Genus/Species: Bacillus (B. subtilis, B. circulans, B. coagulans)
Gram Stain: Positive
Morphology:
- Cell: Rod shaped cells.
- Spore: The species are endospore formers. Shape of the spores varies with the species and maybe oval, ellipsoidal, cylindrical or spherical. The location maybe terminal, sub-terminal or central. The spores formed are quite heat resistant and retain viability even at temperatures as high as 100°C for one minute. Spore formation is usually observed in old cultures. Studies show that formation maybe enhanced in media containing 40mg/L manganese sulfate. Spores also shown to be resistant to 70°C for 10 minutes or to 95% ethanol for 20 minutes. Thus, they survive pasteurization temperatures in the heat stabilization of fortified wines. The spores are also resistant to acid and salt and can persist in the environment for long periods of time.
- Colony: Colony morphology is highly variable and depends on various strains and the species. Large to very large, grey white colonies, maybe dry in appearance or may look wet and blistery have been observed.
- Liquid growth: Growth is uniform and solution appears turbid.
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Physiological traits: Requires oxygen for growth conditions. Some however, maybe facultative anaerobes.
Ecological traits: The species are worldwide soil saprophytes and are secondarily found in water supplies.
Distinguishing features: The species of Bacillus are identified as catalase positive; No clearing of calcium carbonate-ethanol plates is observed.
Role in wine:
Spoilage in wine due to this species is usually under conditions of oxygen exposure as these are obligate aerobes. Reports of Bacillus-associated wine (predominantly dessert wines) and brandy spoilage has been found in literature (Murrell and Rankine, 1979). Isolates of B.subtilis, B. circulans and B. coagulans have been obtained. Presence of this organism has been detected on corks, though at much lower levels than molds. B.subtilis in particular is known to cause ropiness in wines, due to the production of long-chain polysaccharides. Some strains appear to be ethanol-tolerant; spores may contaminate wine; vegetative cells have been found in wine.
Sensitivities:
- SO2: At concentrations similar to those found in wine (150 ppm, pH 3.0-6.5), bisulfite has not been found to be mutagenic to Bacillus subtilis (Khoudokormoff, 1978). Higher concentrations of a sodium sulfite-bisulfite mixture, however, showed mutagenic effects in a B. subtilis test system at concentrations of 0.1 to 0.5 M, pH 7, but not at 0.05 M. Cells treated with adducts of sodium hydrogen sulfite and cytidine monophosphate or uridine monophosphate exhibited mutagenic effects at concentrations of 0.25 and 0.5 M (Chang et al., 1977).
- Sorbate: Potassium sorbate concentrations of 3.5mg/L and greater effectively inhibit spore germination. Greater inhibition is seen at lower pH conditions.
- DMDC: Not very effective in inhibiting growth of the species. Concentrations as high as 1000mg/L cause inhibition of the species.
- pH: Lower pH conditions negatively impact the growth of the species and this coupled with heat, results in more effective increases in heat sensitivity resulting in inactivation of even the spores.
- Acids: Some studies indicate inhibitory effect of teichoic acid on B.subtilis. Benzoic and propionic acids as well as esters of p-hydroxybenzoic acid (parabens) have also shown to inactivate Bacillus though the activity may be pH dependent. Dipicolinic acid in very high concentrations has been found to inhibit germination of spores but has no effect on vegetative growth.
- Ethanol: Studies show that at concentrations of greater than 0.7M ethanol negatively impact growth of the species and can inhibit sporulation.
- Anaerobiosis: Conclusive results could not be obtained though one study indicated that anaerobic conditions increased thermal stability of the cells and spores.
- Heat: Quite resistant to heat. Can survive pasteurization temperatures as mentioned above.