As with most of the experiments in this course, you will be handling a variety of undomesticated organisms of unknown identity or pathogenicity. Handle all cultures with respect and using standard microbiological procedure.

The heating block used to treat the samples is very hot. Do not touch the heating block, and allow your sample to cool before handling.

Gram-positive Bacteria as a group are common soil organisms. Bacillus species are very common mesophilic, aerobic heterotrophs that produce heat-resistant endosopores. The enrichment and isolation of Bacillus is straightforward - a sample of soil (rich in Bacillus) is heated to kill non-spore-forming mesophiles, and then plated on rich media and incubated aerobically at 30C. Thermophiles will not grow at this temperature, and anerobic spore-formers (Clostridium) will not grow aerobically. Other mesophilic aerobic endospore-formers are photosynthetic, e.g. Heliospirillum, scarce, and require lots of light for growth.

• Samples of soil, decaying logs, dead leaves, or other organic matter
• Innoculating loop
• distilled water
• heating block at 80C & microfuge tubes to fit
• PYD plates:

  Add to 1 liter of distilled water:

  • 2g peptone
  • 2g yeast extract
  • 5g dextrose
  • 15g agar

  Mix & autoclave. Cool to about 50C & pour into plates

1. In a microfuge tube, suspend a small amount (a loop full - no more!) of soil in 1 or 2 drops of distilled water.
   
2. Mix well & incubate at 80C for 10 minutes.
3. Use an innoculating loop to streak a sample of this heat-treated soil onto a peptone-yeast extract-dextrose plate.
   
4. Incubate at 30C for 1-2 days.
5. Examine samples of various colonies microscopically. Look for rod-shaped organisms with distinctly mottled appearance and especially the presence of endospores (some example micrographs are shown below). Pick a well-isolated colony that looks like Bacillus and restreak onto a fresh plate. Incubate as before.
   

Make note of colony morphologies and examine cell morphologies in wet mounts. Bacillus colonies are typically white and dry or pasty looking, but some form very mucoid colonies (that can drip onto the lid of the plate!), and relatives of B. cereus form large feathery colonies. Bacillus cells are typically fairly rectangular rods, often occuring in pairs or chains, with a mottled appearance and obvious endospores.

Here are examples of two common examples of the hundreds of species of Bacillus: