A shake-up in cell culturing: Flame sterilization may affect the culture


Date:
July 1, 2020
Source:
University of Tsukuba
Summary:
Researchers have found that flame-sterilizing shake-flasks, to
avoid introducing microbial contaminants, considerably increases
the carbon dioxide concentration in the flasks. This enhanced
carbon dioxide concentration affects the growth of some microbial
species, which may affect the quantity of vaccines or other valuable
substances produced by the microbes.



FULL STORY ========================================================================== Researchers commonly culture bacteria for many purposes, such as
to screen pharmaceuticals and manufacture vaccines. In these cases,
shake flasks have been commonly and generally used for over 90 years to cultivate microbes.


==========================================================================
To keep track of what's going on in the shake flask, researchers must
use stringent sterilization techniques while extracting a sample of
cells. A common sterilization technique is to expose the plug and flask
to a flame at several time points during cell extraction. However,
if this sterilization affects the cell culture in any way, you may inadvertently hinder production of the vaccine or whatever substance
you want from the culture.

In a study recently published in Scientific Reports, researchers
from the University of Tsukuba have shown that flame sterilization
introduces carbon dioxide into shake flasks. This excess carbon dioxide
can considerably affect cell growth.

Carbon dioxide is a product of methane combustion in the flame. The
researchers found that flaming the flask for even a few extra seconds,
or tilting the flask a few extra degrees, considerably increased the
carbon dioxide concentration in the flask.

"For example, at a flame exposure time of only 5 s, increasing
the inclination angle from 15DEG to 25DEG increased the carbon
dioxide concentration in the headspace by approximately 50%," says
Professor Hideki Aoyagi, senior author of the study. "Computational
modeling confirmed our findings." These increases in carbon dioxide concentration are induced over the course of only a few seconds of flame sterilization. But do they substantially affect cell growth? To test
this hypothesis, the researchers needed to add excess carbon dioxide
while keeping the flasks shaking, because interrupting the shaking can
itself affect cell growth.

"We introduced intermittent carbon dioxide at concentrations similar to
those expected by flame sterilization," explains Professor Aoyagi. "The ultimate oxygen demand of Acetobacter pasteurianus -- known to spoil
wine -- increased by up to 70%. Pelomonas saccharophila increased by
up to 35%, whereas the other two microbes were not clearly affected
in terms of growth." The researchers do not yet know how common it is
for flaming a shake-flask to alter cell culture growth. Nevertheless,
seemingly minor experimental sterilization variables -- too subtle
for most researchers to even notice at first glance -- may actually be pertinent. Culturing microbes in shake flasks and producing valuable
products in the culture -- perhaps relevant to COVID-19 research --
may be substantially more complicated than previously appreciated.


========================================================================== Story Source: Materials provided by University_of_Tsukuba. Note: Content
may be edited for style and length.


========================================================================== Journal Reference:
1. Masato Takahashi, Takafumi Honzawa, Ryuichi Tominaga, Hideki Aoyagi.

Analysis of the influence of flame sterilization
included in sampling operations on shake-flask cultures
of microorganisms. Scientific Reports, 2020; 10 (1) DOI:
10.1038/s41598-020-66810-3 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2020/07/200701100018.htm

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