CYTOLOGICAL OBSERVATIONS ON BACT. COLI, PROTEUS VUL- 

 GARIS AND VARIOUS AEROBIC SPORE-FORMING BACTERIA 

 WITH SPECIAL REFERENCE TO THE NUCLEAR STRUCTURES 



By C. F. ROBINOW*, Strangeways Research Laboratory, Cambridge 



(With Plates 5-8, and 3 Figures in the Text) 



INTRODUCTION 



Discrete Feulgen-positive chromatinic bodies, occurring 

 in regular numbers and going through a regular cycle of 

 division, were first demonstrated convincingly by Stille 

 ( 1 937) and Piekarski (1937) in several well-known species 

 of sporing and non-sporing bacteria. The literature on 

 the subject has already been briefly reviewed in a pre- 

 vious communication (Robinow, 1942) in which the 

 nuclear apparatus in the spores and vegetative cells of 

 B. mycoides and various other bacteria was described. 



The first part of the present paper deals with the 

 shape and behaviour of the chromatinic bodies of Bact. 

 coli and Proteus vulgaris, and in particular with the 

 numerical relationships and distribution of these bodies 

 in growing bacteria from young cultures. The technique 

 of fixing, staining and observing the material has been 

 described in some detail because scepticism about the 

 chromatinic bodies in bacteria is usually due to the use 

 of inadequate cytological methods and unsuitable optical 

 equipment. 



The latter part of the paper is concerned with obser- 

 vations on the cellular structure of bacteria from young 

 cultures. Since de Baiy's original description of B. meg- 

 atherium (1884), it has been known that the rod forms of 

 some of the aerobic spore-bearing bacilli are not single 

 units but are composed of several separate cells. The 

 validity of this structural principle for growing bacteria 

 of many species besides B. megatherium is not always 

 realized, though it is recognized by some workers (cf. 

 Migula, 1897; Nakanishi, 1901; Knaysi, 1930; and 

 Badian, 1933). My own observations on suitably stained 

 and plasmolysed bacteria of several species provide further 

 evidence in support of the view that rod forms have a 

 composite structure from the earliest growth stages. 



MATERIAL 



The following organisms were investigated: 



Bact. coli : a strain received from the National Institute 



of Medical Research, Hampstead ; 

 Proteus vulgaris: No. 5821 Nat. Coll. Type Cultures 



and a strain isolated from human faeces in the " 



Pathology Department, Cambridge; 

 and for purposes of comparison : 



B. mycoides: No. 16 Nat. Coll. Type Cultures; 



B. mesentericus : from the collection of Dr W. J. 



Dowson of the Botany School, Cambridge; 



* Of the Cancer Department, St Bartholomew's 

 Hospital. 



B. megatherium : isolated from the soil of the Labo- 

 ratory garden and identified by Dr T. Gibson, 

 Edinburgh ; 



B. cereus: from the Nat. Coll. Type Cultures. 



Although observations were made on only one strain 

 of Bact. coli and two strains of Proteus vulgaris, ex- 

 perience has shown (Piekarski, 1937-40; Neumann, 

 1941; E. Klieneberger, unpublished observations; and 

 Robinow, unpublished observations) that there is such 

 a close similarity between the chromatinic structures in 

 different species that nothing is to be gained by a com- 

 parative study of many strains belonging to the same 

 species. 



TECHNIQUE 

 Nutrient agar (heart) and broth (heart), both from 

 Messrs British Drug Houses Ltd., were used as culture 

 media throughout the investigation. 



The chromatinic structures were differentiated from 

 the cytoplasm by treating the osmium-fixed cells with 

 N/HC1 at 60° C. before staining them with Giemsa solu- 

 tion (Piekarski, 1937; Robinow, 1942). This method 

 gives preparations of uniform clarity and is equally 

 useful for studying nuclear structures in whole mounts 

 of yeasts, moulds and blue-green algae. The effect of the 

 treatment with hydrochloric acid is not to make the 

 chromatinic bodies more stainable as is sometimes sup- 

 posed, but merely to remove from the cytoplasm some- 

 thing with a strong affinity for Giemsa's stain which 

 obscures the chromatinic structures in the interior of 

 the cell. The chromatinic structures can be demonstrated 

 without hydrolysis (Badian, 1933; Neumann, 1941) but 

 less clearly and regularly. 



The details of the procedures adopted were as follows : 



A. Fixation 

 (1) Spore-forming organisms 



Air-dried films of spores made on glass cover-slips 

 from cultures on potato agar, were covered with two or 

 three narrow strips of nutrient agar and incubated in a 

 moist chamber at 37° C. for 2-3 hr. For the staining of 

 nuclear structures the agar strips were removed and the 

 cover-slips quickly transferred to a deep dish, well sealed 

 by a greased glass plate, where 5 ml. of 2% osmium 

 tetroxide wetting three layers of glass balls produced a 

 strong concentration of osmic vapour. After 2-3 min. 

 the films were allowed to dry in air, then treated for 

 12 min. in Schaudinn's sublimate alcohol warmed to 

 45-50°C, and finally rinsed and stored in 70% alcohol. 

 The secondary fixation with sublimate, recommended 



28-2 



[Reprinted by permission of the Cambridge University Press from The Journal of Hygiene 

 43: (0) 413-423, September, 1944] 



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