The following solutions can be ordered commercially:
·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
Leifson flagella stain (14)Solution A:
- Sodium chloride 1.5 g
Distilled water 100 ml
Tannic acid 3.0 g
Distilled water 100 ml
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
Role of microbes in ecosystem tests
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
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.
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.
3 g Beef extract
10 g soluble starch
12 g agar
bring up to 1 liter with distilled or deionized water
develop with Gram Iodine
Methyl red Reagent
dextrose peptone broth
0.5% dipotassium phosphate
VP Reagent A
0.1 methyl red dye
ethanol 00 ml
distilled water to 500 ml
VP Reagent B
5 g alpha naphthol
bring volume to 100 ml absolute alcohol
40g potassium hydroxide
bring to volume with 100 ml distilled water
3 motility test options
SIM agar and Reagent recipes:
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
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)
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.