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From 2010.igem.org

GRAM STAIN PROTOCOL

1. With a wax pencil draw a series of circles about tbe size of dime on a new glass slide and numbeithem "’I, 2, 3.4, 5, 6.

2. Prepare bacteria for staining from broth cultures

a. Flame sterilize a loop and let it cool then obtain a ’loopful "of bacterial culture.

c. Distribute the loopful cells as completely as possible in tbe water on the slide

d. Allow the slide to air dry completely (you can hasten this by placing them on the vei-v bottom [silver surface] of the incubator) -

e. "Heat fix" the cells to the slide witb 3-4 slov,, passes lhrough flame.

f. Place paper tov,,el in the bottom of the staining tray.

3. Cir.’sial violet - cover smear with primary stain let stand 30-60 sec., drain & rinse with squirt bottle of water (squirt above the smear and let water run gently over it). You may dab water next to your section.

4. Gl~lm iodine - cover with (a mordant - crosslinks the dye to pepiidoglycan) for 30-60 sec., drain & rinse as before.

5~ Decolorizer - (alcohol/acetone) drop by drop ( 15 drops) from above the specimen, until color stops easily coming off, drain & rinse.

6. Sa frnnin -cover with conterstain., for 30-60 sec, drain, rinse

7. Blot dr3., orouttd the specimen, you may return to the incubator again to dr,,,.

8. Focus On lower power first (100 or 400x), then view with oil immersion I~ns (1000x)

10. Dispose of slide in sharp biohazard trash

11. Wipe oil from oil immersion lens with LENS PAPER and cleaner (NOT Ki~WIPE!!!)