For the diversity project, my chosen group of bacteria are the Thermophiles, more specifically the hyperthermophilic bacteria for which the optimal growing is achieved above 80 oC. These amazing creatures are able to survive in high temperature habitat, such as volcanic area, hot springs and geothermal vents. In such environments, enzymes of these organisms are able to work at as high temperature as 120 degree celsius. Especially interesting areas are the Black Smokers, found in deep sea near volcanically active regions of the Earth, where a lot of microbial activity has been observed.Isolation to study and fully understand mechanisms and roles of organism in their natural habitat is extremely important and critical step in research. However, the commonly used techniques for separating bacteria (plating on solid surface) can face some difficulties in the case of hyperthermophiles. Solidifying agents as agar can melt above 50 oC, therefore not suitable for plating in this case.
A more stable gelling agent can be used to avoid this problem. Solid GERLITE medium can be used for this purpose which can stay stable and solid upto 120 degree celsius, which is an appropriately high temperature to select hyperthermophilic bacteria as they are the ones that can manage to survive in such conditions. Gellan gum (trademarked as GERLITE) is a polysaccharide produced by Pseudomonas species. After heating and cooling down it forms plain gel with the help of cations that makes colony count of thermophilic bacteria available.
Bacteria from collected samples can only survive if they can tolerate high temperature and hydrostatic pressure. These features are restricted to hyperthermophiles living in deep sea hydrothermal vents. The composition of the media is the following. The amount of materials are per liter of used distilled water: 25 g of NaCl2 g of MgSO40.
8 g of KCl0.5 g of Na2HPO40.01 g of FeSO43.0 ml of a trace elements solution that contains (gl-l): H3BO3, 2.86; MnCI2 x4H20, 1.
81; ZnSO4 x7H2O, 0.222; Na2MoO4x 2H20, 0.390; CuSO4 x 5H20,0.079; Co(N03), .
6H20, 0.0494. 1 g of (NH4)2SO43 g of Na2S2035 g of sodium acetateThe pH level is set to 5.5 with the aid of 4N HCl. After this 8g of GERLITE is added to the medium, followed by immediate autoclaving at 121°C and 18 lb/in2 for 30 min.To create the original habitat of the hydrothermal vents, high hydrostatic pressure should be maintained for optimal growth.
This can be carried out with pressure vessels to reach 265 atm that can be observed at such environmental conditions. The samples can be incubated in oven or silicon oil bath to reach the temperature of above 100 °C. The inoculum for this enrichment culture experiment can be hydrothermal fluid samples taken from deep sea vents like black smokers or chimneys that can be found for example at the East Pacific Rise or in the Atlantic and Pacific ocean below 2000 m.