Methylene Blue Staining Of Viable Adherent Cells
2g Methyene Blue
500 ml DDW
500 ml Methanol
Stir overnight with cap loosened
Reuse bottle and pipet
Used stain can be reused 3 times, each time after fitering though whatman filter to remove debris.
Take sample plate or control plate ( one which should have colonies) out of the incubator and scan for colonies. If contol colonies are 1 mm in diameter, then the whole experiment can be stained.
Remove media with Pasteur Pipet by aspiration. Take care not to disturb the cells on the bottom of the dish.
Cover the bottom of the plate with stain using plastic pipette reused for stain
Stand at room temperature from 5 minutes to 1 hour
Tip stain into bottle containing used stain (kept near sink)
Wash gently by immersing plate bottom up into a bucket with luke warm tap water
Invert dishes on a tray with lids on top and dry in lab
Count colonies , i.e. groups of 30 or more cells, check minimum colony size under the microscope
TO STAIN P-TEST PLATES
Score plates for end point
Tip out the media into pipette container with bleach
Fill wells with stain
Let sit at rt for 10 minutes to 1 hour
Tip out stain into sink
Wash as above and dry with lid secured with rubber band so they dont get mixed up