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w. B. REDMOND et al.
D. Stock solutions and mixtures
T o save time in making RVA and RVB media, make, store and use the
following stock mixtures and solutions. Use distilled water. Store in
refrigerator.
Mixture no. 1 : Sodium pyruvate 7.5 g ; Na2HP04 (anhydrous), 12.0 g ;
KH2P04,5.5 g; NH4CI,5.0 g; NaC1,25*0 g ; water, 1000 ml. Dissolve each
compound before adding the next one. Dispense 100ml amounts into
flasks. Sterilise by autoclaving. Use 100 ml for making 1 litre of RVA-17
or RVB-10.
Mixture no. 2: Nutrient broth, 20.0 g; proteose peptone no. 3 (Difco),
25.0 g ; casein hydrolysate (enzymatic), 20.0 g ; water, 1000 ml. Dissolve by
warming and dispense 100 ml amounts into flasks. Sterilise by autoclaving.
Use 100 ml for making 1 litre of RVB-10.
Mixture no. 3: Nutrient broth, 40.0 g ; casein hydrolysate (enzymatic),
20.0 g; water, 1000 ml. Dissolve by warming and dispense 100 ml amounts
into flasks. Sterilise by autoclaving. Use 100 ml/l litre of RVA-17.
Mixture no. 4 : Glucose, 50.0 g; water, 500 ml; dissolve and dispense
55 ml amounts into flasks. Sterilise by autoclaving. Use one flask per
1 litre of RVB-10 or RVA-17.
Stock solutions
CaClz: 1 M calcium chloride. Dissolve 11.1 g of CaCl2 (anhydrous) in
100 ml water. Dispense 20 mlltube. Sterilise by autoclaving.
Fe3: ferric chloride. Dissolve 100 mg FeC13-6H20 in 100 ml water.
Dispense 20 ml/tube. Sterlise by autoclaving.
Mg4: 1 M magnesium sulphate. T o 24-65 g MgS04.7Hz0 add distilled
water to 100 ml. Dissolve. Dispense 20 ml amounts into tubes. Sterilise
by autoclaving.
ZCI: zinc-copper. Dissolve 10 mg ZnS04 and 10 mg CuS04-5H20 in
200 ml water. Dispense 20 ml amounts into tubes. Sterilise by autoclaving.
TW3 : Tween-80 (polyoxyethylene sorbitan monooleate). Tween-80,
25 ml; water (distilled) 75 ml. Warm and mix thoroughly. Dispense 20 ml
each into tubes. Autoclave for 15 min at 15 Ib. Shake tubes while still hot
to mix Tween with water.
Sodium glutamate: sodium glutamate 10 g; water 100 ml. Sterilise by
Seitz filtration (test for sterility by inoculating 1 ml into 10 ml RVB-10;
incubate). Dispense 20 ml amounts into tubes.
To make RVB-10: add 8-Om1 glycerol; 2.0ml TW3 (stock); 2-Om1
ZC1 (stock) to 200 ml water. Autoclave 15 min at 15 lb pressure.
Add 1.0 ml hlg4 (stock); 1.0 ml CaC12 (stock), 1.0 ml Fe3 (stock) to
450 ml water. Autoclave for 15 min at 15 Ib pressure.
w. B. REDMOND et al.
D. Stock solutions and mixtures
T o save time in making RVA and RVB media, make, store and use the
following stock mixtures and solutions. Use distilled water. Store in
refrigerator.
Mixture no. 1 : Sodium pyruvate 7.5 g ; Na2HP04 (anhydrous), 12.0 g ;
KH2P04,5.5 g; NH4CI,5.0 g; NaC1,25*0 g ; water, 1000 ml. Dissolve each
compound before adding the next one. Dispense 100ml amounts into
flasks. Sterilise by autoclaving. Use 100 ml for making 1 litre of RVA-17
or RVB-10.
Mixture no. 2: Nutrient broth, 20.0 g; proteose peptone no. 3 (Difco),
25.0 g ; casein hydrolysate (enzymatic), 20.0 g ; water, 1000 ml. Dissolve by
warming and dispense 100 ml amounts into flasks. Sterilise by autoclaving.
Use 100 ml for making 1 litre of RVB-10.
Mixture no. 3: Nutrient broth, 40.0 g ; casein hydrolysate (enzymatic),
20.0 g; water, 1000 ml. Dissolve by warming and dispense 100 ml amounts
into flasks. Sterilise by autoclaving. Use 100 ml/l litre of RVA-17.
Mixture no. 4 : Glucose, 50.0 g; water, 500 ml; dissolve and dispense
55 ml amounts into flasks. Sterilise by autoclaving. Use one flask per
1 litre of RVB-10 or RVA-17.
Stock solutions
CaClz: 1 M calcium chloride. Dissolve 11.1 g of CaCl2 (anhydrous) in
100 ml water. Dispense 20 mlltube. Sterilise by autoclaving.
Fe3: ferric chloride. Dissolve 100 mg FeC13-6H20 in 100 ml water.
Dispense 20 ml/tube. Sterlise by autoclaving.
Mg4: 1 M magnesium sulphate. T o 24-65 g MgS04.7Hz0 add distilled
water to 100 ml. Dissolve. Dispense 20 ml amounts into tubes. Sterilise
by autoclaving.
ZCI: zinc-copper. Dissolve 10 mg ZnS04 and 10 mg CuS04-5H20 in
200 ml water. Dispense 20 ml amounts into tubes. Sterilise by autoclaving.
TW3 : Tween-80 (polyoxyethylene sorbitan monooleate). Tween-80,
25 ml; water (distilled) 75 ml. Warm and mix thoroughly. Dispense 20 ml
each into tubes. Autoclave for 15 min at 15 Ib. Shake tubes while still hot
to mix Tween with water.
Sodium glutamate: sodium glutamate 10 g; water 100 ml. Sterilise by
Seitz filtration (test for sterility by inoculating 1 ml into 10 ml RVB-10;
incubate). Dispense 20 ml amounts into tubes.
To make RVB-10: add 8-Om1 glycerol; 2.0ml TW3 (stock); 2-Om1
ZC1 (stock) to 200 ml water. Autoclave 15 min at 15 lb pressure.
Add 1.0 ml hlg4 (stock); 1.0 ml CaC12 (stock), 1.0 ml Fe3 (stock) to
450 ml water. Autoclave for 15 min at 15 Ib pressure.