LYSIS OF FROZEN AND THAWED CELLS OFESCHERICHIA COLIBY LYSOZYME, AND THEIR CONVERSION INTO SPHEROPLASTS

Abstract

Lysozyme had no visible effect on growing or resting cells of Escherichia coli. However, when these bacteria, while exponentially growing in nutrient broth or in a synthetic medium, were rapidly frozen and later thawed, they became highly susceptible to a rapid lysis by lysozyme (3-50 [mu]g/ml). It was necessary to add the enzyme before freezing or immediately after thawing, since the sensitivity of bacteria to lysozyme was rapidly lost when the thawed cells were incubated at 37[degree]C before the addition of lysozyme. When held at 0[degree]C, thawed bacteria retained their sensitivity to lysozyme for a prolonged time. Whereas at 37[degree]C the cells of E coli became completely resistant to lysozyme lysis within an hour of incubation in nutrient broth, they remained sensitive if starved or treated by KCN. This indicated that only metabolically active bacteria would regain their natural resistance to lysozyme after thawing. Frozen and thawed E coli could also be lysed by trypsin, but the character of this lysis was different from that caused by lysozyme. When the freezing was carried out in 0.3 [image] sucrose, the rapid lysis of bacteria by lysozyme was prevented, and the bacterial rods were converted into round, osmotically fragile, spheroplasts. Some of these spheroplasts, when plated in sucrose agar grew into colonies consisting of normal, rod-shaped, bacteria. It was also shown that lysozyme adsorbed to bacteria before freezing, and that chilled, or frozen and thawed E coli treated with lysozyme in sucrose strongly adsorbed deoxyribonucleic acid prepared from phages or bacteria.