The separation, culture and identification of Bifidobacteria

gardener sakuyaandy:
We
culture
Bifidobacteria is cultured in an anaerobic culture tank, if not, with anaerobic bottles or anaerobic cylinders.
the
how did you use it? We have added L-cysteine to MRS, but these are cultured media, if you need to distinguish between bacteria can be used X-GAL improved MRS can play a role in identification, the specific literature name is <>
gardener jerrycs:
anaerobic
microorganisms
in nature is widely distributed, a wide variety, its physiological role is increasingly valued. Bifidobacteria is a specialized anaerobic bacteria, very sensitive to oxygen, therefore, the key to the separation, culture and live bacteria count of Bifidobacteria is to provide anaerobic and low redox potential culture environment.
the most suitable growth temperature of Bifidobacteria is 37 degrees C to 41 degrees C, the minimum growth temperature is 25 degrees C to 28 degrees C, and the maximum growth temperature is 43 degrees C to 45 degrees C. Initially the most suitable pH 6.5 to 7.0, in pH4.5 to 5.0 or pH 8.0 to 8.5 does not grow. Its cells are in various forms, with short rods than normal, slim rods with sharp end shapes, spherical, long rod curved, branched or fork-shaped, stick-shaped or spoon-shaped. A single or chained, V-shaped, fenced arrangement, or clustered into stars. Terrain positive, not acidic, do not form spores, do not move. Bifidobacteria has a smooth, convex, complete edge, creamy to white, sparkle and soft texture. Bifidobacteria is a normal physiological bacteria in the human body, colonized in the intestines, is the dominant bacterium group of the intestine, accounting for 92% of the infant digestive tract plexus.
the bacteria and the human body for life, the number of which is closely related to human health, is currently recognized as a class of representative beneficial bacteria that promote the health of the body. The bacteria can form a physiological barrier on the surface of the intestinal mucosa to resist the invasion of pathogens such as typhoid salmonella, laxative E. coli, dysentery-causing herb, etc., and maintain the normal micro-ecological balance in the body's intestines; Family vitamins, nicotic acid and folic acid and other vitamins, can control endotoxinemia and prevention of constipation, prevention of anemia and rickets, can reduce the formation of nitrosamines and other carcinogens pregeners, have anti-cancer and anti-cancer effects, can antagonize free radicals, hydroxy free radicals and lipid peroxidation, with anti-aging function.
of Bifidobacteria, such as anaerobic tank method, anaerobic bag method, anaerobic tank method. These methods require specific deoxygenation measures, many operating steps, more cumbersome. This experiment introduces a simple test tube culture method - Hengeit anaerobic roller tube technology, Hengeit anaerobic roller tube technology is the United States
biology
Hengert first proposed in 1950 and applied to the study of anaerobic microorganisms in the stomach anaerobic culture technology.
This technology has undergone decades of continuous improvement, so that Hengert anaerobic technology is becoming more and more perfect, and gradually developed into a complete set of research anaerobic microorganisms, and over the years has proved to be a rigorous, specialized anaerobic bacteria research a very effective technology. The advantage of this technique is that the pre-reduced culture base can be used at any time for testing, and the bacteria in the test tube can be observed or examined at any time without interfering with anaerobic conditions.
, there are many ways to identify Bifidobacteria. In recent years, a new raPD and other
merial biology
technology to build the genetic fingerprint map of Bifidobacteria, analysis of different Bifidobacteria species exist between ogeneous and polymorphism, RAPD technology can also be used for Bifidobacteria species identification and genotype. In general, we apply methods suitable for the identification of all anaerobic bacteria, including Bifidobacteria-specific enzyme detection, acetic acid, lactic acid and other organic acid determination, sugar fermentation test and other related indicators.
Experimental appliances
high purity nitrogen anaerobic tube syringe culture tank ice cube water bath pot tweezers marker pen MRS medialized micro-identification tube alcohol cotton ball porcelain disc oscillator copper column deoxygenation system quantitative sampler
string temperature
water bath slide
smoulator
thermostat ultrasonic anaerobic crusher filter tube layer oxygen tank, etc.
methods and steps
1 Copper column system deoxygenation
copper column is a hard glass tube with copper wire or copper chips inside. The tube is 40 to 400mm in size, is processed into a funnel at both ends, has a tropical outer wall around it, and is connected to a transformer to control voltage and stabilize the temperature of the copper column. The hoses are connected at both ends of the copper column, with gas cylinders connected at one end and trachea port at the other end. Since gases such as N2, CO2 and H2 from gas cylinders all contain trace amounts of O2, when these gases pass through copper columns at temperatures of about 360 degrees C, trace O2s in copper and gas are combined to produce CuO, and copper columns change from bright yellow to black. When H2 is passed into the oxidized copper column, the oxygen in H2 and CuO is combined to form H2O, which is reduced to copper and the copper column is bright yellow. This copper column can be used repeatedly and continuously for deoxygenation purposes. Of course, the H2 source can also be produced by a hydrogen generator.
2 Preparation of pre-reduced media and diluents
When making pre-reduced media and diluents, the configured media and dilution are boiled to drive oxygen, and then installed into the screws with a semi-quantitative sampler while hot In anaerobic test tubes, the general
agar
medium is 4.5 to 5.0mL, the dilution is 9mL, and a long needle with N2 gas is inserted to exclude O2. At this point, it is clear to see the redox indicator added to the culture base - edged cyan from blue to red finally turned colorless, indicating that the test tube has become oxygen-free state, and then cover the screw butene plug and screw cover, sterilization standby.
3 separation
(1) number
take five sterile water test tubes, marked with a marker pen 10-1, 10-2 ... 10-5。
(2) Dilution
Under sterile conditions, use a sterile syringe to absorb a liquid sample of 1mL mixed evenly, add an anaerobic test tube filled with pre-reduced physiological saline, mix it evenly with an oscillator to make 10-1 dilution. Use a sterile syringe to absorb 1mL10-1 dilution into another anaerobic test tube filled with 9mL physiological saline to make 10-2 dilution. In this way, 10 times the series dilution, to 10-7, to make different sample dilution. Usually choose 10-5, 10-6, 10-7 three dilution for the tube count.
(3) Roll Tube Separation
1) Roll Tube
Dissolve anaerobic agar medium in a boiling water bath, place it in a water bath at a constant temperature of 46-50 degrees C, to be used, draw 10-4 with a sterile syringe, 10-5, 10-6 three dilution of 0 .1mL each, respectively, injected into the test tube to be used, and then flattened in a porcelain plate filled with ice water quickly rolling, the melting agar with bacteria in the test tube wall will immediately form a solidified layer.
2)
the resulting bacteria need to be picked out, the mirror to check its shape and purity. If pure culture has not been obtained, the tube should be diluted again and the bactericides should be picked again until pure culture is obtained. The single bacteria to be picked are observed and identified under a magnifying glass in advance and marked. The culture base test tube is then secured to a suitable stand, the test tube plug is opened, and a long nitrogen needle with proper airflow and flame-sterilization is quickly inserted into the tube. At the same time, another liquid anaerobic tube removes the plug and inserts it into another sterilized breathable needle. Carefully insert the prepared elbow capillary into the solid culture, find the place to be picked, gently absorb, transfer to the liquid test tube, plug. Culture at 37 degrees C. Check the purity of the separated culture after culture 24h or the turbidity of the pending culture fluid.