A Study of the Division of Saccharomyces cerevisiae 



Using Carbon Replicas 



D. E. Bradley 



Research Laboratory, Associated Electrical Iiuhistries Limited, 

 Aldermaston, Berkshire, England 



DECAUSE bacteria and similar organisms are dense 

 to electrons, their surfaces cannot be satisfactorily 

 studied by direct examination in the electron micro- 

 scope. If the surface detail of the organisms is to be 

 revealed, a replica technique must be employed. The 

 most suitable method found so far uses evaporated 

 carbon as a replicating material, (3), and is carried 

 out as follows: 



A clean aqueous suspension of yeast cells is first pre- 

 pared. This is most satisfactorily obtained from a liquid 

 rather than a solid nutrient medium. Cells grown in 

 6°o malt extract solution were found to be free from con- 

 taminating matter. Cultures were incubated for varying 

 periods up to 72 hours at 33"C, and fixed for 30 minutes 

 in 4 % formalin before being finally washed prior to 

 replication. 



A single-stage replica technique was used in which the 

 cell is coated with a layer of carbon and then dissolved 

 away. The yeast cells were first dried down onto a thick 

 formvar film mounted on an electron microscope speci- 

 men support grid. It was found that a good dispersion 

 could be obtained by spraying techniques, though there 

 was a risk of damage to the yeast cells. The cells were 

 next coated with a layer of carbon 150 to 200 A thick. 

 It was then necessary to wash away the formvar substrate 

 so that the yeast cells could be dissolved from the carbon 

 replica. This was carried out by flowing a few ml of 

 chloroform over the grid from a burette at the rate of 

 1 ml per minute. After the chloroform had evaporated. 



the grid was immersed in a solution of 3 g of a mixture 

 of potassium permanganate and dichromate in concen- 

 trated sulphuric acid for fifteen minutes. After removal, 

 it was washed in water, then in concentrated hydro- 

 chloric acid for a few seconds (this removed the manganese 

 dioxide formed by the decomposition of the acid mixture), 

 and finally in water. After drying, it was ready for shad- 

 owing and examination in the electron microscope. 



Care must be exercised in the interpretation of 

 electron micrographs of replicas produced by this 

 technique, since a number of artifacts, which are 

 easily recognisable, are liable to occur. The most 

 obvious of these is distortion during the dehydration 

 of the cell. This causes a very gross effect which can 

 be clearly seen in figure 1. It is also likely that, in 

 the case of large organisms such as yeast, the replica 

 will become distorted and a similar effect produced. 

 The rim at A in figure 2 is caused by cell dehydration 

 and gives the appearance of the cell having been 

 flattened against the formvar film. It was found that 

 these artifacts were by no means a serious hindrance 

 and undistorted cells could easily be found by 

 systematic scanning of the grid. 



Two kinds of budding scar, illustrated in figure 1, 

 were found on yeast cells. The "birth" scar (marked 

 C), which is the scar formed at the point where a 

 cell was attached to its parent, and the "bud"* scar 

 (marked A and B), which is formed on the parent 

 cell when the daughter becomes detached. These 

 scars are quite different in form as can be seen from 

 the figure. These observations are in agreement with 

 those of Barton (2), who carried out his work opti- 

 cally. The morphology of these scars is of great im- 

 portance in a study of the mechanism of division. 

 Electron micrographs of cells prior to division help 

 in proposing a sequence of events for the process. 



Figure 2 shows the '"neck" connecting two cells 



Fig. 1. Shadowed carbon replica of single yeast cell showing 

 "bud" scars at A and B and "birth" scar at C. (By courtesy 

 oi Research.) Ma.gn\f\ca.Uo:\ 14,000. 



Fig. 2. Internal rims showing in the "neck" joining two cells 

 before division. Shadowed carbon replica. (By courtesy of 

 the /. Roy. Microscop. Soc.) Magnification 11,000. 



