Team:Cambridge/Protocols/SporeImage

Imaging B. Subtilis and its spores

Preparation

To complete this protocol you will need:

• 5% malachite green
• 0.5% safarinin O
• A Bunsen burner and/or heatblock
• slides
• A high quality fluorescence microscope, preferably confocal

NOTE: BE vary careful when preparing and handling dies. They get EVERYWHERE!!!!! and NEVER. LEAVE.

Method

Note: It is best to carry out the following procedure in a petri dish and on top of paper to catch any dye.

• Spread around 100ul liquid culture of your cells/spores on a glass slide. Try and do this evenly so as not to leave splodges
• Fix culture with heat from 50⁰C heatblock or bunsen flame
• Add around 50ul malachite green directly to your culture.
• Allow this to fix for five minutes either on a 50⁰C heatblock or with periodic heating from a bunsen flame. This step must not be rushed as the malachite green needs to penetrate the thick spore membrane.
• Wash off the dye (apply water away from culture and use fingers to move slide around, releasing dye from underneath), initially draining waste into a specific die waste container but once it is dilute enough, the die can be washed down the sink.
• Apply 50ul safarinin O. Allow time to fix but this is not as critical as for the malachite green.
• Wash of VERY WELL
• Apply 10-20ul high concentration gylcerol (>50%) to your culture and fix with cover slip.
• Image under microscope.

• Malachite green should fluoresce with far red excitation and show the spores
• Safarinin O should fluoresce with 490nm excitation and 590nm emission and show vegetative cells.