24.4: Materials and Procedures

Materials

• Day 1:
  • Unknown mixed culture
  • 2 TSA plates
  • Stains
• Subsequent days: Reagents and stains are available at all times. Media is available by request only.
  • TSA plates and slants
  • TSB
  • Stains
• Physiological media:
  • PR Dextrose Broth
  • PR Lactose Broth
  • PR Sucrose Broth
  • MR-VP Broth
  • Simmon’s Citrate Slant
  • TSI Agar Deep Slant
  • Nutrient Gelatin Deep
  • Urea Broth
  • Starch Agar Plate
  • Skim Milk Agar Plate
  • EMB Agar Plate
  • MacConkey Agar Plate
  • Tryptone Agar Plate
  • Mueller-Hinton Plate (Novobiocin 30 disk)
• Test Reagents:
  • Catalase Reagent-3% Hydrogen Peroxide
  • Oxidase Dry slides
  • Indole Dry Slides
  • Methyl Red
  • Barritt’s Reagents A & B

Procedures

Day 1:

1. Receive mixed culture, label it, and record #. DO NOT discard this original culture.
2. Streak culture onto two TSA plates, incubate.
3. Gram Stain mixed culture.

Subsequent Days (activities will be individual in nature, and not everyone will do the same thing on Day 2, Day 3, etc.):

1. Subculture ISOLATED colonies only onto a TSA plate, TSA slant, and into a TSB tube, incubate. After incubation observe and record culture characteristics of your plate, slant, and broth. Request media to re-streak cultures if isolation of both bacteria is not achieved.
2. Re-Gram stain isolated colonies.
3. Request media and run appropriate tests on ISOLATED colonies as needed.
4. Record all results.