MAKING_LIFE 2014–2015

Halophile Cultures
posted by sarah on 23 March 2016

Andy Gracie
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).
Practical guide

  • 5 Tubes

  • Halobacterium medium

  • Forceps

  • Flame

  • Halophile salt crystals

  • Shaker incubator or test tube rack

  • Hypersaline medium: 25% salt agar??

  • 3 Petri dishes / plates

  • Inoculation loop

  • Pipet

  • Spreader

  • 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.