C. F. Robinow 



417 



If the bacteria are studied in the order of gradually 

 increasing length, it is easy to see how the pattern of 

 lines in the long forms is derived from the simple arrange- 

 ment in the short rods by the transformation of fine lines 

 into broad ones and by the appearance of new fine lines 

 midway between those which are beginning to thicken. 

 The contour of the cytoplasm, which is deeply stained, 

 has many irregularities similar to those observed by 

 Knaysi (1930) in cells of B. sublilis: 'The ectoplasm 

 shows thickenings and appendages of various sizes and 

 shapes on its internal surface.' 



When the bacterium divides, the plane of division 

 always coincides with the broad central band. The 

 constriction occurs in the middle of this double band 

 which at this stage has the appearance of a plump x 

 (PI. 7, fig. 24 c, d, e). This suggests that the cytoplasm 

 in the deeply stained region is concerned with the forma- 

 tion of the new transverse cell wall (cf. bacillus (a), PI. 8, 

 fig. 30, and fig. 1 of Knaysi, 1930). Since the dark 

 lines are visible long before the bacterium has begun 

 to show signs of constriction it must be assumed 

 that transverse divisions, either cytoplasmic or of the 

 same nature as the outer cell wall, are laid down long 

 before the bacterium has begun to 'divide' in the ac- 

 cepted sense of the term. 



Text-fig. 2. Semi-diagrammatic drawing of a plasmo- 

 lysed, almost completely divided specimen of B. mega- 

 therium from a preparation made as described under 

 Method 5, p. 414. Dimensions: 12-4xl-5ft. Letters 

 indicate transverse cell walls (septa) in order of 

 decreasing age ; D points to a cytoplasmic membrane 

 which resulted from the division of the protoplast AB. 

 A transverse septum, continuous with the outer cell 

 wall laid down in the plane of this boundary, would 

 have completed the spatial separation of the two 

 sister protoplasts. Compare with PI. 8, figs. 31-34. 



(6) Plasmolysis. In studying the chambered structure 

 of bacteria it would, however, be of advantage to see 

 the cell wall clearly stained together with the cytoplasm. 

 Knaysi (1930) obtained this result in B. subtil is by sus- 

 pending thecellsina dropof 25 % watery solution of NaCl 

 containing one per mille of crystal violet. Using a different 

 method suggested by the experiences of Ruhland & 

 Hoffmann (1925) with Bcggiatoa rnirabilis and staining 

 with either crystal violet or Giemsa (see Method 5, 

 p. 414) 1 have obtained preparations of the much larger 

 cells of H. megatherium and also of H. cereus in which 

 cell wall and cytoplasm were differentially stained and 

 the cytoplasm had shrunk from the cell wall which, as 

 Knaysi (1930) has already pointed out, is a great ad- 

 vantage in studying the mode of division of the bacilli. 

 Unfortunately, I was unable to produce this retraction 

 of the cytoplasm at all regularly in Hart, coli and Proteus, 

 so that the observations made by this method refer 

 almost exclusively to It. megatherium ami B. cereus. 



Although permanent mounts can he made, freshlj 

 stained preparations mounted in water give the sharpest 



and most brilliant pictures. In such material the cell 

 walls are pink and the cytoplasm a dense mauve or 

 purple. The rods consist of two, three or four separate 

 portions divided from each other by transverse septa 

 giving the same staining reaction as the cell wall. Owing 

 to the shrinking of the cytoplasm the protoplasts are 

 retracted from both the outer cell wall and the transverse 

 septa, so that the septa are very clearly seen (Text-fig. 2; 

 PI. 8, figs. 30-34). The youngest cells are nearly cubical; 

 they grow and elongate until their length is about three 

 times their width when a new transverse septum is 

 formed and the growth process repeated. Heavy staining 

 of plasmolysed* films, which is necessary to demonstrate 

 the cell wall, tends to obscure the cytoplasmic boundaries 

 which are the precursors of the transverse septa. Light 

 staining of plasmolysed films, as well as the Os0 4 -HCl- 

 Giemsa and the Bouin-Giemsa technique show that 

 oblong photoplasts, even though they may have re- 

 tracted as a whole, actually consist of two more or 

 less completely divided cells. Thus had the bacillus at 

 the top of PI. 8, fig. 30 been adequately plasmolysed and 

 more heavily stained it would probably have jjresented 

 the same appearance as the right-hand bacillus in PI. 8, 

 fig. 33. Text-fig. 2 gives an example, at (D), of a protoplast 

 that is still only divided by a cytoplasmic membrane, 

 while neighbouring cells in the same bacterium are 

 already separated from this protoplast and from each 

 other by transverse partitions which are continuous 

 with the outer cell wall. 



These results confirm de Bary's original description 

 (1884) of the multiple structure of the large Megatherium- 

 rods. They also show that the jsrocess of annular furrow- 

 ing by which whole bacteria are seen to divide is really 

 the splitting of a preformed transverse partition (Text- 

 fig. 2 A; PI. 8, fig. 33). In the description of bacteria the 

 term 'cell division' should be restricted to the formation 

 of the cytoplasmic membranes — precursors of transverse 

 partitions — which develop in the_ protoplasts after the 

 separation of recently divided chromatinic structures. 



Although it was not possible to make good plasmolysed 

 preparations of Bad. coli and Proteus with the NaOH 

 method, the number of separate cells in the plasmolysed 

 bacteria of B. megatherium, i.e. two, three or four, corre- 

 sponds with the number of sections into which the 

 transverse lines seen in Bouin-Giemsa preparations 

 usually divide the rods in young cultures of Bad. coli 

 and Proteus (cf. PI. 7, fig. 24, and PI. 8, figs. 30-34). 



(c) Chilling. If the average bacterium in a young 

 culture consists of two to four cells as the foregoing 

 results indicate, it was thought that a mildly damaging 

 treatment might not affect all the cells of the same rod 

 equally. To test this, young cultures of Bad. coli were 

 chilled in the refrigerator at 2-4° C. for periods ranging 

 from 20 hr. to several days. The expected result was 

 obtained, and among many hundreds of normal or 

 severely damaged bacteria, a few regularly presented 

 the appearance shown in PI, S. figs. 35 .'IT. 



* In this paper the term 'plasmolysed' is used, for 

 the sake of convenience, in a purely morphological sense 

 and in disregard of the physico-chemical differences be- 

 tween the (irreversible) method of fixation with NaOH 

 and the reversible response of plan* cells to which the 

 term normally refers. 



I 7 6 



