brand name Generic name Antibiotic
category
 BM-Cyclin Tiamulin Macrolide
 Minocycline Tetracycline
 Ciprobay Ciprofloxacin Quinolone
 Baytril Enrofloxacin Quinolone
 Zagam Sparfloxacin Quinolone
Mycoplasma elimination methods
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18. Prevention of mycoplasma contamination
1. Laboratory should be kept clean.
2. Animals should not be kept in the cell culture room.
3. Cell culture materials should be properly disposed off by
central sterilization.
4. Effective house keeping procedures should be followed to
minimize contamination of the environment.
5. Work surfaces should be chemically disinfected prior to
and following work and thoroughly cleaned at regular
intervals.
6. Facility should be designed and equipped for aseptic
culture procedures.
7. Reliable mycoplasma detection methods should be
established and performed.
8. Mycoplasma-positive cultures should be immediately
discarded (or cryopreserved) or treated with
mycoplasmacidal measures.
9. Protective clothing to protect both the culture and the
culturist.
10. Unauthorized persons should not be allowed entry.
11. Incubators should be regularly controlled and cleaned
(monthly).
12. Discarded glass and plastic ware and spent media should
be carefully disinfected.
13. Incoming cell cultures should be kept in quarantine until
proven sterile (or at least separated in time and space
from sterile cultures).
14. Antibiotic-free media should be used whenever possible.
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19. Conclusion
 Many cell lines that are widely used for biomedical research have been contaminated and overgrown by other, more
aggressive cells. This is a list of cell lines that have been cross-contaminated and overgrown by other cells.
 The main sources of mycoplasma contamination in a cell culture laboratory are animal-derived media products,
laboratory personnel and cross contamination of other contaminated cell lines. It is common to have up to 107
mycoplasma with human, bovine or porcine origin per milliliter of cell culture supernatants, but the appearance and the
behavior of cell cultures are quite normal. Mycoplasmas cannot be seen by visual examination or light microscopy.
 Practically, Elimination of mycoplasma is very difficult, with antibiotics or even the newly developed "mycoplasma
elimination reagents". However, some mycoplasma species can escape from elimination procedures and invade cultured
eukaryotic cells. This is due to the mechanism of internalized mycoplasmas that leave the cells and contaminate new
cells. Therefore, the easiest way to avoid mycoplasma contamination in cell cultures is to examine them periodically. A
routine mycoplasma examination can reduce the hazard of concealed mycoplasmas in cell cultures. Regular quality
control and biosafety testing are as important as high GMP standards in biotechnological and biopharmaceutical
industries. The best approach to fighting contamination is for each person to keep records of all cell culture work
including each passage, general cell appearance, and manipulations including feeding, splitting, and counting of cells.
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20. References
1. Barile MF and Rottem S, Mycoplasmas in Cell Culture, Rapid Diagnosis of Mycoplasmas; Plenum Press, New
York, 1993: 155–193.
2. Del Guidice RA and Gardella RS, Mycoplasma Infection of Cell Culture: Effects, Incidence, and Detection, Uses
and Standardization of Vertebrate Cell Cultures – In Vitro Monograph, Tissue Culture Association, Gaithersburg;
5, 1984: 104–115.
3. Olarerin-George AO and Hogenesch JB, Assessing the prevalence of mycoplasma contamination in cell culture
via a survey of NCBI’s RNA-seq archive, Nucleic Acids Research; 5, 2015: 2535-2542.
4. Miller CJ, Mycoplasma infection significantly alters microarray gene expression profiles, Biotechniques; 35(4),
2003: 812-814.
5. Drexler HG and Uphoff CC, Contamination of Cell Cultures, Mycoplasma, The Encyclopedia of Cell Technology;
Wiley, New York, 1991: 609–627.
6. Hay RJ, Macy ML and Chen TR, Mycoplasma infection of cultured cells, Nature; 339, 1989: 487–488.
7. Armstrong SE, The scope of mycoplasma contamination within the biopharmaceutical industry, Biologicals;
38(2), 2010: 211-213.
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