BISC209: Recipes
Misc Recipes for test media and their reagents
LAB 4
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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.
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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
- Malachite green hot plate Safranin
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Congo red
acid-alcohol
fuchsin
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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
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Leifson flagella stain (14)Solution A:
- Sodium chloride 1.5 g
- Distilled water 100 ml
- <LIL
Tannic acid 3.0 g
- Distilled water 100 ml
- Pararosaniline acetate 0.9 g
- Paraosaniline hydrochloride 0.3 g
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Ethanol, 95% (vol/vol) 100 ml
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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
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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.
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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
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0.1 methyl red dye
ethanol 00 ml
distilled water to 500 ml
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5 g alpha naphthol
bring volume to 100 ml absolute alcohol
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40g potassium hydroxide
bring to volume with 100 ml distilled water
3 motility test options
SIM agar and Reagent recipes:
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
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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.
