40 STUDIES IN MICROBIOLOGY (2) 



Exercise VII 



and control. Spread a loop of the bacterial sus- 

 pension on the quadrant marked 150 and expose 

 the uncovered plate to ultraviolet light at a dis- 

 tance of 25 inches for 30 seconds. (Caution: Do 

 not look into the ultraviolet light. Do not 

 expose your skin for more than a few seconds.) 

 Next spread a loop of the suspension on the 

 quadrant marked 120 and expose the plate to 

 ultraviolet light for an additional 30 seconds. 

 Now spread a third loop on the quadrant 

 marked 90 seconds and expose the plate for 

 90 seconds. Finally spread a loop of the sus- 

 pension on the control quadrant, cover, and give 

 the labeled plates to your instructor. He will 

 incubate them for two days at room tempera- 

 ture and will then store them in the refrigerator 

 until next week. 



Microscopic examination of 

 Bacillus megatherium 



Bacillus megatherium is a giant among bac- 

 teria even though it is only 1 micron wide by 

 4 micra long. (A micron is 1/1000 mm, or 

 1/25,000 inch.) The other strains which we have 

 been using are much smaller, and in order to 

 see them more elaborate microscopy is needed. 



Prepare a wet mount of B. megatherium as 

 follows. Place a droplet of water on a clean 

 glass slide. This may be done conveniently with 

 a glass rod. Stir a loopful of B. megatherium 

 culture into the drop. Gently place a cover glass 

 over the drop; try to avoid leaving air bubbles 

 under the glass. Focus on the bacteria under 

 the high-power objective of your microscope. 

 To do this, watching from the side, bring the 

 objective down until it Just fails to touch the 

 cover glass. Now, looking through the ocular, 

 slowly raise the objective by means of the fine 

 adjustment until the field is in focus. To see 

 the bacteria well it will be necessary to close 

 down the diaphragm (with the lever under the 

 stage) so that the field is only dimly illuminated. 



The bacteria may be seen more easily after 

 staining them with a dye. Remove the cover 

 glass. Let the suspension dry. Pass the slide, 

 face up, through a bunsen flame three times. 



The heat will coagulate bacterial proteins and 

 fix the bacteria to the slide. When it is cool, 

 flood the area with a drop of methylene blue. 

 Wait one minute, then rinse the slide with water. 

 Gently blot it dry with a paper towel. Examine 

 the slide once again under the high power. 

 Draw what you see. 



Action of lysozyme: bacterial protoplasts 



The enzyme lysozyme breaks down the com- 

 plex polysaccharides of which the cell walls of 

 many bacteria are composed, leaving the cell 

 covered only by its delicate plasma membrane. 

 Whereas the intact bacterium may have been 

 rod-shaped, it becomes spherical on losing its 

 rigid cell wall. Such naked, spherical cells are 

 called protoplasts. We shall watch cells of B. 

 megatherium being lysed by lysozyme, and form- 

 ing protoplasts. This experiment does not de- 

 mand sterile conditions. 



Quite frequently, as in the present instance, 

 the contents of a cell are considerably more con- 

 centrated than the surrounding medium. As a 

 result, water tends to flow from the medium 

 into the cell, making it swell. In B. megatherium, 

 as in many other bacteria, this tendency to swell 

 is resisted by the rigid cell wall. (Recall the 

 opposite effect of suspending Elodea in strong 

 salt solution, in Exercise I, which made the cell 

 shrink away from the cell wall.) When bacterial 

 cells have lost their cell walls through the action 

 of lysozyme, this restraint is removed. The 

 entrance of water from the medium, swelling 

 the cell, subjects its plasma membrane to great 

 strain. Eventually it ruptures, and the cell con- 

 tents pour out into the medium. 



This is easily seen by adding lysozyme to a 

 turbid suspension of bacteria. The suspension 

 rapidly clears as the cell walls are hydrolyzed 

 away, and the bacteria burst or lyse. If sucrose 

 is added to the medium, so that its osmotic 

 concentration is equal to that of the cell interior, 

 the cell no longer swells, and the result is a 

 stable, spherical protoplast. 



Two suspensions of B. megatherium are pro- 

 vided, identical except that one is suspended in 



