Top Agar Plating

 

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Top Agar Plating allows Mycobacteria to come into contact with mycobacteriaphage.  This protocol is optomized for M. smegmatis. 

Procedure:

A turbid culture of M. smegmatis, grown in an inkwell will be used.  Dilution of phage will be done, see (Day 1 of the diary or Phage Dilution Plating).  100 uL of the appropriately diluted phage is added to 100 uL of the M. smegmatis, and allowed to remain at 37C for 20-30 minutes, if that long.  Ideally, this will be done on a heating block at 37C, using the appropriately sized reusable test tubes with caps (Phage supplies).  After this incubation period, 3 mL of top agar (Media Preparation), cooled to less than 50C (meaning it can be touched to your cheek) will be added to the phage in the same glass test tube and mixed by flicking with the thumb.  This solution (notice how important it is that the test tube is the correct size) is then quickly poured onto the top of the 7H10 plate, gently hand shaken in a radial/rocking manner, and allowed to solidify.  These plates will be incubated at 37C until plaques are visible; check for plaque formation every 24 hours.  The incubation temperature and growth duration may differ for certain phages.