Isolation and Identification of Anaerobic Bacteria

The isolation and identification of anaerobic bacteria is a test for the separation and identification of bacteria with high oxygen sensitivity. Because anaerobic microorganisms are widely distributed in nature and have a wide variety, their physiological functions have received increasing attention. Obligate anaerobic bacteria are very sensitive to oxygen. Therefore, the key to their separation, culture and viable count is to provide a culture environment free of oxygen and low redox potential. Basic Information Specialist classification: growth and development check classification: biochemical examination Applicable gender: whether men and women apply fasting: fasting Tips: If the colony is too small, use a magnifying glass to observe the colonies. Normal value The type and proportion of the flora in the body are normal, and the human body is in a state of dynamic balance and health. Clinical significance Hengate anaerobic rolling tube technology, Hengate anaerobic rolling tube technology is an anaerobic culture technology first proposed by American microbiologist Hengate in 1950 and applied to rumen anaerobic microorganism research. Abnormal results: special diseases caused by Clostridium anaerobics such as gas gangrene, tetanus, botulism, etc. People who need to be examined: patients with diabetes, severe liver disease, cirrhosis, uremia, hemorrhoids ulcers, limb gangrene, botulism and other symptoms. Precautions In the process of separation and identification of anaerobic bacteria, the following matters should be noted: (1) Anaerobic specimens must be isolated from the air during collection and transportation, and must be completed within 30 minutes, while avoiding contamination of normal flora. (2) The medium should be freshly prepared. If it is stored for too long, oxygen is dissolved on the surface or peroxide is in the medium, which is not conducive to the growth of anaerobic bacteria. (3) If the colony is too small, the colony should be observed with a magnifying glass. (4) To perform an oxygen tolerance test, it is required to pick 4 to 5 colonies of different traits from each agar plate, inoculate aerobic and anaerobic blood agar plates, and place them in aerobic, carbon dioxide and anaerobic environments. (5) If you do extracellular enzyme identification, you must have sufficient bacterial concentration. Inspection process Separation (1) number Take five sterile water tubes and mark them with a marker to indicate 10-1, 10-2, ... 10-5. (2) dilution In a non-oxygen sterile ultra-clean anaerobic glove box, use a sterile syringe to draw 1 mL of a well-mixed liquid sample, add it to an anaerobic test tube containing pre-reduced physiological saline, and mix it evenly with an oscillator. Make a 10-1 dilution. A 1 mL 10-1 dilution was pipetted into a separate anaerobic tube containing 9 mL of physiological saline using a sterile syringe to make a 10-2 dilution. Diluted serially by 10 times to 10-6 to make different sample dilutions. The tube count is usually selected by three dilutions of 10-4, 10-5, and 10-6. (3) Roller separation 1) Rolling tube The anaerobic sterile agar medium was dissolved in a boiling water bath, placed in a water bath at a constant temperature of 46-50 ° C, and used, and when the medium was taken out from the bottle, it was inflated with N 2 in the medium. Then inflate with N2 in the test tube, remove all the air inside the tube, then add the medium to the tube and immediately plug the stopper. When the stopper is inserted into the tube, the inflation needle is pulled out in time. Pipette 0.1 mL of each of 10-4, 10-5, and 10-6 dilutions into a test tube to be used, and then place it flat on a porcelain plate containing ice water and roll rapidly. The solubilized agar forms a solidified layer immediately on the inner wall of the test tube. 2) Separation and purification The resulting colonies need to be picked and examined for their morphology and purity. If a pure culture has not been obtained, dilute the tube again and pick the colon again until a pure culture is obtained. The single colonies to be picked are preliminarily observed under a magnifying glass and marked. The medium test tube is then fixed to a suitable holder, and the test tube rubber stopper is opened, and a gas-filled nitrogen needle having a proper air flow and a flame-killed bacteria is quickly inserted into the tube. At the same time, another liquid anaerobic tube is removed from the rubber plug and inserted into another sterilized venting needle. Carefully insert the prepared elbow capillaries into the solid medium, identify the colonies to be picked, gently aspirate them, transfer them to a liquid test tube, and stopper. The culture was cultured at 37 ° C for 24 hours or longer or after the turbidity of the culture solution was checked, and the purity of the isolated culture was examined. 3) Dash separation One end of the test tube rubber stopper is burned on the flame, and the gas suction needle is used to plug the nozzle. Before the needle is quickly removed, the air is ventilated for 15-20 s, and one end of the tube is burned for a while on the flame. Tighten the pipe plug, and the coiled pipe is erected and insulated. Use CO2:H2=80:20. Because CO2 is heavier than air, the pipe plug will not have air residue after opening. The scribing tube can be grown at 34-37 degrees to grow colonies. Identification of strains 1 sugar fermentation test Sugar fermentation experiments are the most commonly used biochemical reactions and are particularly important in the identification of intestinal bacteria. Most bacteria can use sugar as a carbon source and energy source, but they have great differences in their ability to break down sugar. Some bacteria can break down sugar and produce acid (such as lactic acid, acetic acid, propionic acid, etc.) and gas (such as Hydrogen, methane, carbon dioxide, etc.; some bacteria produce only acid and do not produce gas. For example, Escherichia coli can decompose lactose and glucose to produce acid and produce gas; Salmonella typhimurium can decompose glucose to produce acid and not produce gas, and can not decompose lactose; common Proteus decomposes glucose to produce acid and produces gas, which cannot decompose lactose. The production of acid can be determined using an indicator. Bromocresol purple [pH 5.2 (yellow) - 6.8 (purple)] was added in advance when the medium was prepared, and when the acid was produced by fermentation, the medium was changed from purple to yellow. The generation of gas can be evidenced by the presence or absence of bubbles in the inverted Dehan tube in the fermentation tube. Specific experimental steps: 1 The bacterial liquid is appropriately diluted, and then in a sterile environment, a certain amount of the diluted solution is injected into the biochemical identification tube, and sealed with a sterile sealing film. 2 Put the inoculated identification tube into an anaerobic tank, and place it in a 37 ° C constant temperature incubator with sufficient nitrogen. The color change and gas production of each tube were observed after 324 h. 2 protein decomposition experiment 1 The bacterial liquid is appropriately diluted, and then in a sterile environment, a certain amount of the diluted solution is injected into the biochemical identification tube, and sealed with a sterile sealing film. 2 Put the inoculated identification tube into an anaerobic tank, and place it in a 37 ° C constant temperature incubator with sufficient nitrogen. After 324 hours, the tubes were subjected to the Mirren reaction, the hydrazine reaction, the yellow reaction and the mouthwash reaction, respectively. 3 starch hydrolysis experiment 1 The bacterial liquid is appropriately diluted, and then in a sterile environment, a certain amount of the diluted solution is injected into the biochemical identification tube, and sealed with a sterile sealing film. 2 Put the inoculated identification tube into an anaerobic tank, and place it in a 37 ° C constant temperature incubator with sufficient nitrogen. After 324 h, an appropriate amount of Lugol's iodine solution was added to each tube to observe the hydrolysis of the starch. Not suitable for the crowd No relevant information. Adverse reactions and risks No related complications and hazards have been found.