Plating Ms. barkeri 227

TSA 4-14-98
  Available in anaerobic bag:  
  sterile 50 ml conical tubes small tubes with kimwipe 'wick' (minitubes)
  clinical centrifuge dilution media (uninoculated, reduced)
  pre-poured 6 cm plates 20% Na2S
  modified wide mouth Ball® jars1 83 mM Ti-citrate
  sterile spreaders syringes and needles
 
1. In a clinical centrifuge, spin down 50 ml of an actively growing Ms. barkeri 227 culture in a sterile conical tube. 6000 rpm 20 min.
 
2. Pour off spent media into a beaker. The cell pellet is very loose - be prepared to pour off supernatant as soon as the centrifuge stops. Also, the cells are often on the side of the tube. Resuspend cells in 800 ml of dilution media.
 
3. Plate 25 and 50 ml on plates. Optimize the plate humidity for each anaerobic bag. If different bugs are used, the humidity will have to be optimized again.
 
4. Add 0.5 ml of 83 mM Ti-citrate to a minitube. Add 0.4 ml of 20% Na2S (8g Na2S/jar)1 to a minitube. If different bugs are used, the amount of Na2S will have to be optimized.
 
5. Put vacuum grease around the rim of the jar, close it and remove from the bag.
 
6. Vacuum gas out of the Ball® jar, tighten lid and pressurize to 5-8 psi.
 
7. Measure methane at time=0, incubate.
 
8. Measure OD600 of resuspended cells to find the approximate number of cells plated.
     
  Using: 1 OD600 = 7.4x108 cells /ml (for Mc. maripaludis)2
  Ms. barkeri 1.5-2.0 mm diameter3
  Mc. maripaludis 1 mm diameter3
9. Measure methane daily
   
Notes: - The septum on the Ball® jar should be unused.
  - Do steps 2-6 as quickly as possible.
References
1. Apolinario, E.A. and Sowers K.R. (1996) Plate colonization of Methanococcus maripaludis and Methanosarcina thermophila in a modified canning jar. FEMS Microbiol.Lett.145, 131-137.
2. Tumbula, D.L., Makula, R.A. and Whitman, W.B. (1994) Transformation of Methanococcus maripaludis and identificationof a PstI-like restriction system. FEMS Microbiol. Lett. 121, 309-314.
3. Bergey's Manuel of Systematic Bacteriology (1989) 3, 2190, 2203.