Exercise VII 



STUDIES IN MICROBIOLOGY (2) 41 



dilute phosphate buffer alone (pH 7.0) and the 

 other in phosphate buffer to which sucrose has 

 been added to a concentration of 0.15 M, making 

 the medium isosmotic with the cell contents. 



Pour about 2 ml of the suspension of cells in 

 buffer alone into a small test tube. Note the 

 turbidity of the suspension. Add 4 to 5 drops 

 of lysozyme solution and swirl, watching the 

 tube as you do so. You should soon see the 

 suspension clarify, as the cells lyse. Examine 

 the end result under the microscope. 



Repeat this experiment, using the suspension 

 of cells containing sucrose. Do you still note 

 changes in turbidity? Again look at the result 

 under the microscope. The spherical protoplasts 

 should be visible. 



It will be worth preparing a wet mount of 

 B. megatherium in phosphate buffer containing 

 sucrose, and adding one drop of lysozyme on 

 the slide while looking at the cells. The dissolu- 

 tion of the cell wall can be seen, and all the 

 stages in the formation of protoplasts. 



Microorganisms in the air 



Label a plate of nutrient agar with your 

 name, and leave it open, exposed to the air, for 

 30 minutes. Don't place it too close to where 

 anyone is working, lest he spill bacteria near 

 your plate. These plates should be incubated 

 at home for two days at room temperature 

 (about 20° to 25°C), then placed in the refrigera- 

 tor so that you can examine them next week. 



Further microscopy of bacteria 



The microscopes you have been using do not 

 have sufficient magnification to make most bac- 

 teria visible. A few higher-power microscopes 

 may be available, possessing an oil immersion 

 objective lens. Since in this case the light is not 

 required to pass from glass to air and back, 

 greater magnification can be achieved. In addi- 

 tion, this microscope may provide phase con- 

 trast, which enhances the contrast wherever 

 there is a difference in refraction of light within 

 the object or between it and its surroundings. 



Examine wet mounts of Serratia marcescens 

 and Pneumococcus under the highest power avail- 

 able to you, using phase contrast if you have it. 

 After placing the slide on the stage, put a drop 

 of immersion oil on the center of the cover 

 glass. Watching from the side, bring the high- 

 power objective down until it dips into the oil 

 and almost touches the cover glass. With the 

 fine adjustment slowly raise the objective until 

 the bacteria come into focus. 



Bacteria are often divided into two groups on 

 the basis of shape: bacilli (rods) and cocci 

 (spheroids). B. megatherium is clearly a rod. 

 Serratia is more difficult to classify; it is con- 

 sidered to be a short rod. Pneumococcus is, of 

 course, considered to be a coccus. Do you find 

 its shape to be perfectly round ? Cocci that are 

 strung along in chains are called streptococci 

 (strepto, Gr. = chain); those which occur in 

 pairs are diplococci. What would you call Pneu- 

 mococcus 7 



Further reading 



On genes and enzymes: 



K. V. Thimann, Life of Bacteria, Macmillan, 1955, 

 pp. 561-571. 



On radiation and mutation: 



R. Y. Stanier, M. Doudoroff, and E. A. Adel- 

 BERG, The Microbial World, Prentice-Hall, 1957, 

 pp. 264-268. 



K. V. Thimann, op. cit., pp. 662-667. 



On mutation: 



S. P. T., pp. 321-324. 



R. Y. Stanier, et al., op. cit., pp. 380-393. 



On bacteria under the microscope: 



R. Y. Stanier, et al., op. cit.. Chapter 1 and pp. 

 105-109. 



K. V. Thimann, op. cit., pp. 38-57. 



ViLLEE, pp. 132-143. 



On microorganisms in the air: 



R. Y. Stanier, et al., op. cit.. Chapter 5 (fungi); 

 pp. 243-248 (colonial forms); pp. 296-329 (major 

 groups of bacteria). 



