Appendix G

Media Glossary
January 2006

Glossary of agars, broths and other reagents

Section W

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Wagatsuma agar (for Kanagawa test)

Basal Medium
Peptone	10.0 g
Sodium chloride (NaCl)	70.0 g
Yeast extract	3.0 g
Potassium phosphate dibasic (K 2 HPO 4)	5.0 g
Mannitol	10.0 g
Agar	15.0 g
Crystal violet	0.001 g
Distilled water	1.0 L
pH 8.0 ± 0.2
Supplement
Human or rabbit red blood cells, fresh (24 h) with anticoagulant	50.0 mL
Physiological saline	200 - 250 mL

Complete medium:

Suspend basal medium ingredients in 1.0 L of distilled water. Heat to boiling to dissolve agar. Adjust pH to 8.0 ± 0.2. Place in a steam bath for 30 min. DO NOT AUTOCLAVE. Cool to 45-50^oC.

Prepare blood by mixing fresh (within 24 h of drawing) human or rabbit blood with same or larger volume of physiological saline. Centrifuge at 4000 x g at 4^oC for 15 min. Decant the saline and wash 2 more times. After third washing pour off saline and resuspend cells to the original volume with saline.

A dd 50 mL of washed blood cells to the basal medium. Mix and dispense into sterile Petri dishes. Dry thoroughly and use promptly.

Wilkins Chalgren Anaerobe Blood Agar

Basal Medium
Pancreatic digest of casein	10.0 g
Gelatin peptone	10.0 g
Sodium chloride (NaCl)	5.0 g
Yeast extract	5.0 g
Glucose	1.0 g
L-Arginine	1.0 g
Sodium pyruvate (C 3H 3O 3Na)	1.0 g
Hemin	5.0 mg
Menadione (Vitamin K)	0.5 mg
Agar	10.0 g
Tween™ 80	1.0 mL
Distilled water	950 mL
pH 7.1 ± 0.2
Supplement
Lysed horse blood	50.0 mL

Complete medium:

Suspend basal medium ingredients in distilled water and bring the volume to 950 mL. Mix thoroughly and gently heat to boiling to dissolve agar. Autoclave at 121^oC for 15 min. Cool to 50-55^oC. Aseptically add 50.0 mL of lysed horse blood. Mix thoroughly and dispense into sterile Petri dishes.

Wilkins Chalgren Anaerobe Broth

Basal Medium
Pancreatic digest of casein	10.0 g
Gelatin peptone	10.0 g
Sodium chloride (NaCl)	5.0 g
Yeast extract	5.0 g
Glucose	1.0 g
L-Arginine	1.0 g
Sodium pyruvate (C 3H 3O 3Na)	1.0 g
Hemin	5.0 mg
Menadione (Vitamin K)	0.5 mg
Distilled water	1.0 L
pH 7.1 ± 0.2

Complete medium:

Suspend basal medium ingredients in 1.0 L of distilled water. Mix thoroughly. Dispense into tubes or flasks and autoclave at 121^oC for 15 min.