- •Lecture n 7,8
- •Enzymes of bacteria.
- •Bacterial Kinases
- •Methods for studying enzymatic properties for the identification of pure bacterial cultures.
- •Saccharolytic properties.
- •Proteolytic properties.
- •Other properties.
- •Inoculation of microorganisms into depth of solid media.
- •Chemical methods.
- •Biological methods.
- •Methods of creation of anaerobic conditions. Physical methods.
- •Inoculation of media containing reducers and easily oxidative substances.
- •Methods for the isolation of pure cultures of anaerobic bacteria.
- •Isolated colonies are prepared by some methods.
Methods for studying enzymatic properties for the identification of pure bacterial cultures.
The composition of enzymes is determined by the genome and is a permanent species characteristic.
Stability of fermentative systems of bacteria allows using biochemical features of for identification.
For this purpose, culture of bacteria is inoculated in differential media: 1) рrotein-containing media (gelatin, milk, serum, etc.) for detection of proteolytic enzymes; 2) sugar- and polyatomic alcohol containing media for detection of saccharolytic activities.
Differential media usually contain dye, which indicates presence or absence of fermentation, oxidation or reduction of particular compound(s).
Saccharolytic properties.
Saccharolytic properties are capabilities of fermenting sugars and polyatomic alcohols with formation of acid or gas and acid. Saccharolytic properties are studied on Hiss media, which contain different carbohydrates and indicator (so called 'many-colored row’). Hiss media can be liquid or semi-liquid. Liquid media hiss contains Andred indicator (fucsin, discolored with an alkali). Initial color of the medium is yellow.
Under fermentation of carbohydrates with formation of acids, color of medium is changing due to changes of color of Indicator (fucsin becomes red). The color of media changes from yellow to red. In case of production of gases, they are accumulated in ‘floating tube’.
Proteolytic properties.
Presence of proteolytic enzymes in bacteria is studied on media with gelatin, milk, serum and peptone. During the stab inoculation of media some bacteria (Vibrio cholerae, Bacillus anthracis) at room temperature ( +20-220C) are liquefying gelatin. In addition, different bacteria give specific type of liquefaction (in a form of nail, Christmas tree). During the inoculation in milk, there is splitting of casein clot with formation of peptone, leading to yellow discoloration.
Indicators of deeper splitting of protein are formation of ammonia, indole and hydrogen sulphide. For detection of indole, which is formed in case of presence of tryptophanase, by Morel method, filter paper strips are washed with hot fat solution of oxalic acid and allowed to dry off. Indicator paper is placed between tube wall with and plug. If indole is produced on 2- 3 day, lower part of strip becomes pink.
Presence of ammonia is detected by changing color of pink litmus paper, placed between the tube wall and plug, into blue.
Presence of hydrogen sulphide is detected by using filter paper strips with lead acetate, which are placed between the tube wall and plug. If hydrogen sulphide is produced, lower part of strip becomes black.
Other properties.
Presence of catalase in aerobes and facultative anaerobes is detected by placing one loop of bacteria into 1 drop of 3% hydrogen peroxide. In case of presence of catalase, gas (O2) bubbles are formed.
Oxidase detection is done by placing and rubbing loop of culture onto the indicator paper soaked in alcohol solution of α-naphthol and 1% water solution of menthol. In case of the presence of oxidase, paper becomes blue.