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<BR> 0.065 g methylene blue <BR> 15 g Agar<BR> final pH 6.9-7.3 <BR><BR></LI></UL>

LAB 5-9
Catalase Test<BR> fresh hydrogen peroxide<BR> <BR> Oxidase Test<BR> Use commercial kit <BR> positive controls: Bacillus, Neisseria, Micrococcus or Pseudomonas<BR><BR>

Special Stains
Negative stain for cell morphology: Nigrosin<BR></LI></UL>

Endospore Stain: Malachite green hot plate Safranin </LI></UL>

Capsule Stain Congo red<BR> acid-alcohol<BR> fuchsin </LI></UL>

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  </LI></UL>The following solutions can be ordered commercially: <LI> Leifson flagella stain (14)Solution A: <BR> <UL><LI>Sodium chloride 1.5 g<LI> Distilled water 100 ml </LI></UL> Solution B:<UL><LI> Tannic acid 3.0 g<LI> Distilled water 100 ml<LI> </LI></UL>

Solution C:<UL><LI> Pararosaniline acetate 0.9 g <LI> Paraosaniline hydrochloride 0.3 g<LI> Ethanol, 95% (vol/vol) 100 ml<UL><LI> </LI></UL> 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. </LI></UL></LI></UL> <UL><LI> Presque Isle Cultures flagella stain<UL><LI> Solutions are available from Presque Isle Cultures. The components of the flagella stain are proprietary.<LI> Flagella mordant (Solution I)<LI> Silver stain (Solution II) <LI> Solutions I and II can be stored at room temperature for several weeks. </LI></UL></LI></UL>

Acid Fast Stain <ul><LI> Carbolfuchsin<BR> acid-alcohol <BR> methylene blue</LI></UL>

Role of microbes in ecosystem tests
OF-glucose medium<BR> 0.2 per cent peptone<BR> 0.5 per cent NaCl<BR> 0.03 per cent K2HPO4<BR> 0.3 per cent agar<BR> 0.003 per cent bromthymol blue<BR> 1.0 per cent carbohydrate <BR> pH 7.1.<BR><BR>

Cellulose Degradation Test Medium<BR>This medium could be used to screen "total culturables" for cellulose degrading isolates or to test select isolates for their role in the enviroment.<BR> ''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.''<BR> Cellulose Congo Red Agar recipe<BR> 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. <BR> positive control: Streptomyces lividans TK23, non cellulose-utilizing bacteria: Escherichia coli, Proteus mirabilis, Pseudomonas putida<BR><BR> antibiotic plates and broths (use appropriate medium to grow isolates) or THIS YEAR try Nutrient Agar only. No cycloheximide in media. <BR><BR> Ammonification reagent<BR> Nesler's reagent will be purchased commercially. Please be extremely careful handling this solution.<BR> <UL><LI> 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.<BR>

NH4+ + 2[HgI4]2− + 4OH− → HgO·Hg(NH2)I + 7I− + 3H2O <BR> 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.</LI></UL><BR>

starch medium<BR> 3 g Beef extract<BR> 10 g soluble starch<BR> 12 g agar<BR> bring up to 1 liter with distilled or deionized water<BR> pH 7.3-7.7<BR>

develop with Gram Iodine<BR><BR>

LABS 6-9
MRVP<UL><LI> dextrose peptone broth <BR> 0.7% peptone,<BR> 0.5% dextrose<BR> 0.5% dipotassium phosphate<BR> pH 6.7-7.1</LI></UL> Methyl red Reagent <UL><LI> 0.1 methyl red dye<BR> ethanol 00 ml<BR> distilled water to 500 ml<BR></LI></UL>

VP Reagent A <UL><LI> 5 g alpha naphthol<BR> bring volume to 100 ml absolute alcohol</LI></UL> VP Reagent B<UL><LI> 40g potassium hydroxide<BR> bring to volume with 100 ml distilled water</LI></UL><BR>

3 motility test options<BR> SIM Test<BR> 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 <BR><BR>

SIM agar and Reagent recipes:<UL><LI>

Approximate Formula* Per Liter<BR> Pancreatic Digest of Casein - 20.0 g<BR> Peptic Digest of Animal Tissue - 6.1 g<BR> Ferrous Ammonium Sulfate - 0.2 g<BR> Sodium Thiosulfate - 0.2 g<BR> Agar - 3.5 g<LI>

Kovac's reagent: (per liter)<BR> p-Dimethylaminobenzaldehyde 50,0g<BR> Amyl Alcohol 750.0 ml<BR> Hydrochloric acid 250.0 ml<LI>

Indole Spot Test Reagent<BR> p-Dimethylaminocinnamaldehyde 10.0 g<BR> Hydrochloric acid 100.0ml <BR> Water, deionized 900.0 ml<BR> </LI></UL>

Carbohydrate Fermentation Medium
<UL><LI> 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.</LI></UL> <UL><LI> Sugars Available for Carbohydrate Testing: Sherly will tell us which ones she will make each year.  <BR> Glucose<BR> lactose<BR> lysine<BR> ornithine<BR> sorbitol<BR> adonitol<BR> arabinose<BR> mannitol<BR>

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