26 Discussion 



which strikes an occasional part of the plate and gives rise to wide 

 divergences in subsequent behaviour. 



Hinshelwood: Do you keep complete time-size logs of all the colonies 

 on the plate? We have always thought it necessary to do this, i.e. to 

 measure or photograph the plate day by day or every half-day. Other- 

 wise, if you inspect things at arbitrary intervals and arbitrary sizes you 

 do tend to create sharp distinctions which may not exist. These are 

 especially marked if the liquid culture has already begun to multiply. 



Lederberg: No, we have not done this. Nor are we acquainted with such 

 a detailed presentation in the literature. 



Pollock: We have done some work on the original strain of Bad. coli 

 mutabile used by Sir Cyril (see Dean and Hinshelwood, 1954&, loc. cit.) 

 for studying the lactose training phenomenon which he maintains is 

 specifically induced in most of the cells by lactose. Most of our findings 

 correspond to what Lederberg has described for D-arabinose training. 

 Cells untrained to lactose (Lac~) plated into lactose-agar produce 

 small colonies (Fig. la) compared to cells from a lactose-trained culture 

 (Lac+) which form much larger, denser colonies (Fig. lb) in the same 

 period (5-7 days). If you leave the Lac~ colonies for longer, most of 

 them eventually grow into large colonies (Fig. Ic) comparable in size 

 to the Lac+ colonies (as described by Dean and Hinshelwood) but 

 mottled and irregular in appearance. A series of stages in the develop- 

 ment of Lac~ colonies is illustrated in Fig. 2a to / showing clearly 

 the emergence of papillae. Subcultures from the papillae yield nearly 

 100 per cent colonies of the Lac+ type (Fig. lb) in lactose agar. It 

 might be quite easy to miss these papillae in colonies growing deep 

 in agar and they were only easily visible by the use of a binocular 

 plate microscope. With surface-inoculations, the papillae were quite 

 obvious to the naked eye and it was much easier to distinguish between 

 colonies of the Lac+ and Lac~ type. Fig. 3 is a re-analysis of the Hin- 

 shelwood and Dean curve of increase in cell numbers of a glucose-grown 

 culture inoculated into a liquid lactose medium. As well as plating 

 samples out into glucose agar (for total viable count) they were also 

 plated into lactose agar and Lac+ and Lac~ colonies counted. The total 

 viable count corresponds exactly with Dean and Hinshelwood's curve, 

 but it can be seen that the final rise in numbers is due entirely to growth 

 of Lac''' cells (which have, indeed, been increasing logarithmically during 

 most of the experiment). With surface inoculation on lactose agar there 

 is absolutely no difficulty, after 4 days of incubation, in distinguishing 

 the two types of colony. The Lac"'" colonies are opaque and dome-shaped 

 and nearly twice the diameter of the much thinner and flatter Lac~ 

 colonies. Fig. 4a shows the appearance of colonies, after 8 days of incu- 

 bation, from a Lac~ culture inoculated on the surface of lactose agar. 

 Fig. 4<b shows 3 Lac"^ and 3 Lac~ colonies from an artificial 50 : 50 mixture 

 of Lac+ and Lac~ cells inoculated in the same way and incubated for 

 the same period. The Lac~ colonies in Fig. 46 are about twice the dia- 

 meter of those in Fig. 4a; their growth has obviously been greatly 

 stimulated by a substance produced by the Lac+ cells. It might be 

 something as simple as glucose. It seems possible that this effect might 



