BISC209: Recipes

Misc Recipes for test media and their reagents

LAB 4

PEA MEDIUM

• ORDER FROM BBL
• 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
  acid-alcohol
  fuchsin

flagella stain

• MATERIALS
  • ·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
    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 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

  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
  
  

  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.
  • Sugars Available for Carbohydrate Testing: Sherly will tell us which ones she will make each year.
    Glucose
    lactose
    lysine
    ornithine
    sorbitol
    adonitol
    arabinose
    mannitol
    NOTE: glucose fermentation is tested in OF-glucose medium and lactose on EMB

    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