Relationship of cytochrome content to the sensitivity of bacteria to NaCl on freezing and thawing.

Eight species of bacteria representing rod, coccus, gram-positive, and gram-negative forms were tested for their sensitivity to sodium chloride during freezing and thawing. Six of the eight species tested were salt-sensitive, though to different degrees, while Lactobacillus casei and Streptococcus faecalis were resistant. Escherichia coli grown anaerobically exhibited only 38% of the salt sensitivity of aerobically grown cells. Analysis of cytochrome pigments in the organisms revealed that the six sensitive organisms all contained these pigments but in varying amounts, while the two resistant ones were devoid of them. Anaerobically grown E. coli contained 50% of the cytochromes of aerobically grown cells. A relationship between cytochrome content of the organisms and salt sensitivity during freezing and thawing was demonstrated with a correlation coefficient of 0.76 (P less than 0.05); the higher the cytochrome content, the more salt-sensitive the organism. This indicated that 58% of the salt sensitivity was due to the cytochrome content. Using a model organism E. coli, the effect of salt during freezing and thawing on the respiratory activity was examined. Freezing and thawing in water or saline decreased the respiration by whole cells of substrates expected to be NAD-linked while NADH-stimulated respiration was increased. In cell-free extracts derived from unfrozen cells or those frozen and thawed in water or saline, the respiration of ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) was constant. The respiration of NADH, succinate, and lactate in cell-free extracts derived from cells frozen and thawed in saline was reduced compared with those extracts derived from unfrozen cells or cells frozen and thawed in water. Studies with E. coli showed that the decreased respiratory activity caused by disruptions in the electron-transport chain could not account for the salt sensitivity on freezing and thawing. More likely, salt sensitivity is related to the presence of bonds between cytochromes and other membrane components which are disrupted by sodium chloride on freezing and thawing. This would then result in loss of membrane integrity and function.