Agar, preparation

J. N. BeMiller, Agar. Preparation of agar and agarose methanolysis mercaptolysis, Methods, Carbohydr. Chem., 5 (1965) 65-69. 

Culture and Inoculum. The test organism is Micrococcus flavus (F. D, A. strain) which is maintained at refrigerator temperature on slants of nutrient agar prepared as follows peptone 6.0 g., pancreatic digest of casein 4.0 g., yeast extract 3.0 g., beef extract 1.5 g., glucose 1.0 g., agar 15.0 g., and distilled water 1 liter. The pH is 6.5. 

It should be emphasized here that certain European manufacturers provide very good preparations of agar, yeast extract, and peptone. A competent yeast geneticist should be consulted, however, because not all agar preparations are pure enough for synthetic media. 

D (+) Galactose is a constituent of numerous polysaccharides It is best obtained by acid hydrolysis of lactose (milk sugar) a disaccharide of d glucose and d galactose L (—) Galactose also occurs naturally and can be prepared by hydrolysis of flaxseed gum and agar The principal source of d (+) mannose is hydrolysis of the polysaccharide of the ivory nut a large nut like seed obtained from a South American palm... 

Reprint methods where the developed dried chromatogam is laid on the prepared agar layer detector with the exclusion of air bubbles. In this and in the... 

A 100 ml aliquot of this medium is placed In a 500 ml Erlenmeyer flask and sterilized by autoclaving for 20 minutes under 15 psi pressure. Spores of mutant strain S. aureofaciens S1308 (ATCC No. 12,748) are washed from an agar slant Into the flask with sterile distilled water to form a suspension containing approximately 10 spores per milliliter. A 1.0 ml portion of this suspension is used to inoculate the fermentation media in the example which follows. A fermentation medium consisting of the following ingredients was prepared. 

An incubated culture of Actinomyces antibioticus was prepared using a medium consisting of 1% tryptone-peptone, 0.5% starch, 0.2% K HPO, 0.2% NaCI and 0.25% agar in distilled water, grown at a temperature of approximately 25° to 35°C, the incubation being complete after 6 to 10 days. 50 liters of this incubated culture are extracted approximately six times with ether, using 20 liters of ether for each extraction. 

Methylprednisone 21-acetate (0.5 g), when hydrolyzed by means of aqueous alcoholic potassium bicarbonate yields 16 fnethylprednisone. An alternative method of the preparation of the compound of this example is as follows. Bacillus sphaericus var. fusifermis (A.T.C.C. 7055) is incubated on a nutrient agar (composed of Bacto-beef extract, 3 g Bacto-peptone,... 

Sterile agar slants are prepared using the Streptomyces sporulation medium of Hickey and Tresner, J. Bact., vol. 64, pages 891-892 (1952). Four of these slants are inoculated with lyophilized spores of Streptomyces antibioticus NRRL 3238, incubated at 28°C for 7 days or until aerial spore growth is well-advanced, and then stored at 5°C. The spores from the four slants are suspended in 40 ml of 0.1% sterile sodium heptadecyl sulfate solution. A nutrient medium having the following composition is then prepared 2.0% glucose monohydrate 1.0% soybean meal, solvent extracted, 44% protein 0.5% animal peptone (Wilson s protopeptone 159) 0.2% ammonium chloride 0.5% sodium chloride 0.25% calcium carbonate and water to make 100%. 

Estimation The above medium is reinforced with lOg/i of thiocyanate, sulphur is omitted and it is prepared as pour plates by the addition of 3% agar. Organisms other than Thiobacilli will grow from spread samples, but the Thiobacilli are easily distinguished by sulphur haloes (see Fig. 2.19). 

Prepare an approximately 0.1 M silver nitrate solution. Place 0.1169 g of dry sodium chloride in the beaker, add 100 mL of water, and stir until dissolved. Use a silver wire electrode (or a silver-plated platinum wire), and a silver-silver chloride or a saturated calomel reference electrode separated from the solution by a potassium nitrate-agar bridge (see below). Titrate the sodium chloride solution with the silver nitrate solution following the general procedure described in Experiment 1 it is important to have efficient stirring and to wait long enough after each addition of titrant for the e.m.f. to become steady. Continue the titration 5 mL beyond the end point. Determine the end point and thence the molarity of the silver nitrate solution. 

To clear slant tubes, culture agar solution is prepared. To have clear agar solution, it has to boil. Agar is a polysaccharide. It is not soluble in cold water, so heating helps to dissolve it in water. Once the solution is dispensed in the tube, it goes for sterilisation. Agar is in the solid state at room temperature, about 35 °C. 

The first official test was published by the Food, Drug and Insecticide Administration of the US Department of Agriculture, in which portions of the preparation were placed on the surface of nutrient agar inoculated with Staph, aureus. After incubation the zones of inhibition, if ary, around the preparation were measmed. This test was modified later by incorporating 10% of horse serum in the agar to simulate conditions in a woimd and a control consisting of unmedicated base was also used in each experiment. This test is known as the cup-plate test (see also section 3.6.3 and Fig. 11.5). 

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