48 STUDIES IN MICROBIOLOGY (4) 



Exercise IX 



(To, T4, and T4r), along with one unlabeled 

 sample of one of these three phages. We will 

 plate out all four samples, and by examining 

 the plaque types identify the unknown phage. 



One of the most characteristic features of 

 viruses is their small size. This was appreciated 

 very early when it was observed that they pass 

 through filters which have pores fine enough to 

 retain bacteria. For this reason these minute 

 infective agents were called "filterable" viruses. 

 We shall test the filterability of viruses and bac- 

 teria with a porcelain filter. 



EXPERIMENTS 



Bacterial transformation (continuation) 



Examine the blood agar plate from last 

 week's experiment on the genetic transformation 

 of Pneumococcus. Colonies of Pneumococcus 

 have a characteristic appearance on blood agar 

 plates, so they can easily be distinguished from 

 contaminants. The Pneumococcus colony is very 

 small, a fraction of a millimeter in diameter. 

 Around the colony is a zone of hemolysis, a 

 clear area where substances released by the cells 

 have lysed the blood cells in the agar. Any 

 colonies which you find on the plates are 

 streptomycin-resistant, since streptomycin had 

 been added to the agar. 



Hold the plate up to the light. Do you find 

 any colonies of Pneumococcus in the control or 

 DNA sectors? in the sector corresponding to 

 the transformed culture? Count the number of 

 resistant colonies. 



Reproduction of bacteriophage 



In sterile, wide test tubes obtain 5 ml of 

 nutrient broth. One student should prepare a 

 dilution series of the phage for himself and his 

 partner as follows. Transfer 1-drop portions of 

 the E. coli culture to each of 8 small tubes con- 

 taining 1 ml of soft agar (4 per student). (The 

 soft agar is kept in the water bath at 45°C.) 

 With a sterile dropper, add 1 drop of the phage 

 suspension to the nutrient broth. Mix. Now 

 prepare to determine the number of virus par- 



ticles, by making a dilution series of the phage 

 in broth in the 4 tubes containing bacteria in 

 soft agar. With the sterile dropper add 2 drops 

 of the phage in broth to the first tube, 2 drops 

 of that to the second, and so on. (Do not let 

 the soft agar harden; keep the tubes in the 45° 

 bath as much as possible during these transfers.) 



Obtain a 4-quadrant nutrient agar plate, and 

 label appropriately. Now quickly pour the con- 

 tents of the dilution tubes onto the appropriate 

 quadrants, one at a time. Rock the plate slightly 

 each time to obtain a thin, even layer of liquid 

 over the quadrant surface, and let harden. Be 

 careful not to spill over onto the neighboring 

 quadrants. 



We shall now repeat this experiment after 

 allowing the virus a period of growth. Add 3 

 drops of E. coli cells to the phage suspension 

 in broth. Insert an aerator tube. Incubate at 

 37° for 60 minutes with aeration. Dilute out 

 the virus in a second series of 4 tubes containing 

 E. coli cells in soft agar, as you did before. 

 Plate out the dilutions on agar as above. 



Take both these plates home with you, and 

 keep them in a warm spot. By the following 

 morning you should be able to count the blank 

 areas, or plaques, on the plates. On quadrants 

 where many virus particles were plated, plaques 

 will run together ("confluent lysis"). Where no 

 viruses were plated, there will be smooth, con- 

 fluent growth of bacteria. Count the plaques in 

 those quadrants where they appear clearly. 

 Hold the plates against a black background or 

 up to the light in order to facilitate counting. 



From your counts calculate the number of 

 virus particles initially present in the suspension. 

 Calculate also the number of virus particles 

 present at the end of the growth period. How 

 many times greater than the initial count was 

 the final count? How does viral reproduction 

 compare with bacterial reproduction in rate? in 

 its essential mechanism? 



Plaque morphology and identification of an 

 unknown phage 



Obtain a 4-quadrant nutrient agar plate and 

 label appropriately. Add 1 drop of the E. coli 



