The accelerating effect of sublethal heat on spore germination in mesophilic aerobic bacteria

Abstract

In their early studies on disinfection, Koch, Gaffky and Loeffler (1881) re-ported that anthrax spores which had been heated to 900 and 95°C. required longer: to produce visible plate colonies than unheated spores. Since that time the growth-delaying action of sublethal heat has been recorded by many in-vestigators, both for spores and vegetative cells: Bredemann (1909), for Bacillus amylobacter; Weiss (1921), Esty and Meyer (1922), Esty and Williams (1924), Dickson (1928) and Sommer (1930), for Clostridium botulinum; Schultz (1940), for. mesophilic aerobes; Allen (1923), for sporulating and non-sporulating meso-philic aerobes; Eijkman (1908) and Hershey (1939), for Escherichia coli. There is considerable evidence to indicate that the germination of thermophilic spores is -not retarded by non-killing heat treatment; Bigelow and Esty (1920), Esty and Williams (1924), and Feier (1927). According to Sommer this also is true for Bacillus subtilis. Earlier observations by Williams (1929) lend support to this contention. As a result of these reports the belief has become firmly established that sub-lethal heat has either a delaying or negligible action upon the germination of spores; the possibility that heat might provide a stimulus to germination has been generally overlooked. Eckelmann (1917), in a discussion of the causes of the heat-inhibition of spores, suggested that in some instances stimulation of ger-mination might occur but offered no experimental proof. Allen noted that heat-shock reduced the subsequent generation time of one sporing culture, but it is our observation that a factor may have no influence upon the time required for spores to become heat-labile, and yet may change the rate of subsequent vegetative proliferation. In this paper we will show that germination of the spores of the mesophilic aerobes may be consistently accelerated by exposure to heat through a wide range of temperatures. Attention has been given to some of the factors which exert an influence upon this reaction. Because of its potential usefulness in the processing of food and other materials this phenomenon possesses more than academic interest. METHODS AND MATERLkLS The test organisms were the following: Bacillus megatherium (N. R. Smith #696), Bacillus cereus (#369, #720 N. R. Smith), Bacillus subtilis (A.T.C.C. #6051, #6634), Ba4iUus cohaerens, Bacillus fusiformis (Bureau Dairy Industry Collection), CC (National Canners Association S 4149), # 9499 (National Canners Association #9499). CC is closely allied to Bacillus mesentericus. The position of # 9499 in the genus is not known. The plating medium was nutrient agar of the following composition: Difco peptone, 5 g.; Difoo beef extract, 3 g.; sodium chloride, 5 g.; glucose, 3 g.; agar, 13 g.; water, 1,000 ml. pH 7.0. The broth was similar, except for the omission of agar. The evapo-rated milk was prepared from whole milk concentrated 2 to 1 and was not sterilized before use. Total bacterial counts of the evaporated milk prior to use varied between 300 and 700 per ml., of which less than 5 per ml. survived heat at 85°C. for 10 minutes. The spores were produced on plain nutrient agar slopes; when sporulation was complete, the growth was washed off with distilled water, filtered through cotton, and centrifuged. The water was decanted and the washing process repeated twice. When clumping occurred the clumps were largely broken up by moderate shakig of the suspension with small gass beads. The concentrated stock sus-pensions thus prepared, practically 100 per cent spores, were plated to determine purity and count and were then held at 6°C. until used. Germination was determined by the following procedure: A small quantity of the diluted stock suspension of spores was seeded into the test medium and the two thoroughly mixed. The uniformly dispersed suspension of spores was then divided into a series of equal portions, one of which, the control, received no preincubation heat, and was stored at 8°C., while the several remaining portions were preheated at selected temperatures for various periods. These, together with the unheated control, were then adjusted to and incubated at 370C. for 3 to 5 hours. When the preheating medium was water or buffer solutions, small equal volumes of the heated spore suspensions were subseeded into glucose broth prior to incubation. Following incubation, all samples were heated at 85°C. for 10 minutes in order to kill the spores which had become heat-labile during the incubation interim. Subculturing of the final heated suspensions was carried out in glucose agar plates which were counted after 48 hours at 37°C. Asump-tion by the spores of the heat-lability characteristic of vegetative cells was accepted as evidence of germination. There was no appreciable change in the pH of the cultures during'incubation.