BISC209: Recipes

Misc Recipes for test media and their reagents

LAB 4

PEA MEDIUM

• 10g tryptose
  3g Beef extract
  5g Sodium Chloride
  2.5g Phenylethyl alcohol
  15g Agar to 1 liter distilled or deionized water
  pH 7.1-7.5.

EMB medium

• 10g peptone
  10g Lactose
  2g dipotassium phosphate
  0.4g eosin Y
  0.065 g methylene blue
  15 g Agar
  final pH 6.9-7.3
  
  

Lab 5 (and available through lab 9
catalase test
fresh hydrogen peroxide

Oxidase test
order fresh commercial kits
positive controls: Bacillus spp, Neisseria, Micrococcus or Pseudomonas

Special Stains

• Negative stain for cell morphology:
  • Nigrosin
    

  
  

  Endospore Stain:
  • Malachite green hot plate Safranin
  Capsule Stain
  • Congo red
    acid-alcohol
    fuchsin
  Acid Fast Stain
  • Carbolfuchsin
    acid-alcohol
    methylene blue

  Role of microbes in ecosystem tests

  OF-glucose medium
  0.2 per cent peptone
  0.5 per cent NaCl
  0.03 per cent K2HPO4
  0.3 per cent agar
  0.003 per cent bromthymol blue
  1.0 per cent carbohydrate
  pH 7.1.
  
  

  cellulose degradation medium
  (use melted agar of the appropiate enrichment media choices
  or try Nutrient agar for all???
  
  

  antibiotic plates and broths (use appropriate medium to grow isolates
  
  

  Ammonification reagent
  Nesler's reagent will be purchased commercially. Please be extremely careful handling this solution.
  

  • Recipe: 0.09 mol/L solution of potassium tetraiodomercurate(II) (K2[HgI4]) in 2.5 mol/L potassium hydroxide. A yellow coloration indicates the presence of ammonia: at higher concentrations, a brown precipitate may form. The sensitivity as a spot test is about 0.3 μg NH3 in 2 μL.
    NH4+ + 2[HgI4]2− + 4OH− → HgO·Hg(NH2)I + 7I− + 3H2O
    It is toxic if swallowed, inhaled or absorbed through the skin. It presents a neurological hazard and may act as a carcinogen and be a reproductive hazard. It is corrosive and causes burns. Wear gloves and consider performing this assay in a hood.
  
  

  starch medium
  3 g Beef extract
  10 g soluble starch
  12 g agar
  bring up to 1 liter with distilled or deionized water
  pH 7.3-7.7
  

  develop with Gram Iodine
  
  

  LABS 6-9

  MRVP
  • dextrose peptone broth
    0.7% peptone,
    0.5% dextrose
    0.5% dipotassium phosphate
    pH 6.7-7.1
  Methyl red Reagent
  • 0.1 methyl red dye
    ethanol 00 ml
    distilled water to 500 ml
    
  VP Reagent A
  • 5 g alpha naphthol
    bring volume to 100 ml absolute alcohol
  VP Reagent B
  • 40g potassium hydroxide
    bring to volume with 100 ml distilled water
  
  

  One of 3 motility test options and the metabolic tests for the ablility to produce hydrogen sulfide (indicating the presence of either cysteine desulfurase or thiosulfate reductase causing the production of H₂S gas and the presence of the enzyme tryptophanase to produce Indole
  
  

  SIM agar and Reagent recipes:
  • Approximate Formula* Per Liter
    Pancreatic Digest of Casein - 20.0 g
    Peptic Digest of Animal Tissue - 6.1 g
    Ferrous Ammonium Sulfate - 0.2 g
    Sodium Thiosulfate - 0.2 g
    Agar - 3.5 g
  • Kovac's reagent: (per liter)
    p-Dimethylaminobenzaldehyde 50,0g
    Amyl Alcohol 750.0 ml
    Hydrochloric acid 250.0 ml
  • Indole Spot Test Reagent
    p-Dimethylaminocinnamaldehyde 10.0 g
    Hydrochloric acid 100.0ml
    Water, deionized 900.0 ml
    

  Carbohydrate fermentation medium

  This medium is available commercially
  

  • Purple broth base (1% peptone, 0.1% beef extract, 0.5% NaCl, 20 µg/ml bromcresol purple) plus 1% of the desired carbohydrate.
  • sugars we might test:
    NOTE: glucose is tested in OF-glucose medium and lactose in EMB. Let's repeat lactose, and add some or all of these: lysine, ornithine, sorbitol, adonitol and arabinose.
    

    Links to Labs

    Lab 1
    Lab 2
    Lab 3
    Lab 4
    Lab 5
    Lab 6
    Lab 7
    Lab 8
    Lab 9
    Lab 10
    Lab11
    Lab 12