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President, Vaccination News, A Non-Profit Corporation

Re: problems with detection of cell culture contamination

Return to: Scandals: On "mad cows" and sick monkeys: From the people who brought you SV40 in vaccines....

Methods Mol Med. 2004;88:319-26. Related Articles, Links
Click here to read 
Detecting Mycoplasma contamination in cell cultures by polymerase chain reaction.

Uphoff CC, Drexler HG.
Contamination of cell cultures with bacteria, fungi, and yeasts represents a major problem in cell culture. Whereas these microorganisms are easily detected during routine cell culture by the turbidity of the culture and observation under the inverted microscope, one class of bacteria regularly evades detection. These bacteria belong to the class of Mollicutes, commonly known as mycoplasma. Mycoplasma may persist undetected in cell cultures for a long time without visibly affecting the culture. Nevertheless, mycoplasma can cause extensive alterations in the cell cultures. The frequency of contamination is about 10-30% with pronounced variations in series regarding the laboratory, type of cell culture examined, and the origin of the culture, for example. The infecting mycoplasmas are limited to a few species of the genera Mycoplasma and Acholeplasma with human, swine, and bovine as predominant natural hosts. The ubiquitous use of cell cultures and their transfer from one laboratory to another has lead to widespread dissemination of such infections. Hence, adequate detection methods need to be established and frequently employed in every laboratory applying cell cultures. Every incoming cell culture should be kept in quarantine until mycoplasma detection assays are completed and the infection status is determined. Positive cultures should either be discarded and replaced by clean cultures or cured with specific antibiotics. Only clean cultures should be used for research experiments and for the production of biologically active pharmaceuticals. Additionally, stringent rules for the prevention of further mycoplasma contamination of cell cultures should be followed (2).

DSMZ German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany.
Comment:  Although this and many other articles on contamination with mycoplasma are directed to the effect it has on cells for research purposes, mycoplasma has the potential to cause disease.  Here is what Joseph Tully wrote in Mycoplasma Infection of Cell Cultures (1978): "Mycoplasmas were initially recognized as important animal-pathogens.  They were found to involve a variety of organs and tissues, and to be be associated with acute and chronic respiratory disease, mastitis, polyarthritis, and genitourinary tract infections (113).  The concept of a vast noormal flora of mycoplasmas in animals (and including man) was not well appreciated until major advances occurred in the cultivation of the more fastidious mycoplasmas, such as M. pneumoniae (3). From that time in the early 1960s, we have seen a continual extension of the number of mycoplasmas in each animal host selectively surveyed.  The great majority of these mycoplasmas appear to be strictly part of the normal microbial flora.  However, it is important to stress that limited information is available on the interrelationships of normal mycoplasma flora and other microbial agents, including virulent as well as avirulent bacteria, viruses, etc.  Thus, normal flora might still play some role in human or animal diseases when acting in concert with other microbial agents or when the host is compromised in some way."
PMID: 14634244 [PubMed - in process]

Biologicals. 2003 Sep;31(3):203-8. Related Articles, Links
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Bovine viral diarrhoea virus antigen in foetal calf serum batches and consequences of such contamination for vaccine production.

Makoschey B, van Gelder PT, Keijsers V, Goovaerts D.

Virological R&D Department, Intervet International b.v., Wim de Korverstraat 35, NL-5831 AN, Boxmeer, The Netherlands.

A protocol to test foetal calf serum (FCS) for contamination with bovine viral diarrhoea virus (BVDV) is described. Following this protocol, which combines cell culture methods and detection of pestivirus RNA, seven batches of FCS were tested. Infectious BVDV was detected in four of those batches. One of the remaining batches contained a relatively high number of non-infectious BVDV particles. A sample of this batch was formulated with aluminium hydroxide and aluminium phosphate as adjuvant into an experimental vaccine preparation. This product was injected twice into BVDV seronegative cattle with a 4 week interval. Blood samples taken 4 weeks after the second application were negative for BVDV specific antibodies. Our data stress that detection of BVDV RNA is not sufficient for a complete risk assessment on FCS. Discrimination between infectious and non-infectious BVDV is essential. This can only be achieved by cell culture methods.

PMID: 12935809 [PubMed - in process]

Chang Gung Med J. 2003 Apr;26(4):250-8. Related Articles, Links

Detection and treatment of mycoplasma contamination in cultured cells.

Jung H, Wang SY, Yang IW, Hsueh DW, Yang WJ, Wang TH, Wang HS.

Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Taipei, ROC.

BACKGROUND: Mycoplasmas, the smallest and simplest prokaryotes that reside in endosomes of mammalian cells, are widespread contaminants found in cell cultures. About 30% of all cell cultures, varying from 15 to 80%, are reportedly contaminated with mycoplasmas. Here, we present our experience in successfully detecting and treating mycoplasmal infection in various cell lines. METHODS: The nested polymerase chain reaction (PCR) detection and microscopic examination, including phase-contrast, fluorescent, as well as differential interference contrast, were used for detecting potential mycoplasma contamination of cell lines used in our laboratory. As soon as mycoplasma was identified, antibiotic treatment was initiated. RESULTS: Mycoplasmal contamination was detected in six of 15 cell lines using the nested PCR amplification of mycoplasma DNA, which was further demonstrated using 4, 6-Diamidino-2-phenylindole (DAPI) staining and fluorescent microscopy. Alternate treatment with two antibiotics, macrolide (tiamulin) and tetracycline (minocycline), effectively eliminated mycoplasma, which was validated by both PCR and microscopic studies. CONCLUSIONS: The nested PCR using genomic DNA extracted from cultured cells as templates is a rapid and sensitive method for detecting mycoplasma contamination. Treatment with combined antibiotics can completely eradicate mycoplasmal infection from cultured cells. For the ease of use, PCR and/or DAPI staining appear suitable for detecting potential mycoplasmal contamination in laboratories that rely heavily on the cell culture system.
PMID: 12846524 [PubMed - in process]

J Vet Diagn Invest. 2002 Mar;14(2):120-5. Related Articles, Links

Detection of bovine viral diarrhea virus by TaqMan reverse transcription polymerase chain reaction.

Mahlum CE, Haugerud S, Shivers JL, Rossow KD, Goyal SM, Collins JE, Faaberg KS.

Department of Veterinary Diagnostic Medicine, University of Minnesota, St. Paul 55108, USA.

Detection and elimination of calves and cows persistently infected with bovine viral diarrhea virus (BVDV) is important for the control of this pathogen. Historically, BVDV detection involved cell culture isolation followed by virus detection through immunofluorescence or immunoperoxidase monolayer assay (IPMA) methods. More recently, immunohistochemistry (IHC) has been added as a routine test for BVDV detection. The detection of BVDV by gel-based reverse transcription polymerase chain reaction (RT-PCR) is more sensitive and rapid than by cell culture isolation, but test results can be compromised by sample contamination during nucleic acid amplification. This study was designed to develop a closed-tube format of BVDV nucleic acid amplification and detection, TaqMan RT-PCR. The results of this new technique were compared with those obtained with virus isolation, IPMA, and IHC. With TaqMan RT-PCR, BVDV was detected in many samples negative by IPMA, IHC, and virus isolation with the exception of 1 sample that was positive by IHC. TaqMan RT-PCR in a closed-tube format offers a rapid, economical, high volume, and sensitive method for BVDV detection without the concerns of amplified cDNA product contamination associated with open-tube gel-based PCR tests.

PMID: 11939332 [PubMed - indexed for MEDLINE]

In Vitro Cell Dev Biol Anim. 2002 Feb;38(2):79-85. Related Articles, Links
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Comparative PCR analysis for detection of mycoplasma infections in continuous cell lines.

Uphoff CC, Drexler HG.

Department of Human and Animal Cell Cultures, DSMZ-German Collection of Microorganisms & Cell Cultures, Braunschweig.

Mycoplasma contamination of cell lines is one of the major problems in cell culturing. About 15-35% of all cell lines are infected with a limited number of mycoplasma species of predominantly human, swine, or bovine origin. We examined the mycoplasma contamination status in 495 cell cultures by polymerase chain reaction (PCR) assay, microbiological culture method, and deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization, and in 103 cell cultures by PCR and DNA-RNA hybridization, in order to determine the sensitivity and specificity of the PCR assay in routine cell culture. For those two cohorts, results for the three or two assays were concordant in 92 and 91% of the cases, respectively. The sensitivity (detection of true positives) of this PCR detection assay was 86%, and the specificity (detection of true negatives) was 93%, with positive and negative predictive values (probability of correct results) of 73 and 97%, respectively. PCR defined the mycoplasma status with 92% accuracy (detection of true positives and true negatives). The mycoplasma contaminants were speciated by analyzing the PCR amplification fragment using several restriction enzymes. Most of the cultures (47%) were infected with Mycoplasma fermentans, followed by M. hyorhinis (19%), M. orale (10%), M. arginini (9%), Acholeplasma laidlawii (6%), and M. hominis (3%). To sum up, PCR represents a sensitive, specific, accurate, inexpensive, and quick mycoplasma detection assay that is suitable for the routine screening of cell cultures.

PMID: 11928999 [PubMed - indexed for MEDLINE]


J Vet Diagn Invest. 2001 May;13(3):261-2. Related Articles, Links

Evaluation of a new sandwich enzyme-linked immunosorbent assay for detection of bovine viral diarrhea virus in unprocessed fetal bovine serum.

Plavsic MZ, Prodafikas G.

Life Technologies, Grand Island Cell Culture R&D, NY 14072, USA.

A new sandwich enzyme-linked immunosorbent assay (S-ELISA) kit that uses raw (unprocessed) fetal bovine serum (FBS) as the testing sample was evaluated for upstream bovine viral diarrhea virus (BVDV) testing. Pooled FBS samples (n = 84) were tested using the S-ELISA. Thirty serum samples originating from persistently infected (PI) calves that had been confirmed by virus isolation (VI) as BVDV positive and another 30 samples previously confirmed by VI as BVDV negative were also evaluated. Of the 84 field samples, the S-ELISA detected 13 (15.5%) BVDV-positive specimens. When these 13 positive samples were tested by VI and immunofluorescent assay, 11 (84.6%) were positive and 2 (15.4%) were negative. The S-ELISA was positive for all 30 PI samples (100%) and negative for all 30 negative samples (100%). These data indicate that the new kit is a relatively reliable diagnostic tool and can be considered for upstream detection of BVDV-contaminated raw FBS pools.

Publication Types:
  • Evaluation Studies

PMID: 11482608 [PubMed - indexed for MEDLINE]

PDA J Pharm Sci Technol. 2001 Nov-Dec;55(6):337-45. Related Articles, Links

A method for the rapid detection of microbial contaminants in animal cell culture processes.

Onadipe A, Ulvedal K.

Lonza Biologics plc, Slough, Berkshire, UK.

The early detection of microbial contamination is an important issue in the production of biopharmaceuticals using animal cell culture systems. A new method, based on the ChemScan RDI analyser, was evaluated for the rapid detection and enumeration of low concentrations of microorganisms within a large population of animal cells and in cell culture media. The method was tested with suspension cell cultures and is applicable to adherent cell cultures. In both cases, the method uses an initial step to eliminate the animal cells followed by collection and fluorescent labelling of the viable microbial contaminants on a filter membrane. Total counts of the viable microorganisms were obtained after analysis of the membrane by the ChemScan RDI analyser. The results showed that the ChemScan RDI detected individual bacterial cells after filtration of the pre-treated animal cell culture. The detection limit of the ChemScan RDI was less than 10 bacteria/ml in cell culture containing 10(6) mammalian cells/ml and one bacterium in 500 ml of cell culture medium. A strong correlation between the standard plate count and the ChemScan RDI was observed, even at low bacterial concentrations. The total time for each analysis was less than two hours.

PMID: 11766821 [PubMed - indexed for MEDLINE]

Rev Argent Microbiol. 2000 Jan-Mar;32(1):27-32. Related Articles, Links

[Contamination of bovine fetal serum with bovine viral diarrhea virus]

[Article in Spanish]

Zabal O, Kobrak AL, Lager IA, Schudel AA, Weber EL.

Instituto de Virologia, CICVyA, INTA Castelar, Buenos Aires, Argentina.

Fetal bovine serum (FBS) used in cell culture may be contaminated with viruses, among them bovine viral diarrhea virus (BVDV) affecting the production of biological reagents and the results of diagnosis. The filtration process used in the preparation of commercial FBS abrogates most viral agents that may be present in raw FBS, but BVDV may pass through the filters because of its small size and its pleomorphism. While detection of bovine herpes virus-1 and parainfluenza-3 (PI-3) is determined by observation of the cytopathic effect, and also by hemadsorption in the case of PI-3, the most frequently isolated BVDV is non cytopathic, and infects cells without morphological alterations, inducing problems that arise after several cell generations. Batches of raw and processed FBS were analyzed. Frequencies of BVDV detection in raw serum in Argentina were similar to those published for USA. By conventional methods for BVDV detection, only 2 of 20 commercial batches of FBS had BVDV. Using cell cultures maintained with high concentrations of the serum under study for at least 2 weeks, with detection of viral antigen by indirect immunofluorescence, the percentage of BVDV detection was 80%. This method shows that most lots of commercial FBS contain BVDV. RT-PCR allows faster detection of the viral genome, but it must be validated, as it does not show viral replication. To eliminate the problem of BVDV contamination in FBS, only gamma irradiated FBS is used in our laboratory.

PMID: 10785940 [PubMed - indexed for MEDLINE]

Br J Biomed Sci. 2000;57(4):295-301. Related Articles, Links

Mycoplasma detection in cell cultures: a comparison of four methods.

Garner CM, Hubbold LM, Chakraborti PR.

Oncology Research Laboratory, Derby Cancer Centre, Derby City General Hospital, Uttoxeter Road, Derby DE22 3NE, UK.

Mycoplasma is a common contaminant of tissue culture samples. Infection is persistent, difficult to detect and diagnose, and very difficult to cure. The concentration of mycoplasma in infected cultures can be as high as 10(7) colony-forming units per mL, and their presence can change many of the cell reactions, including altering cell growth rate, inducing morphological changes or cell transformation, and mimicking virus infection. Therefore, it should be assumed that a mycoplasma-contaminated cell line may be significantly influenced in every respect, and, thus, experimental data derived from such a cell line is likely to be invalid. Contamination is not obvious, either macroscopically or microscopically; thus, routine mycoplasma testing is essential for any cell culture laboratory. Many of the testing procedures developed so far are time-consuming, expensive, inconclusive and unsuitable for screening large numbers of test specimens. This study compares DNA staining, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) and PCR ELISA, to determine which is the best procedure for routine assessment of cell cultures. All four methods gave reproducible results with both infected and non-infected cell lines. Both ELISA methods were easy to perform, reproducible and easily interpreted.

Publication Types:
  • Evaluation Studies

PMID: 11204859 [PubMed - indexed for MEDLINE]

Hum Cell. 1999 Dec;12(4):229-36. Related Articles, Links

Detection of mycoplasma contaminations in cell cultures by PCR analysis.

Uphoff CC, Drexler HG.

DSMZ-German Collection of Microorganisms & Cell Cultures, Department of Human and Animal Cell Cultures, Braunschweig, Germany.

Mycoplasma contamination is still one of the main problems in using cell cultures in biological and medical research and in the production of bioactive substances, because mycoplasma can alter nearly all parameters and products of the cell. They can persist undetected in the culture if no special detection methods are applied. In recent years, the PCR technology has become a commonly used method to analyze genomic DNA and the expression of genes, with both high specificity and sensitivity. This technique can be effectively employed for the detection and even the identification of mycoplasma contaminations in cell cultures applying primers complementary to the 16S rDNA region. Although this technique, once established, is characterized by simplicity and speed, PCR is still a complex process and its sensitivity and specificity can be influenced by a number of different parameters, e.g. inhibiting compounds originating from the preparation process of the DNA, RNA or cDNA, contamination of the solutions with PCR products, and the selection of a primer pair which does not cover all the mycoplasma species occurring in cell cultures. Thus, adequate controls have to be included to obtain reliable results. The present review examines the use of different primers of the 16S rDNA region including their specificity, the sensitivity applying various DNA or RNA preparation procedures, and the methods to detect finally the amplicons. In conclusion, basic nucleic acid preparation and PCR product detection methods offer a simple, fast and reliable technique for the examination of mycoplasma contaminations in cell cultures, provided that the indispensable control assays are implemented.

Publication Types:
  • Review
  • Review, Tutorial

PMID: 10834110 [PubMed - indexed for MEDLINE]

Methods Cell Biol. 1998;57:49-65. Related Articles, Links

Cell culture contamination: sources, consequences, prevention, and elimination.

Lincoln CK, Gabridge MG.

Bionique Testing Laboratories, Inc., Saranac Lake, New York 12983, USA.

The subject of the chapter is cell culture contamination. Contamination may enter the cell culture system as a physical, chemical, and/or biological component of the environment. The potential sources and consequences of cell culture contamination are unique to the cell culture system and the contaminant. A basic understanding of cell culture contamination is necessary to appreciate the need to develop and practice standardized cell culture procedures. General sources, consequences, and preventative measures are discussed for physical and chemical contamination based on current technology. Mycoplasmal contamination is the focus of the discussion on biological contamination and its impact on cell cultures. The introduction of other biological contaminants should be controlled by the institution of cell culture management procedures needed to minimize the incidence of mycoplasmal contamination. The need to eliminate the routine use of antibiotics in cell culture systems and institute routine testing to detect contamination is emphasized. More rapid detection of contamination should reduce the incidence of cross-contamination and minimize the consequences of any contamination event.

Publication Types:
  • Review
  • Review, Tutorial

PMID: 9648099 [PubMed - indexed for MEDLINE]

Rev Sci Tech. 1998 Dec;17(3):733-42. Related Articles, Links

Use of polymerase chain reaction to simultaneously detect and type bovine viral diarrhoea viruses isolated from clinical specimens.

el-Kholy AA, Bolin SR, Ridpath JF, Arab RM, Abou-Zeid AA, Hammam HM, Platt KB.

Veterinary Sera and Vaccines Researches Institute, Genetic Engineering Research Unit, Cairo, Egypt.

The techniques of indirect immunofluorescence (IF), immuno-peroxidase (IP) staining and the one-step reverse transcriptase polymerase chain reaction (RT-PCR) were compared for detection of 102 isolates of bovine viral diarrhoea virus (BVDV) in infected cell cultures. The BVDV was obtained from bovine clinical specimens, including sera, buffy coats and tissues, submitted from farms located in the States of Iowa and Wisconsin, United States of America. The IF technique detected 88/102 (86.3%) of the viral isolates, whereas IP staining detected an additional 4 isolates (92/102; 90%). The one-step RT-PCR using primers derived from the 5' untranslated region of the BVDV genome detected 102/102 (100%) of the BVDV isolates. A second-round PCR utilising another pair of PCR primers from the 5' untranslated region, allowed rapid genotyping of BVDV. The procedure used showed that the PCR assay based on the 5' untranslated region of the virus genome is the most sensitive indicator for BVDV detection in cell culture, and is also of considerable epidemiological importance since it allowed rapid genotyping of BVDV isolated from clinical specimens. In addition to detection and genotyping of BVDV isolated from clinical specimens, the RT-PCR procedure can be used for routine screening of locally produced and imported biologicals for BVDV contamination. However, the procedure requires further refinement to enable direct application on the clinical specimen.

PMID: 9850544 [PubMed - indexed for MEDLINE]

Methods Mol Biol. 1997;75:305-11. Related Articles, Links

Routine testing of cell cultures and their products for mycoplasma contamination.

Stacey A, Doyle A.

European Collection of Cell Cultures, Centre for Applied Microbiology and Research, Salisbury, Wilts, UK.

Publication Types:
  • Review
  • Review, Tutorial

PMID: 9276280 [PubMed - indexed for MEDLINE]

Folia Microbiol (Praha). 1997;42(2):113-6. Related Articles, Links

Standardization in animal cell technology.

Stacey GN.

European Collection of Cell Cultures, Centre for Applied Microbiology and Research, Porton Down, Salisbury, UK.

Continuous cell lines offer a level of reproducibility, and thus standardization, which cannot normally be achieved using primary cells. However, even with continuous cell lines adoption of correct cell banking and appropriate quality control procedures are critical to the provision of reliable, reproducible and safe cell stocks. These procedures enable establishment of cryopreserved stocks of pure cultures of correct identity and phenotype which are free from adventitious agents. In addition to quality control techniques, culture conditions and growth medium used often require standardization. In particular different sources of serum, growth factors and cell attachment substrates may lead to significant variation in the 'performance' of cell lines. To ensure a high degree of reliability it is essential to obtain cells from authenticated and quality controlled sources. In culture collections, the principles of correct cell banking should be applied with appropriate quality control for which the minimum standard should be confirmation of viability and mycoplasma testing. Such approaches will afford increased confidence in research data and avoid the waste of time and resources which result from the use of cross-contaminated or infected cells.

Publication Types:
  • Review
  • Review, Tutorial

PMID: 9306654 [PubMed - indexed for MEDLINE]

J Vet Diagn Invest. 1997 Oct;9(4):427-31. Related Articles, Links

A method to detect bovine viral diarrhea virus contamination in cell cultures using immunoperoxidase staining.

Castro MD, Stoffregen WC, Brigman GP, Hillard KA.

Department of Veterinary Sciences, College of Agricultural Sciences, Pennsylvania State University, University Park 16802, USA.

PMID: 9376437 [PubMed - indexed for MEDLINE]


Biologicals. 1996 Jun;24(2):131-5. Related Articles, Links
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Detection of bovine polyomavirus contamination in fetal bovine sera and modified live viral vaccines using polymerase chain reaction.

Kappeler A, Lutz-Wallace C, Sapp T, Sidhu M.

Biologics Evaluation Laboratory, Animal Diseases Research Institute, Agriculture and Agri-Food Canada, Nepean, Ontario, Canada.

A nested polymerase chain reaction (PCR) assay has been developed for the detection of bovine polyomavirus (BPyV) DNA. The assay has been used to screen commercial lots of fetal bovine serum and modified live veterinary vaccines for the presence of the agent. A PCR product of the expected size was detected after the first round of PCR for eight out of 20 serum lots, but in none of the 14 vaccines tested. The subsequent nested assay revealed that four more serum lots were positive for BPyV DNA, as well as two vaccine lots. When hybridized with a labelled probe, blots of the PCR products from vaccines revealed that in one of the two positive samples a specific product was present after the first PCR at a level not detectable in gel electrophoresis. Nested PCR appears to be a useful tool for the detection of low level contamination with BPyV DNA of products used in, and derived from cell culture.

PMID: 8889060 [PubMed - indexed for MEDLINE]

J Histochem Cytochem. 1996 Jan;44(1):75-6. Related Articles, Links

Rapid screening and detection of cellular cross-contamination in cell cultures: a new application of cytochemistry.

Markovic O, Hay RJ, Steenbergen K.

Publication Types:
  • Letter

PMID: 8543786 [PubMed - indexed for MEDLINE]

Dev Biol Stand. 1996;88:163-5. Related Articles, Links

A comparison of methods for the estimation of retroviral burden.

Bierley ST, Raineri R, Poiley JA, Morgan EM.

IEM Section, Pathology Associates International, Durham NC, USA.

The presence of retroviral contamination is of vital concern in the manufacture of cell culture-derived biopharmaceuticals. These cell lines usually have A- or C-type retrovirus-like particles which are visible by transmission electron microscopy (TEM) even when infectivity (IF) or reverse transcriptase activity (RTA) cannot be demonstrated. The supernatant of the post-production cell cultures, therefore, also needs to be evaluated by TEM for viral burden. A major question, however, is how to establish a quantitative viral load estimate for the evaluation of a purification process. The FDA recommends that a purification process for viral contaminants remove or inactivate 3-5 logs over the estimated viral burden. Viral particles are difficult to identify and quantify, however, by conventional negative staining. We present a comparison of infectivity assay, reverse transcriptase assay, negative staining, and thin sectioned TEM. These assays were performed on four samples. Ultracentrifuged sediments of cleared cell-culture media were measured, fixed and processed. Thin sections were evaluated by TEM and the number of viral particles estimated by morphometric derived quantification. Retrovirus particles were easily identified and quantified when examined by TEM as compared to negative staining and correlated with the other viral assays (IF, RTA). These results demonstrate that the TEM thin section method was a superior technique to negative staining for estimating viral particle load in cell-culture supernatant. To validate further the plastic embedding with thin sectioning, we evaluated cell culture supernatants (pellets) for retroviral burden at various dilutions, from two cell lines. Morphometric determinations were made of the number of viral particles present per unit volume and compared to results obtained by infectivity assay. Since the morphometric calculation for viral density assumes even distribution of viral particles, we also evaluated and calculated viral counts on multiple thin sections taken throughout selected pellets.

PMID: 9119132 [PubMed - indexed for MEDLINE]

PCR Methods Appl. 1995 Feb;4(4):199-208. Related Articles, Links

Advances in PCR-based detection of mycoplasmas contaminating cell cultures.

Rawadi G, Dussurget O.

Departement de Bacteriologie et Mycologie, Institut Pasteur, Paris, France.

Publication Types:
  • Review
  • Review, Tutorial

PMID: 8574187 [PubMed - indexed for MEDLINE]

Pathobiology. 1995;63(1):9-11. Related Articles, Links

Detection by polymerase chain reaction of all common Mycoplasma in a cell culture facility.

Pruckler JM, Pruckler JM, Ades EW.

Biological Products Branch, Centers for Disease Control and Prevention, Atlanta, Ga 30333, USA.

The identification of cell cultures contaminated with organisms from the class Mollicutes has led us to examine the effectiveness of polymerase chain reaction (PCR) for detecting these organisms in genomic DNA. We developed a previously identified nested PCR primer set and compared its ability to detect Mycoplasma with that of a commercially available PCR kit for detecting Mycoplasma. We found that although the commercial system detected and identified a few of the most common Mycoplasma species, the primer set (GPO-1, GPO-2, MGSO) detected the presence of all the common Mycoplasma species and many of the rare mycoplasma species previously encountered in tissue culture.

PMID: 7546276 [PubMed - indexed for MEDLINE]

J Vet Med Sci. 1995 Aug;57(4):769-71. Related Articles, Links

Rapid detection of mycoplasma contamination in cell cultures by enzymatic detection of polymerase chain reaction (PCR) products.

Kobayashi H, Yamamoto K, Eguchi M, Kubo M, Nakagami S, Wakisaka S, Kaizuka M, Ishii H.

National Institute of Animal Health, Ibaraki, Japan.

Enzymatic detection of polymerase chain reaction (ED-PCR) was applied for rapid and easy identification of mycoplasmas from contaminated cell culture. This method was based on the capture of amplified products via biotin-streptavidine affinity and the detection of an incorporated hapten in amplified products with enzyme-linked antibody. Primers corresponding to common sequence of Mollicutes in 16S ribosomal RNA dominated gene was used. Nineteen of twenty Mollicutes so far reported as cell contaminants appeared positive by ED-PCR, whereas remaining one, Acholeplasma axanthum, appeared negative. Samples from sixty-two cell culture were tested for contamination of mycoplasmas by means of ED-PCR, cultivation, and electronmicroscopy. The results of ED-PCR were the same as those of cultivating method. The time required for all the detection process in ED-PCR was about 5 hr for 20 samples. We suggest that ED-PCR can be used in the rapid detection of mycoplasms from cell culture.

PMID: 8519917 [PubMed - indexed for MEDLINE]

Tanpakushitsu Kakusan Koso. 1995 Nov;40(15):2361-8. Related Articles, Links

[Simple detection of the contamination in animal cell cultures]

[Article in Japanese]

Harasawa R, Mizusawa H, Takeuchi M.

Animal Center for Biomedical Research, Faculty of Medicine, University of Tokyo, Japan.

Publication Types:
  • Review
  • Review, Tutorial

PMID: 8532895 [PubMed - indexed for MEDLINE]

J Reprod Fertil. 1995 Sep;105(1):17-24. Related Articles, Links

Detection of bovine viral diarrhoea virus antigen and RNA in oviduct and granulosa cells of persistently infected cattle.

Booth PJ, Stevens DA, Collins ME, Brownlie J.

Institute for Animal Health, Compton, Newbury, Berkshire, UK.

Large-scale in vitro bovine embryo production systems commonly use genital tracts obtained from an abattoir as a source of both cumulus-oocyte complexes and co-culture feeder cells. Tissues derived from this source may be contaminated with non-cytopathogenic bovine viral diarrhoea virus (BVDV) since, in several countries surveyed, approximately 1% of animals tested are persistently infected with this pathogen. Therefore, the use of such material in in vitro fertilization systems presents a potential risk for the transmission of BVDV to bovine embryos and via embryo transfer. This potential was investigated by obtaining oviduct epithelial cells and granulosa cells, which are commonly used as feeder cells, from cattle persistently infected with BVDV and examining them for the presence of BVD viral antigen (p80 non-structural protein and gp53 envelope glycoprotein) by indirect immunofluorescent histochemistry, and also viral RNA (encoding the p80 region) by in situ hybridization. In addition, titres of virus present in oviduct, ovary and blood were assayed by immunodetection on calf testis cell cultures. Luminal epithelial cells from the oviduct and primary cultures of granulosa cells and oviduct epithelial cells from such cattle were shown to contain both viral antigen and RNA. The susceptibility of both cell types to BVDV infection was further established by inoculating primary cell cultures of cells derived from cattle not infected with BVDV with a cloned isolate of non-cytopathogenic BVDV (Pe515). RNA encoding BVDV and the antigen were detected 12 h after inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 7490710 [PubMed - indexed for MEDLINE]

Appl Environ Microbiol. 1994 Jan;60(1):149-52. Related Articles, Links

Detection of mycoplasma contamination in cell cultures by a mycoplasma group-specific PCR.

van Kuppeveld FJ, Johansson KE, Galama JM, Kissing J, Bolske G, van der Logt JT, Melchers WJ.

Department of Medical Microbiology, University of Nijmegen, The Netherlands.

The suitability of a 16S rRNA-based mycoplasma group-specific PCR for the detection of mycoplasma contamination in cell cultures was investigated. A total of 104 cell cultures were tested by using microbiological culture, DNA fluorochrome staining, DNA-rRNA hybridization, and PCR techniques. A comparison of the results obtained with these techniques revealed agreement for 95 cell cultures. Discrepant results, which were interpreted as false negative or false positive on the basis of a comparison with the results obtained with other methods, were observed with nine cell cultures. The microbiological culture technique produced false-negative results for four cell cultures. The hybridization technique produced false-negative results for two cell cultures, and for one of these cell cultures the DNA staining technique also produced a false-negative result. The PCR may have produced false-positive results for one cell culture. Ambiguous results were obtained with the remaining two cell cultures. Furthermore, the presence of contaminating bacteria interfered with the interpretation of the DNA staining results for 16 cell cultures. For the same reason the hybridization signals of nine cell cultures could not be interpreted. Our results demonstrate the drawbacks of each of the detection methods and the suitability of the PCR for the detection of mycoplasmas in cell cultures.

PMID: 7509584 [PubMed - indexed for MEDLINE]

Trends Biotechnol. 1993 Apr;11(4):143-51. Related Articles, Links

Beware of mycoplasmas.

Rottem S, Barile MF.

Department of Membrane and Ultrastructure Research, Hebrew University-Hadassah Medical School, Jerusalem, Israel.

Mycoplasma infection of cell cultures is widespread and has major detrimental effects on cellular physiology and metabolism. Since cell culture is used extensively, both in research and in industrial production processes, questions of primary concern arise, such as: how can mycoplasma contamination be detected; what are the effects of such contamination on cellular functions; what methods are available for eliminating contamination?

Publication Types:
  • Review
  • Review, Tutorial

PMID: 7763647 [PubMed - indexed for MEDLINE]

Folia Biol (Praha). 1993;39(5):270-6. Related Articles, Links

Comparison of methods used for detection of mycoplasma contamination in cell cultures, sera, and live-virus vaccines.

Benisheva T, Sovova V, Ivanov I, Opalchenova G.

National Drug Institute, Sofia.

Two methods for detection of mycoplasma contamination in cell cultures, sera, and live-virus vaccines were compared: the direct culture test and the DNA staining method employing bisBenzimide (Hoechst No. 33258). Contamination by different species of mycoplasma was found in 39% samples tested. It is recommended to use both techniques for a reliable detection of mycoplasma contamination.

PMID: 8206173 [PubMed - indexed for MEDLINE]

FEMS Microbiol Lett. 1992 Nov 15;78(1):89-94. Related Articles, Links

Detection of bacterial and mycoplasma contamination in cell cultures by polymerase chain reaction.

Spaepen M, Angulo AF, Marynen P, Cassiman JJ.

Center for Human Genetics, University of Leuven, Belgium.

A fast and simple method to detect bacterial and especially mycoplasma contamination in tissue culture by means of polymerase chain reaction (PCR) amplification is described. In a first step the universal primer pairs P1/P2 (190-bp fragment) and P3/P4 (120-bp fragment) directed to different conserved parts of the prokaryotic 16S rRNA gene are used. A positive signal after amplification on cell culture DNA with these primers provides an indication of bacterial infection. Using the internal primers IP1, IP3 and IP'3 complementary to a part of the V4 and V8 variable regions of the 16S rRNA gene, in combination with a universal primer, cultures contaminated with mycoplasma could be identified. Six mycoplasma species, typical contaminants in tissue cultures, were investigated: Mycoplasma orale, M. fermentans, M. arginini, M. hyorhinis, M. hominis and Aeromonas laidlawii. This mycoplasma test is an easy, specific and sensitive assay which should be extremely useful in any tissue culture setting.

PMID: 1468621 [PubMed - indexed for MEDLINE]

Bioprocess Technol. 1990;10:483-94. Related Articles, Links

Methods for the detection of adventitious viruses in cell cultures used in the production of biotechnology products.

Poiley JA.

Hazleton Laboratories America, Inc., Kensington, Maryland.

The possibility for viral contamination exists in established cultures as well as in primary cultures. The use of established genetically engineered cultures in the production of biologicals for human use requires that these cultures be monitored for adventitious viral agents. Among the methods used for this purpose are animal inoculation and in vitro assays which provide a broad-spectrum screen for viral agents.

Publication Types:
  • Review
  • Review, Tutorial

PMID: 1367069 [PubMed - indexed for MEDLINE]

Bioprocess Technol. 1990;10:483-94. Related Articles, Links

Methods for the detection of adventitious viruses in cell cultures used in the production of biotechnology products.

Poiley JA.

Hazleton Laboratories America, Inc., Kensington, Maryland.

The possibility for viral contamination exists in established cultures as well as in primary cultures. The use of established genetically engineered cultures in the production of biologicals for human use requires that these cultures be monitored for adventitious viral agents. Among the methods used for this purpose are animal inoculation and in vitro assays which provide a broad-spectrum screen for viral agents.

Publication Types:
  • Review
  • Review, Tutorial

PMID: 1367069 [PubMed - indexed for MEDLINE]


Dev Biol Stand. 1989;70:59-66. Related Articles, Links

Detection and elimination of adventitious agents in continuous cell lines.

Erickson GA, Landgraf JG, Wessman SJ, Koski TA, Moss LM.

National Veterinary Services Laboratories, Ames, IA 50010.

The National Veterinary Services Laboratories (NVSL) routinely monitors continuous cell lines (CCL's) used for veterinary biologicals and diagnostic virology. All veterinary biologicals produced in CCL's must follow the master seed concept which limits the use of the master seed CCL to up to 20 passages beyond the passage level characterized and deposited at NVSL. All CLL's are evaluated for the presence of adventitious agents such as mycoplasma, bovine viral diarrhea virus, and other bacteria and viruses. Previously, CCLs were evaluated for tumorigenicity by the Syrian hamster cheek pouch method; however, this procedure has now been eliminated. The adventitious agents most frequently detected in CCL's have been bovine viral diarrhea virus and mycoplasma. Our laboratory has consistently found that the source of bovine viral diarrhea contamination of CCLs has been the use of contaminated fetal bovine cell culture enrichment serum. Gamma irradiation at 2.5-3.5 megarads at -40 degrees C of carefully screened fetal bovine serum has been used in the Diagnostic Virology Laboratory for over 10 years. If the irradiated serum is used at a final concentration of 10 percent, there is no untoward effect on cell susceptibility for virus propagation or cell culture growth. Gamma irradiation has also been demonstrated to be a very efficient inactivator of mycoplasma. Specific conditions utilized by our laboratory to preserve fetal bovine serum cell culture growth factors while eliminating adventitious bovine viral diarrhea virus will be presented.

PMID: 2759356 [PubMed - indexed for MEDLINE]

Dev Biol Stand. 1986;64:195-8. Related Articles, Links

Development of in vitro tests for detection of extraneous agents.

Thornton DH, Nicholas RA, Wood GW.

The use of animals in tests to detect extraneous agents is not only undesirable from the ethical viewpoint but also because of the expense and length of time involved in such tests. We have carried out tests on a variety of potential contaminating avian pathogens to determine whether tests in chicks offer any advantage over tests in embryos or cell cultures. In many cases, but not all, in vitro tests were shown to be more sensitive. The use fluorescent antibody or enzyme-linked assays serves to enhance the sensitivity of the tests. In the future it may be possible to adapt techniques such as nucleic acid hybridisation to the detection of extraneous agents.

PMID: 3025038 [PubMed - indexed for MEDLINE]

J Virol Methods. 1985 Aug;11(4):347-55. Related Articles, Links

An enzyme-linked immunosorbent assay, ELISA, for SV40 antigen detection.

Edevag G, Grandien M, Mares I.

The exclusion of SV40 contamination in poliovaccine produced in Cynomolgus monkey kidney cell cultures is routinely done by inoculation of the inactivated vaccine into Cercopithecus monkey kidney cell cultures where a cytopathic effect reveals the presence of the virus. An ELISA is described for the detection of SV40 antigen and the efficiency of antigen detection was compared with the development of cytopathic effect in Cercopithecus tissue cultures. The assay shortened considerably the time for production control and was in full agreement with the conventional test method.

PMID: 2997255 [PubMed - indexed for MEDLINE]

Res Vet Sci. 1984 Nov;37(3):371-3. Related Articles, Links

Detection of avian leukosis virus: comparison of five techniques.

Nicholas RA, Thornton DH.

Five techniques were compared for their ability to detect decreasing dilutions of RAV-I, an avian leukosis sarcoma virus, in serially passaged chick embryo fibroblast cell cultures. The indirect fluorescent antibody test, sandwich enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase assay were equally sensitive in detecting the virus. The indirect immunoperoxidase and complement fixation avian leukosis tests were less sensitive. It is recommended that the sandwich ELISA be used for routine detection of the avian leukosis sarcoma virus group as possible vaccine contaminants because it is rapid and simple to perform and may be carried out on a large number of samples conveniently.

PMID: 6084274 [PubMed - indexed for MEDLINE]

Vopr Virusol. 1982 Mar-Apr;27(2):199-203. Related Articles, Links

[Use of a cocultivation method for detecting cytomegalovirus contamination of cell cultures of simian origin]

[Article in Russian]

Karetnyi IuV, Dzagurov SG, Shalunova NV, Elekoev KA.

At present, nonanthropoid primates are widely used as sources of cell cultures for manufacture of live viral vaccines. Simian cell cultures, particularly kidney cell cultures are also known to be frequently contaminated with cytomegaloviruses. The isolation of the latter is rather difficult due to the late appearance of the cytopathic effect in cell cultures of natural hosts. In the present study, the sensitivity of 4 methods virus isolation from the test cells was compared: the method of long-term cultivation of cells; the method of long-term cultivation with one subpassage of the cells; the method of cocultivation of the test cells by mixing with sensitive cells; and the method of co-cultivation by overlaying the test cells on an incomplete monolayer of sensitive cells. The latter method shortened the observation period and yielded a higher percentage of isolation of contaminating viruses from African green monkey kidney cell cultures. This method is supposed to be used in future for the detection of viral contamination of African group monkey kidney cell cultures utilized in manufacture of live viral vaccines.

PMID: 6283741 [PubMed - indexed for MEDLINE]

Ann Microbiol (Paris). 1978 Aug-Sep;129B(2):245-65. Related Articles, Links

[Problems in detection of mycoplasma contamination in cell cultures (author's transl)]

[Article in French]

Bonissol C, Gilbert M, Ivanova LM.

A total of 135 cell lines was examined for mycoplasma contamination using two techniques: isolation and specific DNA-staining with DAPI or "Hoechst 33258". The two techniques showed similar results in 64% of the cases of cell contamination while the remainder was detected only by one or the other techniques: 12,82% by the staining technique and 23% by the isolation technique, which shows that the 2 techniques are complementary. The staining technique is very quick, easy to execute, very sensitive, and should be the method of choice to detect contamination when the mycoplasma do not grow in acellular media.

PMID: 82418 [PubMed - indexed for MEDLINE]

In Vitro. 1975 Jan-Feb;11(1):20-34. Related Articles, Links

Comparison of methods for the detection of Mycoplasmal contamination of cell cultures: a review.

Schneider EL, Stanbridge EJ.

Several reviews in recent years have emphasized the problems created by mycoplasmal contamination of cultured cells (1-5). Because of the hazards of interpreting data derived from mycoplasma contaminated cells, most cell biologists routinely screen their cultures for the presence of these organisms. In recent years, the limitations of standard microbiological testing for mycoplasmas have become increasingly apparent and have led to the development of several new biochemical techniques for detection of these organisms. The aim of this review is to describe and compare available detection techniques and to evaluate their relative efficacy. Those properties of mycoplasmas that are relevant to their role as cell culture contaminants will be briefly discussed.

PMID: 1092606 [PubMed - indexed for MEDLINE]

In Vitro. 1977 Jun;13(6):357-65. Related Articles, Links

Mycoplasma in African green monkey kidney cell cultures: biochemical detection and effects in virus-infected cells.

Van Roy F, Fiers W.

Among a number of techniques for the detection of mycoplasmal contamination in African green monkey kidney (AGMK) cell lines, the assay of uridine phosphorylase activity is unsuitable because of the presence of high levels of endogenous enzymatic activity. A thymidine phosphorylase test, however, based on the chromatographic analysis of radiolabeled thymidine breakdown, turned out to be a simple and sensitive mycoplasma detection method. We found, using the latter technique, that 0.22-micrometer-filtered virus inocula could still transfer mycoplasma unless treated with diethyl ether. The effect of mycoplasmal contamination on the synthesis of simian virus 40 and adenovirus in AGMK cells was negligible under the conditions used (no depletion of arginine). Incorporation of radioactive thymidine in viral macromolecules, however, was inhibited severely by the presence of mycoplasma.

PMID: 195895 [PubMed - indexed for MEDLINE]

Dev Biol Stand. 1976 Dec 13-15;37:191-200. Related Articles, Links

The need for non-cultural methods for the detection of mycoplasma contaminants.

Stanbridge EJ, Schneider EL.

Mycoplasmas are common contaminants of cell cultures and are of great importance because of the deleterious effects they have upon the infected host cells. Routine detection testing usually relies upon cultural methods and demonstration of characteristic colonies on agar. Questions as to the efficacy of this method have been raised because of increasing realization of the presence of "non-cultivable" mycoplasmas. Several non-cultural detection methods have been developed to aid in the detection of these fastidious microorganisms. These methods include morphology by light and electron microscopy, immunofluorescence, enzyme assays, autoradiography, sucrose gradient separation, altered transport of nucleic acid precursors and altered nucleic acid profiles. A comparison of the relative sensitivities of these methods will be outlined. The need for, and the feasibility of non-cultural detection methods as quality control tests in human virus vaccine production will be discussed.

PMID: 1031685 [PubMed - indexed for MEDLINE]

Am J Vet Res. 1991 Aug;52(8):1237-44. Related Articles, Links

Monitoring bovine viral diarrhea virus vaccines for adventitious virus, using T1 ribonuclease viral RNA oligonucleotide fingerprinting.

Kelling CL, Kennedy JE, Rump KK, Stine LC, Paul PS, Partridge JE.

Department of Veterinary Science, University of Nebraska, Lincoln 68583-0905.

Viral RNA oligonucleotide fingerprinting was used to discriminate 3 cytopathic vaccine bovine viral diarrhea viruses (BVDV) grown in medium supplemented with serum contaminated with noncytopathic BVDV from the same 3 viruses grown in cell culture free of BVDV. Oligonucleotide fingerprinting also effectively discriminated between reference Singer BVDV, NADL BVDV, and New York-1 BVDV grown in BVDV-free noncontaminated or BVDV-contaminated cell cultures. Oligonucleotide fingerprint mapping of viral RNA maybe used to determine the purity of virus stocks, as well as that of BVDV vaccines.

PMID: 1656821 [PubMed - indexed for MEDLINE]

Dev Biol Stand. 1991;75:177-81. Related Articles, Links

Bovine viral diarrhea virus contamination of nutrient serum, cell cultures and viral vaccines.

Levings RL, Wessman SJ.

National Veterinary Services Laboratories, Animal and Plant Health Inspection Service, USDA, Ames, IA 50010.

Bovine viral diarrhea virus (BVDV) infection is common in the bovine population. Infection in utero leads to virus and antibody contamination of the fetal bovine serum used in cell cultures. These contaminants can interfere with diagnosis of viral infection. The high frequency of virus and antibody detection in individual animal or small pool samples suggests that any large pool of unscreened sera will be contaminated. Infection of cell cultures with BVDV can lead to interference with the growth of other viruses. Vaccine produced on contaminated cells may in turn be contaminated, leading to seroconversion or disease in the vaccine. The safety, purity, and efficacy of viral vaccines require BVDV testing of ingredients, cell substrates and final product. Methods for detection of BVDV in nutrient serum, cell cultures, seed viruses, and viral vaccines, and the frequency of their detection at the National Veterinary Services Laboratories are discussed.

PMID: 1665461 [PubMed - indexed for MEDLINE]

Nature. 1977 Apr 28;266(5605):835-7. Related Articles, Links

Viral contamination of bovine foetal serum and cell cultures.

Nuttall PA, Luther PD, Stott EJ.
From the article: The presence of adventitious viruses in cell cultures is well recognized, and when the cultures are of primate origin there are serious hazards for the production of human viral vaccines.  This is one reason for the increasing use of bovine cell cultures.  These cultures, however, are not free from viral contamination.  We found that calf kidney (CK) and calt testis (CT) cells were often infected by non-cytopathic mucosal disease virus (MDV): the cells seemed morphologically healthy but nearly all showed fluorescence with MDV antiserum and rabbit-anti-bovine conjugate.  We report here that both the cells and foetal calf serum, an essential growth factor of cell culture medium, are sources of the virus...These results indicate a much higher incidence of contamination than reported previously, and illustrate the inadequacy of commercial screening methods...Infection of bovine cell cultures by non-cytopathic MDV has particular significance for the production of viral vaccines...The importance of contamination by MDV with regard to human viral vaccines is unknown, but measles virus vaccine and a potential respiratory syncytial virus vaccine are produce in bovine kidney cells grown in the presence of unheated commercial FCS...Regular screening of bovine cell cultures is essential if stocks are to be kept free from MDV.

PMID: 194159 [PubMed - indexed for MEDLINE]

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