Factors Affecting the Germination of Thick Suspensions of Bacillus subtilis Spores in L-Alanine Solution

Abstract

SUMMARY:The rate of germination (defined as loss of heat-resistance accompanied by change in staining properties under specified conditions, with maintenance of viability) of thick puspensions of'Bm*ZZus mbtilis spores in phosphate-buffered L-alanine solution increased with the time from harvesting. The maximum rate of germination was reached after about 20 days' storage in water at 20°. This effect could be retarded, but not reversed, by storage at low temperatures. The rate of germination may be-temporaqly accelerated by heat treatment. Germination was considerably retarded after treatment with mercuric chloride, and was completely inhibited by 8-hydroxyquinoline (oxine) and by 2 :$-dimercaptopropanoI (BAL) at 10 mlcl concentration. The latter effect was partially reversed by the addition of metals.Using dilute spore suspensions of Bacillzls subtilis (104 spores/ml.) Hills (1950) found that 80 yo loss of heat-resistance occurred during 80 min. incubation a t 35' in 0-5 m~ L-alanine buffered with 38 m~ phosphate a t pH 7-8. The present report concerns germination in thicker suspensions, i,e. 5 x lo@ sporea/ml., with a view to the study of the biochemical changes which<accompany loss of heat-resistance in the above simple medium. # EXPERIMENTALA laboratory strain of B. subtilis was used throughout. Suspensions containing at least 95 yo spores were obtained by growth on CCY agar (Gladstone & Fildes, 1940) a t 87' for 12 days. The spores were washed five times with distilled water after harvesting. Their age was measured from the day of reaping;and storage was a t room temperature (20') unless otherwise stated. No heat treatment was given after harvesting except that specifically stated in an experiment.The percentage germination was a t first determined by plate counts on peptone agar before and after heating at 60' for 15 min. (Hills, 1950), Later, it was found that when films of the L-alanine-treated spores were made, fixed and stained with hot carbol fuchsin and methylene blue, most of the cells appeared elongated and stained uniformly bluish purple or showed a central bluestaining body (Pl. 1). These were readily distinguishable from the unchanged spores, and their percentage occurrence could be determined by direct counting.The details of the staining procedure were as follows. Films were treated for 5min. with hot carbol fuchsin, then washed thoroughly with hot water.Nigrosin (1 yo) was then poured on, and left for 2 min. After washing in cold water, the slide was stained with methylene blue for 2 min. It was found important to avoid overstaining with carbol fuchsin since differentiation between the two forms was then very poor. Twenty-two samples of partially germinated suspensions were stained and counted in this way, and the proportion of the blue-staining forms determined. The value obtained agreed very well with that '

The Swelling of Bacterial Spores during Germination and Outgrowth

“…This was stained by a modified Ziehl-Neelsen stain (Powell, 1950), and viewed with a light microscope. Another drop of the suspension was viewed with a phase-contrast microscope.…”

“…The term germination is thus now reserved for the first stage, and the transformation of the germinated spore into the vegetative form is called outgrowth (Campbell, 1957) or post-germinative development . Germination involves loss of resistance to heat and other injurious agents, loss of refractivity when examined by the phase-contrast microscope (Pulvertaft & Haynes, 1951), decrease in optical density of suspensions and increase in stainability of cells (Powell, 1950), decrease in dry weight due to loss of dipicolinic acid, calcium and a non-dialysable mucopeptide (Powell & Strange, 1953) and the initiation of respiration (Murrell & Scott, 1958). Germination is induced by simple nutrients and may be differentiated from outgrowth by certain inhibitors (Murty & Halvorson, 1957;Halvorson, 1959).…”

L'origine des spores de thermophiles dans le lait stérilisé en bouteilles

“…Un ml de cette suspension est transvasé dans un tube de verre de 10 mm de diamètre intérieur, plongé dans un bain-marie de glycérine à 65°C. A partir de prélèvements périodiques à l'aide d'une pipette Pasteur, on effectue des frottis de lait qui sont colorés par une modificabion de la méthode de POWELL [12] : le frottis après fixation à l'alcool. à 95°est recouvert avec la solution de bleu de méthylène alcoolique, après 2 minutes de contact, le frottis est flambé, lavé et séché.…”

Germination of Thermoactinomyces vulgaris Endospores: Microscopic and Optical Density Studies Showing the Influences of Germinants, Heat Treatment, Strain Differences and Antibiotics

“…W. ATTWELL, T. CROSS AND G. W. GOULD species. Furthermore, these differences do not parallel established lines of classification (Thorley & Wolf, 1961 ;Foerster & Foster, 1966;Sacks & Thompson, 1971).…”

“…A comparison of results in Tables 2 and 3 show that in most instances 'ageing' enhanced the extent of germination achieved by a spore suspension, a phenomenon first described for Bacillzis spp. by Powell (1950).…”