Sporulation media

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%. 

Fig. 3 CHEF gel of meiotic time courses in the wild-type SKI and radSOS mutant strains. Times (hours) in sporulation medium are shown at the top of each lane. Meiotic double-strand breaks occur as smears, indicating multiple fragmentation of the chromosomes. The maximum density of the smears is at around 220 kb, which (incidentally) is the size if the smallest chromosome of the complement. Due to its transient nature in the wild type, the smear appears only faintly in the period between. 3 and 4.5 hr of sporulation. In the radSOS mutant, where double-strand breaks are not repaired (see text), the intensity of the smear is much stronger at later time points. Notice the size differences of some chromosomes for the wild type and the isogenic mutant.

Take 10-ml samples from a sporulating culture and add to them l0x concentrated formaldehyde solution to a final concentration of 4% (w/v). Mix and keep at room temperature for 30 min. If several samples from a time-course experiment are treated together, store them in the refrigerator. Collect cells by centrifugation (4 min at 700 g) and resuspend them in sterile water or sporulation medium. Wash them twice and dissolve the pellet in 1 ml of the zymolyase cocktail as above. Spheroplasting may take somewhat longer than for unfixed cells. 

Probably the most significant stumbling block in successful routine laboratory identification of Brettanomyces and Dekkera lies in the fundamental requirements of taxonomic guides to demonstrate the presence (or absence) of a sexual phase in the life cycle of the yeast. Dekkera requires a sporulation medium that includes augmentation with several vitamins (see Procedure 3.5.2). Required in microgram and milligram amounts, these nutrients are not easily and routinely supplied in most production-oriented laboratories. Hagan (1979) notes that even under ideal conditions, relatively poor sporulation (<1%) is observed. As a result, suspect isolates are often reported as Brett-like or Brettanomyces/Dekkera. ... 

G. YM (Vitamin Supplemented) Dekkera sp. may be sporulated on vitamin-supplemented YM agar (van der Walt and van Kerken, 1961). The components and their final concentration (in sporulation medium) are summarized below. Because some components are present at microgram concentrations, its necessary to prepare several-fold stock solutions which are appropriately diluted during media preparation. 

The inhibition of sporulation by netropsin, an antibiotic interacting with AT groups in the chromosome and suppressing the synthesis of several enzymes related to sporulation [13,14], caused an Inhibition of protein turnover but stimulated the rate of exocellular proteinase formation [15], Netropsin not only increased the synthesis of the enzyme at the beginning of incubation in sporulation medium but delayed also its late suppression (Table 1), It must be stated, however, that the antibiotic stimulated the proteinase formation also during growth and its effect thus does not seem to be specific for sporulation [15],... 

Fig. 8. Effect of temperature on proteinase formation during growth and sporulation. A - empty circles, p - specific growth rate, full circles, % percentage of spores formed after 6 h in the sporulation medium. B - circles, r - specific rate of enzyme formation columns - capacity to form the proteinase in the... 

Table 1. Effect of Netropsin on Specific Rate of Enzyme Synthesis in Sporulation Medium.

Group during growth Enzyme in sporulation medium Phenotype Number of mutants... 

Fig. 1. Effect of netropsin on the formation of an exocellular proteinase during growth and sporulation. A - growth medium, B - sporulation medium o - no netropsin, - with netropsin (1 jjg/mL) ... 

For sporulation medium, a simple medium containing 0.4% potassium acetate gives very good results with cells taken from a late log phase culture from a YEP dextrose medium. A more sophisticated medium is as follows ... 

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