Differential staining of living and dead bacterial spores

Differences in the staining properties of living and dead bac-terial cells have been occasionally mentioned in the literature and recently several special staining methods have been suggested to bring out this differentiation more clearly. Seiffert (1922) and Henrici (1923) employed a weak Congo red solution to dis-tinguish living vegetative forms from the dead cells. Cells killed by heat take the stain whereas living cells remain colorless. Burke (1923) has described a difference in the permeability to carbol-fuchsin which is shown by living and heat-killed spores of Clostridium botulinum. The dead spores are penetrated by the dye and appear as solid staining forms, whereas unheated cultures show only about 1 to 2 per cent of the solidly stained type while the remainder present an unstained center surrounded by a narrow stained border, the so-called "ring " forms. Evi-dence is also presented of a definite relationship between penetra-tion by carbol-fuschsin and death of the spore by heat. As a spore suspension of Clostridium botulinum is heated the propor-tion of solid staining spores increases until it becomes 100 per cent and this point is correlated in a general way with the thermal death point as determined by cultivation. In the present investigation an attempt was made to apply this differential spore stai to the common aerobic spore-formers such as B. megatherium, B. cereus, B. subtilis, etc. The method suggested by Burke (1923) for the spores of Cl. botulinum is as follows: Smears are made on glass slides and air dried, heat fixa-tion is omitted. The slides are then stained for two minutes in steaming carbol-fuchsin (1.5 grams basic fuchsin, Grubler