The effect of oxidation-reduction potential on the outgrowth and chemical inhibition of Clostridium botulinum type E spores

The effect of oxidation-reduction potential (Eh) and oxygen concentration on the outgrowth of Clostridium botulinum type E spores was studied. An airtight flask containing electrodes for continuous measurement of Eh and pH and attached tubing for the exchange and mixing of various gases (i.e. prepurified nitrogen, anaerobic carbon dioxide, hydrogen, oxygen) were utilized. Prereduced media and anaerobic procedures were employed to insure controlled conditions and reproducible results.

Eh measurements were made of Trypticase Soy Broth containing several concentrations of the following reducing agents which are listed in what was found to be the order of their reducing intensities: sodium formaldehyde sulfoxylate, 2-mercaptoethanol, cysteine hydrochloride, sodium thioglycolate, and ascorbic acid. Outgrowth of C. botulinum type E spores was shown to be directly related to a low Eh and a low concentration of added reducing agent.

Outgrowth and Eh changes were measured in the electrode vessel described above for several growth conditions. Eh levels in the media were poised by sparging prereduced media with nitrogen gas or 0.1% oxygen in nitrogen gas or by sparging with nitrogen gas in oxidized media (media which was autoclaved under air). Under uninhibited growth conditions there were no significant growth differences between the oxidized (Eh = -80 mv), oxygen poised (Eh = -80 mv), and reduced (Eh = -190 mv) media. However, inhibition by sodium chloride (3.5% and 4.0%), hydrogen ions (pH 5.11 and 5.05), and sucrose (30% and 35%) revealed an increased lag phase and a decreased maximum absorbance for growth in the oxidized medium compared to growth in the reduced medium. Media poised by dissolved oxygen also exhibited retarded growth when compared to the reduced media. The addition of exogenous hydrogen gas to the media reduced or eliminated the growth and lag time differences described above for sucrose inhibition.

It was shown that the Eh drop observed during growth of the test organism probably occurred during emergence and outgrowth of the spores and not during germination. In addition, this drop was coincident with the production of metabolic hydrogen gas by the organism. Data indicated that the presence of hydrogen gas in the medium would reflect an artificially low potential as a result of the local ionization of the hydrogen at the platinum tip of the Eh measuring electrode. This effect may obscure any actual reducing in Eh.

An increased lag phase was observed when a small inoculum (10² spores/ml) was used compared to a large inoculum (10⁴ spores/ml). This difference in lag was similar to that observed between the reduced and the oxidized or oxygen poised media. It is hypothesized that the variations in growth are due to a distribution of sensitivity within each spore inoculum population to adverse conditions (e.g. low pH, high sucrose, high salt, high Eh).