Halophiles are organisms that love salt. For example bacteria that flourish in salty conditions. In this practical session halobacterium is used, coming in a kit by Carolina Biological Supplies. Halobacterium is a prokaryote, same as bacteria, but has evolved differently, belonging to the Archaea kingdom, and it is an extremophile. In other words halobacterium is a distinctive single-celled microorganism, thriving in an extreme environment (a very salty one, where most organisms would not grow).
- 5 Tubes
- Halobacterium medium
- Halophile salt crystals
- Shaker incubator or test tube rack
- Hypersaline medium: 25% salt agar??
- 3 Petri dishes / plates
- Inoculation loop
- Eye protection, heat-resistant gloves and a lab coat are recommended
Part 1, Halophile Salts
The halophiles we use are stored in salt crystal form. They have the gvpA gene which forms gas vesicles (Vac+). This determines the colour of the growth.
- Pour 5 ml of halobacterium medium into each tube, 25ml in total for 5 tubes.
- Sterilise tops of the open tubes and forceps over flame.
- Using the sterilised forceps, add 2-3 salt crystals into each tube of medium, and cap.
- Incubate tubes at 42°C in an incubator that shakes if available, otherwise place them in a test tube rack and shake by hand each (taking longer for the culture to grow).
- The solution is ready to use when it turns pink, but it may also turn red.
Part 2, Halophile Plates
Using 25% salt medium to grow halobacterium NRC-1.
- Prepare at least three plates of hypersaline (25% salt) agar.
- Mark one plate each: streak, spread, and control group.
- For the streak plate, use an inoculation loop, dipping into the solution (of bacteria and salt from part 1) to streak the plate, forming single colonies.
- For the spread plate, pipette some solution onto the plate, then spread it using a spreader.
- On the control plate, just put anything (to prove that nothing grows).
- Incubate tubes at 42°C if possible, for culture growth results after 1 week, or at room temperature expect results after 2+ weeks.