2g Methyene Blue

500 ml DDW

500 ml Methanol

Stir overnight with cap loosened

No filtration

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

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