C. F. Robinow 



415 



of crystal violet. The shrunk cytoplasm stains deep blue 

 to purple and the cell wall a light pink. The sodium 

 hydroxide solution was prepared by dissolving one pellet 

 of the reagent in 10-30 ml. of water. 



Optical methods 

 In all staining operations much 'trial and error' can 

 be avoided by using a water immersion lens before the 

 preparations are mounted. Economy in the use of fixing 

 and staining reagents can be achieved by employing 

 cover-slips instead of slides. The cover-slips should be 

 mounted on slides of a thickness not greater than that 

 tolerated by oil-immersed achromatic substage con- 

 densers of high numerical aperture. The light source 

 should be minute, at the correct distance from the 

 microscope and fitted with a field stop. For work on the 

 bench the Koehler principle of illumination has been 

 found convenient and has been used throughout. The 

 immersion objectives should be of a quality permitting 

 the use of a 15 times eyepiece, since the magnification 

 of about a 1000 times, commonly employed in bacterio- 

 logical routine, is not high enough for a detailed study 

 of the chromatinic structures. 



RESULTS 



(1) The chromatinic structures of the cells of young 

 cultures of Bact. coli and Proteus vulgaris 

 Impression preparations made from agar plates during 

 the first few hours of growth show masses of transparent 

 bacteria containing brilliantly stained, sharply defined 

 and regularly spaced chromatinic structures. 



The 18-24 hr. slant cultures from which the plate 

 cultures are seeded consist chiefly of three different types 

 of bacteria. Small coccoid elements, short plump bacilli 

 and small slender rods. This diversity of form persists 

 during the first few hours of growth on the plate (PI. 5, 

 figs. 1, 2). 



The division processes of the chromatinic structures 

 during the development of the coccoid forms (type 1) of 

 Bact. coli into the familiar rod-shaped bacteria follow 

 the simple pattern previously outlined for B. mycoides 

 (Robinow, 1942) (Text-fig. 1). The first visible changes 

 in the chromatinic structures of coccoid elements from 

 18 to 24 hr. slant cultures awakening on a fresh nutrient 

 medium are: increase in size, in the depth of staining 

 and in the intensity of the Feulgen reaction. Next the 

 chromatinic structure divides into two closely contiguous 

 dumbbells. The daughter dumbbells increase in width, 

 their contours become asymmetrical and often before 

 completing their separation, they in turn proceed to 

 split longitudinally (PI. 5, figs. 1, 2). A small round 

 granule or strands of some faintly coloured or of some 

 chromatinic material often persist for some time between 

 separating dumbbell hodies (PI. 5, figs. 1 e,f, 2 b, c, 9 a). 

 After the first division of the original chromatinic struc- 

 ture, the cell may divide directly by constriction into 

 two separate ovoid daughter cells. Usually, however, 

 division of the young bacterium does not take place until 

 after the .second or third division of the chromatinic 

 bodies. The early development of coccoid forms of Proteus 

 vulgaris, whether from an 18 hr. agar slant or from the 

 first belt of confluent growth proximal from the zone of 

 swarming in an agar plate culture, is essentially the same 



as that of Bact. coli (PI. 5, figs. 10-12). In the two strains 

 of Proteus used in this study awakening of the cells was, 

 however, much more protracted than in Bact. coli, and 

 consequently a higher proportion of cells with single 

 chromatinic bodies were found in young subcultures. 



If the early growth stages of Bact. coli and Proteus are 

 compared with those of B. mycoides, there is found to 

 be a close similarity (compare PI. 5, figs. 1, 2 with PI. 7, 

 figs. 25—27). The smallest elements of Bact. coli and 

 Proteus resemble the first vegetative generation of 



(SXD< 



€TnJD 



Text-fig. 1. Diagram of successive division stages of the 

 chromatinic bodies from the beginning of the lag 

 phase, after transfer to a fresh nutrient medium, to 

 the first division of the growing bacterium. The dia- 

 gram is based mainly on preparations of Bact. coli 

 but applies equally well to the early development of 

 Proteus vulgaris, c-c' and c-f are alternative modes 

 of development, c-/ being that most commonly fol- 

 lowed. 



PI. 5, figs. 1, 2, 10 

 (c) in PI. 5, figs. 2. 12 

 PI. 5, fig. 4 

 PI. 5, figs. 5-7 

 dividing bacterium 

 (c) in right-hand 

 bottom corner of 

 PI. <), fig. 20, and (d) 

 PI. 7, fig. 24 

 Compare stage c' with constricted bacterium (c) near 

 the top in PI. 5, fig. 11. 



Cell boundaries in (/) inferred from Bouin-Giemsa 

 preparations. 



B. mycoides in their ovoid shape and in the double 

 structure and variable position in the cell of the chroma- 

 tinic body. In all three species the mode of division of 

 the chromatinic body is essentially the same and the 

 products of one division frequently split precociously 

 for the next division before separation is complete 

 B. mycoides (as well as B. megatherium) differs from 

 Proteus and Bact. coli, however, in so far as division of the 

 bacterium does not occur until after the third division of 

 the chromatinic bodies (stage of four double chromatinic 

 structures), whereas in the two non-sporing organisms, 



174 



