## 3 Results and Discussion
### Spectral Marker Inspection
The data from our study demonstrate that lipid and protein contents are sensitive, biochemical components during mycoplasma contamination. The analysis of second derivative spectra and PCA showed that lipid and protein regions were responsible for the discrimination between decontaminated and contaminated cells. Our result is in agreement with previous studies indicating that the lipid and protein regions, but not the DNA region, were the determinants for mycoplasma contamination [13]. This possibly explains why the PCR and DNA staining methods, such as DAPI, which depend on the alteration of nucleic acid, could sometimes yield false-positive or negative results and are possibly unreliable. The pattern of biomolecular changes from our study was varied from the previous study. The difference mycoplasma strains, as well as infected cell-type, could contribute to the observed difference in effects. Some mycoplasma species affect no cytopathic changes, whereas others could interfere with several intracellular processes [5]. Mycoplasma can interfere with the host cells and disrupt several molecular processes, which consequently result in cellular biomolecular alteration. Our study demonstrates the application of FTIR microscopy for the cell line inspection of mycoplasma contamination because FTIR spectra reflects the biomolecular alteration. Notably, there is still the possibility that the antibiotic used for mycoplasma treatment, as well as the mycoplasma itself, could cause permanent cytopathic changes [36; 40], reflecting in the IR spectra of the cell sample. A medium- and long-term study collecting FTIR spectra of mycoplasma-free cells, with and without antibiotic treatment, should be performed to spot the irreparable alteration that possibly takes time to notice. Nevertheless, our result primarily demonstrates that FTIR spectroscopy is as effective as conventional PCR to detect mycoplasma contamination before and after antibiotic treatment. Many mycoplasma detection methods have been developed, such as DNA-RNA hybridization, immunofluorescence staining, biochemical detection utilizing 6-MPDR, ELISA, and PCR. Among them, PCR has been reported to be more rapid and sensitive than the other methods. PCR also shows better sensitivity even with a diluted sample and, therefore, has been used as the standard method for mycoplasma detection [41]. The drawback of a conventional PCR assay is that it requires many steps to confirm the result, as well as being a time-consuming process [42]. FTIR microspectroscopy offers the advantages because it is a fast, simple, and sensitive method that is nondestructive to the sample, label-free, and inexpensive, while providing accurate and reliable results. FTIR microspectroscopy should be considered as an alternative application to detect whether cells are infected with mycoplasma. Consequently, our study demonstrates the IR technique for monitoring biomolecular alteration in a cell model for biomedical research as well as detecting mycoplasma contamination. With the practical advantages of FTIR microspectroscopy, this technique has proven to be a useful and complementary approach to study biological samples regularly when there is a FTIR facility.