860 CLOSTRIDIUM 



in the tissue fluids of injected animals. The usual coverslip method is satisfactory 

 in most instances, but if this fails, examination should be conducted in a closed 

 capillary tube that has been inoculated with a young broth culture and kept at 

 37° C. for about half an hour. As is customary with large organisms, motility 

 is rarely well marked, and is usually of the slow and stately variety, in contrast 

 to the rapid, darting movements of smaller organisms such as Salm. typhi. 



CI. hutyricum and CI. welchii are the only members possessing a capsule ; the 

 capsule of CI. welchii is noticeable in the animal body, and sometimes in cultures 

 containing serum. 



Staining Reactions. — All members stain readily with the usual dyes. Great 

 irregularity is noticeable in the depth of staining, especially in cultures more than 

 a day or two old. Sometimes metachromatic granules are noticeable, or points 

 of more intense coloration. Provided young cultures are examined, the bacilli 

 are all Gram-positive. Some species rapidly lose this property, and some can be 

 decolorized if the alcohol is applied for too long. In the early stages of spore 

 formation, the position of the spore is often marked by an area of intense staining ; 

 as it matures, however, the spore presents a colourless centre surrounded by a 

 peripherally stained ring. 



Cultural Reactions. — On solid media growth is relatively slow, and takes the 

 form of a thin, effuse, often spreading film, which may be difficult to distinguish 

 from the underlying medium. 



The tendency to film formation is promoted by moisture. On first isolation 

 CI. septicum, and particularly CI. tetani, tend to spread rapidly over a moist surface. 

 If CI. teta,ni is inoculated into the condensation water of an agar slope, it will in 

 the course of a day spread over the whole medium ; the film is so thin that, were 

 it not for the dentate edge presented at the upper end of the slope, where the 

 medium is drier, it might easily escape detection. Advantage may be taken of 

 this fact in the isolation of this organism (Fildes 1925a). The spreading of Clostridia 

 can be inhibited by the incorporation of certain chemicals in the solid media. 

 The majority of these, however, are to some extent bacteriostatic ; inhibition of 

 spreading without bacteriostasis, as Hayward (Miles and Hayward 1943) has shown, 

 may be achieved by increasing the concentration of agar up to about 6 per cent. 

 Certain Clostridia also produce on agar motile daughter-colonies, which rotate and 

 wander over the surface of the medium (Turner and Bales 1941). Concentrated 

 agar is less effective as an inhibitor of this type of spreading. 



Agar Plates. — Single colonies are rounded, generally effuse, and present 

 crenated, fimbriate, or rhizoid edges. CI. welchii, which is one of the less strict 

 anaerobes, forms low convex colonies with an entire edge ; CI. sporogenes and CI. 

 histolyticum may form umbonate colonies with a raised centre and a flat periphery. 

 The colonial appearances are often characteristic, but some species give rise to 

 variants which not only are unlike the typical colony, but which strongly suggest 

 the occurrence of contamination. Several different types of colony may be formed, 

 for example, by CI. sporogenes. 



Glucose Agar Shake Cultures. — These are commonly employed for studying 

 the form of deep colonies, and by many workers for the preparation of pure cultures. 

 Except near the surface, growth occurs throughout the medium ; this is frequently 

 disrupted and blown upwards by the development of gas. Single colonies are 

 rounded or lenticular in shaj)e, and lenticular forms may later develop irregular 

 tufts of growth, sprouting from the edge or the poles of the lenses ; sometimes 



