Mycobacteriophage Lysogeny Assay

Gregory Broussard

Revision: 02/24/11

(Please use, change and disseminate freely!)

• Use a culture of M. smeg grown to an O.D.₆₀₀ of 0.8-1.0.
• Dilute phage lysate to 10¹⁰ pfu/ml
• Ex:  (1.6 x 10¹¹pfu/ml) X = (2.0 x 10¹⁰pfu/ml)(1ml)
  • X = 125 µl lyate into 937.5 µl Phage buffer/CaCl₂
• Plate 100 µl diluted lysate onto plates for a total of 2 X 10⁹ pfu/plate
• Make 10^-1 to 10^-7 dilutions of M. smeg strains
• Plate 100 µl of diluted M. smeg onto phage seeded plates and plates not seeded with phage (I usually plate 10^-4 to 10^-7 dilutions).
• Incubate at 37°C for ~4 days (wrapped in parafilm or bagged).
• Count cfu’s and divide cfu’s on phage seeded plates by those without phage and multiply times 100 to get percent lysogeny.