- •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 the isolation of pure cultures of anaerobic bacteria.
The principle and the main stages are the same as for the isolation of pure cultures of aerobic bacteria.
One of the main requirements in cultivating anaerobic bacteria is removal of oxygen from the nutrient medium. The content of oxygen can be reduced by a great variety of methods: immersing of the surface of the nutrient medium with petrolatum, introduction of microorganisms deep into a solid nutrient medium, the use of special anaerobic jars.
First day. Inoculate the studied material into Kitt-Tarozzi medium (nutrient medium): concentrated meat-peptone broth, glucose, peaces of liver (pH 7.2-7.4).
Prior to inoculation place the medium into boiling water for 10-20 min in order to remove air oxygen contained in it and then let it cool. Upon isolation of spore forms of anaerobes the inoculated culture is reheated at 80 C for 20-30 min to kill non-spore-forming bacteria. The cultures are immersed with petrolatum and placed into an incubator.
Second day. Take note of changes in the medium, namely, the appearance of opacification or opacification in combination with gas formation. Take broth culture with a' Pasteur pipette and transfer it through a layer of petrolatum onto the bottom of the test tube. Prepare smears on a glass slide in the usual manner, then flame fix and Gram-stain them. During microscopic examination record the presence of Gram-positive rod forms (with or without spores). Streak the culture from the medium onto solid nutrient media.
Isolated colonies are prepared by some methods.
1. Zoisslcr's method: prepare three plates with blood-sugar agar. Take a drop of the medium with microorganisms into the first plate and spread it along the surface, using a glass spatula. Use the same spatula to streak this culture onto the second and then third plates and place them into an anaerobic jar at 37 C for 24-48 hrs.
2. Veinberg's method of sequential dilutions: anaerobic microorganisms are grown deep in a solid nutrient medium (). The culture from the medium is taken with a Pasteur pipette with a soled tip and transferred consecutively into the 1st, 2nd, and 3rd test tubes with 10 ml of isotonic sodium chloride solution. Continue to dilute transferring the material into the 4th, 5th. and 6th thin-walled test tubes with melted and cooled to 50 °C meat-peptone agar. Alter agar has solidified, place the inoculated culture into an incubator.
3. The Vinyale-Veyone’s method is used for mechanical protection from oxygen. The seeding are made into tube with melting and cooling (at 42 0C) agar media (see above).
On the third day, study the isolated colonies formed in third plate and make smears from the most typical ones. The remainder is inoculated into Kitt-Tarozzi medium. The colonies in the test tubes are removed by means of a sterile Pasteur pipette or the agar column may be pushed out of the tube by steam generated upon warming the bottom of the test tube. Some portion of the colony is used to prepare smears, while its remainder is inoculated into Kitt-Tarozzi medium to enrich pure culture to be later identified by its morphological, cultural, biochemical, toxicogenic, antigenic, and other properties.
So Kitt-Tarozzi medium is used for accumulation of anaerobes under primary inoculation and support of growth of pure cultures.