Glucose-yeast Extract-carbonate medium

Glucose (dextrose) 50 g Yeast extract 10 g CaCOg 30 g Agar 25 g 

Bring to 1 L volume with distilled water and autoclave. 

When autoclave cycle is finished, transfer flask to 50°C (122°F) waterbath, and pour plates once temperature has equilibrated. 

Gluconobacter growing on GYCM over time (3 to 5 weeks) produces water-soluble brown pigments, which are not seen in the case of any similarly cultivated Acetobacter species. Grown on this medium, Acetobacter will produce clear zones or halos around colonies because the acid being produced will neutralize the CaCOs. Unlike the lactic acid bacteria, acetic acid bacteria are obligate aerobes and so it is necessary to use spread plates. 

Once ingredients are dissolved, dilute medium to volume (lOOOmL). 

---

The suspension thus obtained was used to inoculate 100 ml of a sterile vegetative medium having the following composition (g) glucose 5.0 dextrin 20.0 soybean meal 5.0 yeast extract 2.5 calcium carbonate 1.0 tap water 1 L. 

A shake flask culture may have the following composition in g/L meat extract 3.0 yeast extract 10.0 calcium carbonate 4.0 Starch 25.0 tap water q.s. to 1000 ml. The flasks are shaken for about 24 h at about 28°-30°C an then the pre-cultures 1 L are used to inoculate jar fermentors each containing 10 L of the following nutrient medium, g meat extract 40.0 peptone 40.0 yeast extract 10.0 sodium chloride 25.0 soybean meal 100.0 glucose 500.0 calcium carbonate 50.0 tap water q.s. to 10 L. 

Media used for ARA-rich SCO production vary depending on the process/strain used however, they tend to be relatively simple complex media composed of a base of yeast extract and glucose (51), although the ion composition is known to be crucial for optimal productivity as well as the carbon to nitrogen ratio in the medium (48, 52, 53). 

The culture medium for toxin production consists of 3 % nutrient broth (Difco Laboratories, Detroit, Ml, USA), 1.5% yeast extract (Difco Laboratories), 1 % ammonium sulfate, 1 % glucose, 0.4 % calcium carbonate, 0.4% soluble starch and 0.4% cysteine hydrochloride. Adjust the pH of the medium to 7.5 with 4 N NaOH and autoclave at 1 kg/cm for 15 min. After the temperature of the autoclave has fallen below 100 °C, cool the medium as soon as possible to 40-45 °C in tap water to avoid a decrease in reduction state of the medium, and then inoculate the culture of the toxin producing strain prepared as in Section 9.3.2. 

Fig. 7. Culturing of yeast Saccharomyces cerevisiae strain AH22 in rich medium YEP (yeast extract and peptone) with a carbon source (Glucose) or YEP alone without a carbon source (glucose) [15]...

Note YPG media (0.2% yeast extract, 1.0% peptone, and 2.0% glucose) MSM, molasses salt medium (0.2% yeast extract and molasses as carbon source) PDB, potato dextrose broth (20% potato extract and 2% dextrose) YM media (0.3% yeast extract, 0.3% malt extract, 0.5% polypeptone, and 1.0% glucose) AIF, alkali insoluble fraction DA, degree of acetylation DDA, degree of deacetylation DP, degree of polymerization M, molecular weight SmF, submerged fermentation SSF, solid-state fermentation. 

---