Research Article: Detection of mycoplasma in contaminated mammalian cell culture using FTIR microspectroscopy

Date Published: March 17, 2018

Publisher: Springer Berlin Heidelberg

Author(s): Katia Wehbe, Marzia Vezzalini, Gianfelice Cinque.

http://doi.org/10.1007/s00216-018-0987-9

Abstract

Mycoplasma contamination represents a significant problem to the culture of mammalian cells used for research as it can cause disastrous effects on eukaryotic cells by altering cellular parameters leading to unreliable experimental results. Mycoplasma cells are very small bacteria therefore they cannot be detected by visual inspection using a visible light microscope and, thus, can remain unnoticed in the cell cultures for long periods. The detection techniques used nowadays to reveal mycoplasma contamination are time consuming and expensive with each having significant drawbacks. The ideal detection should be simple to perform with minimal preparation time, rapid, inexpensive, and sensitive. To our knowledge, for the first time, we employed Fourier transform infrared (FTIR) microspectroscopy to investigate whether we can differentiate between control cells and the same cells which have been infected with mycoplasmas during the culturing process. Chemometric methods such as HCA and PCA were used for the data analysis in order to detect spectral differences between control and intentionally infected cells, and spectral markers were revealed even at low contamination level. The preliminary results showed that FTIR has the potential to be used in the future as a reliable complementary detection technique for mycoplasma-infected cells.

Partial Text

Mycoplasmas are types of bacteria of class Mollicutes that lack a cell wall which makes them unaffected by common antibiotics. Mycoplasma contamination is a significant problem to the culture of mammalian cells used for research and the rate could be as high as 70% [1]. The contamination can cause disastrous effects on eukaryotic cells as it tends to alter the cells at a molecular level and compromises the value of the contaminated cell lines in providing accurate data for life science research. It can induce alterations in cellular parameters (e.g., chromosome aberrations, changes in metabolism and cell growth) leading to unreliable experimental results and potentially unsafe biological products [2, 3]. In cell culture laboratories, infection usually occurs with the same mycoplasma species, and this proves that mycoplasma infections are often spread from one culture to another [4, 5]. The sources of mycoplasma contamination in the laboratory are very challenging to completely control. Since certain mycoplasma species are found on human skin, they can be introduced in the cultures through poor aseptic technique. They can also originate from contaminated supplements such as fetal bovine serum (FBS) and certainly from other contaminated cell cultures. For these reasons, good aseptic technique should be followed and new cell lines received from other laboratories should be considered suspicious and quarantined till the proof of mycoplasma absence.

FTIR micro-analysis was able to differentiate between the control and the mycoplasma-infected cells. Spectral markers were identified in the lipid and fingerprint regions due to molecular changes in the host cells and/or the presence of mycoplasma. Major differences are in the membrane phospholipid ester, proteins and nucleic acid regions which could be due to the cytopathic effects of mycoplasma on the host cells (e.g., disrupting the integrity of the host cell membrane and cleaving DNA and/or RNA of the host cells). Further investigations are in progress to better interpret these spectroscopic markers. The preliminary results showed that micro-FTIR has the potential to be used in the future as a complementary detection technique for mycoplasma-infected cells and could be very useful in detecting if cells are cleared from mycoplasmas following a treatment to eliminate the bacterial contamination.

 

Source:

http://doi.org/10.1007/s00216-018-0987-9

 

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