Early Biochemical Detection of Low-Level Bacterial Contamination in Mammalian Cell-Culture Medium Using pH, Glucose Depletion, Lactate Accumulation, Conductivity, and Nitrite Shift

Sura Razzaq  khudhair; Shujon Hassan  Jadaan; Jaafar Faez Kadhim  Alsadooni

doi:10.51699/cajmns.v7i3.3264

Authors

Sura Razzaq khudhair Ministry of High Education, Department of Microbiology and Parasitology, College of Veterinary Medicine, University of Al- Qadisiyah
Shujon Hassan Jadaan Ministry of High Education, Department of Microbiology and Parasitology, College of Veterinary Medicine, University of Al- Qadisiyah
Jaafar Faez Kadhim Alsadooni Ministry of education, Al-Qadisiyah director education, Iraq

DOI:

https://doi.org/10.51699/cajmns.v7i3.3264

Keywords:

bacterial contamination, mammalian cell culture, glucose depletion, lactate, resazurin

Abstract

Background: Bacterial contamination remains a major problem in mammalian cell culture because it can alter cell physiology, medium chemistry, and experimental reliability before obvious turbidity appears. Simple early-warning biochemical markers may help detect contamination without microscopy, gel electrophoresis, or image-based confirmation.

Aim: This study evaluated whether low-level bacterial contamination in mammalian cell-culture medium could be detected early using non-image biochemical and spectrophotometric markers.

Methods: Sterile Dulbecco’s Modified Eagle Medium was experimentally exposed to low inocula of safe laboratory strains, including non-pathogenic Escherichia coli K-12 and Bacillus subtilis. Four groups were included: sterile control medium, low-level E. coli contamination, low-level B. subtilis contamination, and mixed bacterial contamination. Medium samples were incubated at 37 °C and tested at 0, 6, 12, and 24 h. pH, glucose concentration, lactate concentration, conductivity, nitrite, optical density at 600 nm, and resazurin metabolic activity were measured.

Results: At 12 h, contaminated media showed clear biochemical shifts before strong turbidity was observed. Mixed contamination reduced pH from 7.42 ± 0.03 to 6.78 ± 0.05, decreased glucose from 4.50 ± 0.18 to 2.94 ± 0.16 g/L, increased lactate from 0.42 ± 0.04 to 1.86 ± 0.13 mmol/L, and increased conductivity from 13.8 ± 0.4 to 16.7 ± 0.5 mS/cm. Resazurin reduction increased significantly in contaminated groups, especially in mixed contamination. A combined marker index using pH, glucose, lactate, and resazurin activity detected contamination earlier than optical density alone.

Conclusion: Low-level bacterial contamination produced measurable biochemical changes in mammalian culture medium before marked turbidity. This model provides a simple, low-cost microbiology study based entirely on non-image verification.

References

H. G. Drexler and C. C. Uphoff, “Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention,” Cytotechnology, vol. 39, no. 2, pp. 75–90, 2002, doi: 10.1023/A:1022913015916.

E. J. Fratz-Berilla, P. Angart, R. J. Graham, and J. R. Cora, “Impacts of intentional mycoplasma contamination on CHO cell bioreactor cultures,” Biotechnol. Bioeng., vol. 116, no. 12, pp. 3242–3252, 2019, doi: 10.1002/bit.27161.

X. Huang, M. Li, Y. Xu, and Y. Zhang, “Prevention, diagnosis and eradication of mycoplasma contamination in cell culture,” STAR Protoc., vol. 4, no. 4, p. 102608, 2023.

J. M. Burstain, M. E. Brecher, K. Workman, M. Foster, G. H. Faber, and D. Mair, “Rapid identification of bacterially contaminated platelets using reagent strips: Glucose and pH analysis as markers of bacterial metabolism,” Transfusion (Paris)., vol. 37, no. 3, pp. 255–258, 1997, doi: 10.1046/j.1537-2995.1997.37397240205.x.

F. Razjou, S. Haghpanah, and H. A. Abedi, “Evaluation of the sensitivity and specificity of use of glucose and pH for bacterial screening of platelet concentrates,” Iran. J. Pathol., vol. 11, no. 1, pp. 35–42, 2016, doi: 10.1007/s12288-016-0660-4.

H. Weichert, N. Lehmann, and K. F. Reardon, “Online glucose-lactate monitoring and control in cell culture and microbial fermentation,” Biotechnol. Bioeng., vol. 110, no. 7, pp. 1984–1992, 2013, doi: 10.1186/1753-6561-7-S6-P18.

J. Shi, X. Chen, Y. Wang, and Y. Li, “Pollution-free and highly sensitive lactate detection in cell culture medium using a microfluidic chip,” Biosensors (Basel)., vol. 13, no. 5, p. 499, 2023.

S. J. Reyes, X. Xu, and C. Chen, “Modern sensor tools and techniques for monitoring, controlling, and improving cell culture processes,” Processes, vol. 10, no. 2, p. 189, 2022, doi: 10.3390/pr10020189.

Y. Yang, F. Ricci, and K. W. Plaxco, “Dual-purpose aptamer-based sensors for real-time monitoring of cellular metabolism,” ACS Sens., vol. 9, no. 9, pp. 4511–4522, 2024.

B. Wacogne, H. P. Herzig, and S. Perrin, “White light spectroscopy for sampling-free bacterial contamination monitoring during mammalian cell cultivation,” Sensors, vol. 25, p. 4721, 2025.

L. Nikfarjam and P. Farzaneh, “Prevention and detection of mycoplasma contamination in cell culture,” Cell J., vol. 13, no. 4, pp. 203–212, 2011.

J. Chen, “Does the National Early Warning Score 2 system serve its purpose?,” Resuscitation, vol. 134, pp. 145–146, 2019, doi: 10.1016/j.resuscitation.2018.11.006.

P. Jaikang, P. Paengnakorn, and K. Grudpan, “Simple colorimetric ammonium assay employing well microplate with gas pervaporation and diffusion for natural indicator immobilized paper sensor via smartphone detection,” Microchemical Journal, vol. 152, p. 104283, 2020, doi: 10.1016/j.microc.2019.104283.

A. M. Otto, C. Janzon, and J. Hutterer, “163: Metabolic activity of tumor cells in a variable microenvironment: combinations of glucose, glutamine, lactate and pH,” Eur. J. Cancer, vol. 50, p. S36, 2014, doi: 10.1016/s0959-8049(14)50136-0.

“signaling rate,” in SpringerReference, Springer-Verlag. doi: 10.1007/springerreference_24778.