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 5-9

Catalase Test
fresh hydrogen peroxide

Oxidase Test
Use commercial kit
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 Test Medium
Use appropriate melted agar from enrichment media choices
or Nutrient agar

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

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

• Purple broth base 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:
  lactose
  lysine
  ornithine
  sorbitol
  adonitol
  arabinose
  NOTE: glucose is tested in OF-glucose medium and lactose in 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