Reactives and solutions used for media preparations
Aniline crystal violet solution

Aniline ..................................................................................................................................... 40 ml
Water .................................................................................................................................. 1000 ml
Crystal violet, saturated alcoholic solution (about 10 g per 100 ml 95% alcohol)...114 ml
Used for Gram stain.
Gram's iodine

Iodine ........................................................ 1 g
Potassium iodide ....................................... 2 g
Water ................................................... 300 ml

Grind the iodine and the potassium iodide in a mortar until thoroughly  mixed. Add water slowly.
Used for Gram stain.
Hank's balanced salt solution (modified)

2 stock solutions needed:
Stock A
NaCl ............................................................................... 80 g
KCl .................................................................................... 4 g
MgSO4  7 H20 ................................................................ 1 g
MgCl2 6 H2O .................................................................. 1 g
dissolve in 400 ml water CaCl2 (anhydrous) 1.4 g; add water to 500 ml
Stock B
Na2HPO4 12 H2O ........................................................ 1.5 g dissolved in 400 ml water
KH2PO4 .......................................................................... 0.6 g
water ad 500 ml

For use, add 25 ml of stock A to 400 ml of water, followed by 25 ml of stock B; then  water ad 500 ml.

Used in Friis medium for Mycoplasma hyopneumoniae and M. flocculare isolation.
Safranin

Safranin, saturated alcoholic solution  (about 2.5 grams per 100 ml 95% alcohol) ............. 10 ml
Water ..................................................................................................................................................... 90 ml

Used for Gram stain.
Vitamin K1 solution

Dissolve 0.15 ml of vitamin K1 in 30 ml of 95 % ethanol, do not sterilize since it is added to the
medium before autoclaving, keep under refrigeration, store in brown bottle, discard after 1 month.
Yeast extract solution

Dry yeast Saccharomyces cerevisiae ................................... 125 g
Demineralized water ............................................................ 750 ml

Place it at 37 'C for 20 min and thereafter heat it to 90-100 'C for 5 min.
Centrifuge at 1000 x g for 30 min. Harvest the supernatant and dispense it in sterile recipients.
Store at -20 'C for 3 months.
Urea - glucose solution

10 g urea + 1 g glucose + distilled water ad 100 ml. Used for MIU medium preparation.
Phenol solution 5%

Phenol crystalline  ..............................................................  50 g
Water to make  ..................................................................... 1000 g

Warm water to about 70'C, admix the phenol and make up to 1 kg. Filter through paper and dispense
as required.
Blood extract

Blood, defibrinated, sterile (sheep or horse) ................................................................................. 100 ml
Sodium chloride sol. 0.85%............................................................................................................... 400 ml
Mix the ingredients in a flask, heat in a water bathand hold at 80'C for 15 min., shaking frequently.
Filter through paper then centrifuge 15-20 min., remove supernatant and sterilize by filtration.
Ox bile

Get bile from a recently killed ox, heat in steam at 100'C for 20 min. and filter through paper. dispense
as required and sterilize fractionally for three 20-minute periods.
(c) Costin Stoica
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