Separation and culture of specialized anaerobic bacteria

Specialist anaerobic bacteria are bacteria that can grow and multiply in an oxygen-free environmentSuch bacteria lack a perfect respiratory enzyme system, can only carry out oxygen-free fermentation, not only can not use molecular oxygen, but also free oxygen to its toxic effectSuch as tetanus, Botox, gas-producing mecobacteria and so on1 Purpose1.11.2 To understand the growth characteristics of anaerobic microorganisms1.2 Observe the morphological characteristics of anaerobic microorganisms (Bifidobacteria)1.3 Master the principle of the rolling tube separation, culture and countingtechnology of anaerobic microorganismsthe simple and effective techniques of cultivating anaerobic microorganisms include: anaerobic tank culture technology; Henggett anaerobic roller technology is introduced hereHengate anaerobic tube technology was first proposed by Hungate, an American microbiologist, in 1950 and applied to the study of anaerobic microbes in the stomachSince then, the technology has undergone decades of continuous improvement, which has made Hengate's anaerobic technology more sophisticated and gradually developed into a complete set of techniques for the study of anaerobic microorganismsAnd over the years of practice has proved that it is a very effective technique for studying strict, specialized anaerobic bacteriaHengate's anorexia-like tube culture technique can be used not only for the separation of beneficial anaerobic bacteria such as Bifidobacteria, and with live bacteria culture count, but also for the separation and identification of harmful bacteria (such as tyrosic acid) or pathogens (such as Clostridium difficile)3 material3.1 sample sifhex (liquid), Bifidobacteria preparation (solid)3.2 Media Improvement MRS Medium, PTYG Medium 3.3 instruments and appliances Hengate anaerobic roller unit set, anaerobic tube, anaerobic bottle, roller, quantitative sampler 4 Process Copper Column Deoxygenation , Pre-Reduced Medium , Dilution Liquid Preparation , Dilution Samples , Rolling Tubes , Cultures , Counting 5 Methods 5.1 Copper Column System Deoxygenation Copper Column is a hard glass tube with copper wire or copper chips inside The size of the tube is 40-400mm, two sections are processed into funnel spouts, the outer wall is surrounded by tropical, and connected with a transformer to control the voltage and stabilize the temperature of the copper column The copper column is connected to the hose at both ends, with the gas cylinder at one end and the outlet at one end Since gases such as N2, CO2 and H2 usually contain O2, when these gases pass through a copper column at a temperature of about 360 degrees C, the trace O2 in the copper and gas is combined to produce CuO, which changes from bright yellow to black When H2 binds to the oxygen in CuO to form H2O when it is pushed into H2 to the oxidized copper column, the CuO is reduced to copper, which in turn is bright yellow This copper column can be used repeatedly and continuously for the purpose of deoxygenation Of course, the H2 source can also be generated by a hydrogen generator 5.2 Pre-reduced medium and dilution preparation Production of pre-reduced medium and dilution, first the preparation of the medium and dilution liquid boiling oxygen, and then with a semi-quantitative sampler while the heat into the snail anaerobic test tube, the general agar medium 4.5-5 ml, dilution liquid 9ml, and inserted through the N2 gas long needle to exclude O2 At this time, it can be clearly seen that the medium added to the redox indicator - the edge of the sky blue from blue to red finally become serotrial, indicating that the test tube has become anaerobic state, and then cover the screw butene plug and screw cap, sterilization backup 5.3 Bifidobacteria sample siluent preparation in sterile conditions accurately called 1g solid or with sterile syringe to absorb 1 ml of mixed and uniform liquid samples, and then added with a pre-reduced physiological saline anaerobic test tube, with a shocker to shake it evenly, to make 10-1 dilution Absorb 1 ml of 10-1 dilution from a sterile syringe into another test tube containing 9 ml of physiological saline to make a 10-2 dilution In order to follow this method, 10 times series dilution to 10-7, to make different concentrations of sample dilution The rolling tube is usually counted with 10-5, 10-6, 10-7 dilutions 5.4 anaerobic tube culture method put melted aseptic axastatic agar medium test tube in a constant temperature water bath of about 50 degrees C, with 1 ml of sterile syringes to absorb 10-5, 10-6, 10-7 three The dilution of the dilution is 0.1 ml each in the melted agar medium test tube, which is then flat in a plate filled with ice cubes or rolled quickly on a special roller machine, so that the melted media with bacteria immediately solidifies into a thin layer on the inner wall of the test tube Repeat each dilution 3 times and then culture in a thermostatic incubator at 37 degrees C (42 degrees C for yogurt samples) Generally cultured 24-48 hours later, can be in the anaerobic tube agar layer or surface can grow visible to the naked eye of the colonies 5.5 Bifidobacteria (separation) count Choose to spread evenly, the number of dozens to hundreds of colonies of anaerobic test tube for live bacteria counting, you can get the number of Bifidobacteria per gram or per milliliter sample 5.6 calculating the number of live bacteria of Bifidobacteria cfu/g(ml) sample s 0.1 ml of roller count of the actual average x 10 x dilution multiple 6 Results 6.1 observed the form of Bifidobacteria and described its morphological characteristics 6.2 calculate the amount of Bifidobacteria per gram or milliliter of sample and record the results