BISC209: Recipes

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Wellesley College-BISC 209 Microbiology -Spring 2010

Misc Recipes for test media and their reagents


  • 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 5-9

Catalase Test
fresh hydrogen peroxide

Oxidase Test
Use commercial kit
positive controls: Bacillus, Neisseria, Micrococcus or Pseudomonas

Special Stains

Negative stain for cell morphology:
  • Nigrosin
Endospore Stain:
  • Malachite green hot plate Safranin
Capsule Stain
  • Congo red
flagella stain
    • ·New microscope slides
    • ·95% ethanol and Kimwipes to clean slides
    • ·Heat—provided by a Bunsen burner, alcohol lamp, ceramic heater, or other appropriate heat source
    • ·Micropipettors to deliver 5-20μL
    • ·Sterile disposable tips for micropipettors
    The following solutions can be ordered commercially:
    • Leifson flagella stain (14)Solution A:
      • Sodium chloride 1.5 g
      • Distilled water 100 ml
      Solution B:
      • Tannic acid 3.0 g
      • Distilled water 100 ml
      Solution C:
      • Pararosaniline acetate 0.9 g
      • Paraosaniline hydrochloride 0.3 g
      • Ethanol, 95% (vol/vol) 100 ml
        Mix equal volumes of solutions A and B; then add 2 volumes of the mixture to 1 volume of solution C. The resulting solution may be kept refrigerated for 1 to 2 months.
    • Presque Isle Cultures flagella stain
      • Solutions are available from Presque Isle Cultures. The components of the flagella stain are proprietary.
      • Flagella mordant (Solution I)
      • Silver stain (Solution II)
      • Solutions I and II can be stored at room temperature for several weeks.

    Acid Fast Stain
    • Carbolfuchsin
      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 Test Medium
    This medium could be used to screen "total culturables" for cellulose degrading isolates or to test select isolates for their role in the enviroment.
    Recipe from Hendricks, Charles W., J.D. Doyle, B. Hugley. 1995. A new Solid Medium for Enumerating Cellulose-Utilizing Bcteria in /soil. Applied and Environmental Microbiology. 2010-2019.
    Cellulose Congo Red Agar recipe
    0.5g K2HPO4 (Fisher), 0.25g MgSO4 (Sigma), 1.88g ashed, acid washed cellulose Powder (J.T. Baker), 0.20g Congo Red (J. T. Baker), 5.0g Noble Agar (DIFCO), 2.0g gelatin, 100 ml soil extract, 900 ml H20.
    positive control: Streptomyces lividans TK23, non cellulose-utilizing bacteria: Escherichia coli, Proteus mirabilis, Pseudomonas putida

    antibiotic plates and broths (use appropriate medium to grow isolates) or THIS YEAR try Nutrient Agar only. No cycloheximide in media.

    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

    • 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

    3 motility test options
    SIM Test
    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

    • Phenol red broth base order from BBL as powder. Recipe: pancreatic digest of Casein 10g, sodium chloride 5 g, phenol red 18 mg. carbohydrate 5g.
      Dissolve 15 g of the powder in 1 L of purified water add 5-10 g or carbohydrate <BR) (Purple broth base is an alternative) is available commercially(1% peptone, 0.1% beef extract, 0.5% NaCl, 20 µg/ml bromcresol purple) plus 1% of the desired carbohydrate.