Flaming as part of aseptic technique increases CO2 (g)and decreases pH in freshwater culture media

Aseptic technique has historically served as a fundamental practice in microbiology, helping maintain culture purity and integrity. This technique has been widely encouraged and employed for use with cultures of heterotrophic bacteria as well as freshwater and marine algae. Yet, recent observations have suggested these approaches may bring their own influences. We observed variations in growth amongst replicate experimental cyanobacterial cultures upon flaming of the culture tube opening during sample transfer and collection. Investigation revealed the pH of culture media had decreased from the initial pH established during media preparation. Flaming of sterile culture media alone confirmed a significant decrease, by as much as 1.7 pH units, and correlated with increased flaming events over time. We hypothesized that the causative factor was the introduction of carbon dioxide into the media. To test this hypothesis, qualitative and quantitative analyses were used to identify the primary driver of pH decline. We further assessed the direct effects of flaming and subsequent pH changes on Microcystis aeruginosa cultures, showing flame-driven pH changes and/or the introduction of carbon dioxide influenced experimental results. Our observations provide a cautionary tale of how classic and well-accepted approaches may not always provide the results promised, suggesting new approaches may be necessary in research areas assessing pH or carbon related-effects on microbial communities.