Exercise IX 



STUDIES IN MICROBIOLOGY (4) 49 



culture to each of 4 tubes of soft agar with a 

 sterile dropper. Add 2 drops of the various 

 phage suspensions to the tubes, mix, and pour 

 the contents onto the appropriate quadrants, as 

 you did earlier. 



Take the plates home with you, and the next 

 day study the various plaque shapes, sizes, and 

 appearances. In your notes describe the various 

 plaque morphologies and decide which of the 

 phages matches your unknown. 



Fillerability of viruses and bacteria 



Do the filtration in pairs, but each student 

 should have his own agar plate. Probably several 

 pairs of students will have to take turns using 

 each set of filters. The porcelain filters will be 

 identical, but since four experiments have to be 

 carried out without stopping to sterilize the 

 filters, use only the filter marked "P" for phage 

 and only the one marked "C" for E. coli. Also, 

 do not contaminate the porcelain part of the 

 filter by touching it with your finger or placing 

 it on the bench. 



Obtain a 4-quadrant nutrient agar plate and 

 label clearly. Two quadrants are for the E. coli 

 suspension, before and after filtration, the other 

 two for filtered and unfiltered phage, plus bac- 

 teria. Streak a loopful of E. coli across one 

 quadrant. (Streaking is a single linear passage 



of the loop on the surface of the agar.) Place a 

 sterile test tube under the filter marked "C," 

 and apply suction. Pass 20 drops of E. coli 

 suspension through the filter. Streak a drop of 

 the filtrate on the appropriate quadrant of your 

 agar plate. 



Add 2 drops of T4 phage suspension to a soft 

 agar tube containing 5 drops of E. coli. Pour 

 the tube contents onto the suitable quadrant. 

 Now filter 20 drops of phage and add a drop 

 of the filtrate to a soft agar tube containing 

 1 drop of E. coli. Again plate out the tube 

 contents. 



Take this plate home with you and keep it 

 with the others in a warm spot. Examine them 

 the next day, and, if necessary, the day after 

 that. Record your observations. 



Further reading 



C. A. Knight and D. Fraser, "The Mutation of 



Viruses," Sci. Am. 193, No. 1, pp. 74-78, July 



1955, Reprint No. 59. 



S. E. LuRiA, "The T2 Mystery," Sci. Am. 192, No. 



4, pp. 92-98, April 1955, Reprint No. 24. 



H. Fraenkel-Conrat, "Rebuilding a Virus," Sci. 



Am. 194, No. 6, 42-47, June 1956, Reprint No. 9. 



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



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



pp. 365-371. 



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



pp. 85-94. 



EQUIPMENT 



Per student 



5-ml pipet, sterile 



8 dropper pipets, sterile 



4 nutrient agar plates 

 2 wide tubes, sterile 

 aerator assembly, sterile 

 bacteriological loop 

 bunsen burner 



14 small test tubes with 1 ml soft agar 



5 ml nutrient broth 



Per 8 students 



suspension of phage T4 in broth at lO'* particles/ml 

 (for phage reproduction experiment) (5 ml) 



suspension of T2, T4, T4r, and unlabeled phage, in 

 broth at 10^ particles/ml (for phage morphology 

 experiment) (5 ml each) 



dropping bottle of E. coli culture in logarithm phase 

 of growth (10-20 ml) 



2 porcelain filter cylinders assembled in suction 

 flasks, sterile 



Per laboratory (30 students) 



water bath at 37°C, with aeration assembly 



2 water baths at 45°C containing small tubes with 

 soft agar 



