The detection and determination of the expansion of microbial strains and pollution

The scale of production in modern industry is growing, with the volume of each fermentation tank in the tens or even hundreds of cubic metersTherefore, in order to complete fermentation in a short period of time, it is necessary to have a large number of microbial cells to doThe task of seed expansion is to obtain a sufficient number of robust microorganismsSeed expansion culture refers to the pure seeding process in which the production strains, which are kept dormant in the sand tube and freeze-drying tube, are activated by the cast slope of the test tube, and then expanded by the flat bottle or shaker bottle and seed tank step by step, and finally obtained a certain quantity and quality of the thoroughbred processThese thoroughbred cultures are called seeds"text-align:center;"img src""alt""expanded culture and pollution detection and identification of microbial strains" title"""the detection and identification of the expansion of microbial strains",the microbial industry since the adoption of pure-bred fermentation, the yield has been greatly improved, but the requirements for preventing hybrid contamination are also higherPeople have accumulated a lot of valuable experience in the fight against the pollution of the hybrid bacteriaStandardized management measures, designed a series of equipment (such as closed fermentation tanks, medium sterilization equipment, sterile air preparation equipment, etc.) and pipes, the application of sterile rooms and even sterile workshops, the establishment of sterile operating technology, thus greatly reducing the rate of fermentation and dyeingBut some fermentation industry is also suffering from the threat of bacteria, after the bacteria light affect the yield, product extraction yield and product quality, heavy caused by "dumping", not only waste a large number of raw materials, causing serious economic losses, but also cause damage to the surrounding environmentAll microorganisms that invade non-inoculated bacteria in fermentation liquid or fermentation containers are collectively referred to as hybrid contamination, and early detection of the bacteria and taking appropriate measures are essential to reduce the losses caused by the contamination of the bacteriaTherefore, the method of checking requires accuracy and speedFermentation contamination can occur at various timesSeed culture of the bacteria is the most harmful, because strictly preventedOnce seed contamination is found, it should be sterilized and discarded, and the seed tank and its pipes are thoroughly sterilizedFermentation pre-nutrient rich, easy to infect bacteria, at this time nutrient consumption is not much, the fermentation liquid should be replenished the necessary nutrients after rapid sterilization, and re-inoculated fermentationIn the middle of fermentation, the bacteria not only seriously interfere with the metabolism of the production strain, but also affect the production of products, and even make the products formed to decompose Since the fermentation of mid-term nutrients has been consumed a lot, the production of metabolites is not a lot, rescue treatment is more difficult, can consider adding the appropriate amount of antibiotics or fungicides If it is late fermentation dye bacteria, at this time the product accumulation has been more, sugar and other nutrients have been nearly exhausted, if the bacteria is not serious, can continue to ferment; The heavier the degree of infection, the greater the harm, and the less bacteria, the less impact on fermentation Therefore, in actual production, it should be treated differently according to the degree of pollution and fermentation period 1, experimental equipment and reagents 1.1 equipment high-pressure steam sterilization pot, ultra-clean workbench, incubator with shaker, microscope, balance, triangular bottle, test tube, pipette, petri dish, vaccination needle, flat coater, alcohol lamp, slide 1.2 reagent nutritional audry, glucose, dihydrophosphate, magnesium chloride, sodium chloride, sodium chloride, Two potassium, beef paste, protein, 0.4% phenol red solution, distilled water, saffron dye liquid, balm, mirror liquid 1.3 medium (1) nutritional agar medium: direct weighing of the nutritional agar powder 4.5g, dissolved in 100m L distilled water preparation, pH7.2, divided into test tubes, after the sterilization squint (2) Seed Medium: Glucose 0.5%, Dihydromine phosphate 0.1%, magnesium seven hydrated sulfate 0.02%, sodium chloride 0.5%, potassium phosphate 0.1% 3) phenol red broth medium: beef paste 0.3%, protein 0.8%, glucose 0.5%, chlorination Sodium 0.5%, 0.4% phenol red solution, pH7.2 2, experimental method 2.1 seed expansion culture 2.1.1 sloped medium preparation the preparation of nutritional agar medium, subassembled, sterilization (121 degrees C condition seisido30min), inverted slope 2.1.2 species of activation (1) the E coli using sterile operation method inoculated on the slope, 37 degrees C culture 18h ; Detection methods commonly used dyeing mirror inspection, if after detection, no pollution, can be expanded culture, if the detection of bacteria, to repeat the above steps, until sterile, otherwise, can not continue the next operation 2.1.3 expanded culture under sterile conditions, from the tested activated medium to pick about 10 colonies, with vaccination needles to the expanded medium (i.e seed media), at 37 degrees C oscillating 16-18h, to observe the colony pattern Then in conjunction with the microscope pattern observation, based on the results to determine whether the next step can be carried out 2.2 contamination detection and identification 2.2.1 microscope inspection (1) sampling: using sterile operation to take a little fermentation fluid, coated on the slide ; 2.2.2 plate check (1) formulated nutritional agar medium, sterilized, inverted plate; (2) take a small amount of fermentation liquid to be tested after dilution (about 10-6 to 10-7) coated on the nutritional agar plate, cultured 24h at 37 degrees C; (3) observed the form of the colony 2.2.3 Brod Culture Check (used to detect whether the media sterilisation is thorough) 1 ml of the seed medium to be tested (seed medium after sterilization) is connected to the glucosol red broth medium, cultured at 37 degrees C, and observed the state and color of the medium 3, the experimental results 3.1 seed expansion culture observed in the activated medium on a large number of colonies, and the colonies color is single, are pale yellow Pick the edge colonies and the pattern of the same small number of colonies, make a small number of fillings, dyeing under the microscope observation, found that many visions are bacteria, can initially draw a activated species without contamination of the bacteria Pick the colonies that do not pollute the bacteria, use sterile operation technology to inoculate, expand the culture After culture dispensing 18h under appropriate conditions, the seed fluid of E coli was obtained, and a large amount of E coli was observed under the microscope 3.2 microscope examination mirror examination, many of the field of vision observed are bacillus, chain or individual presence, no cocci or other forms of bacteria observed, so it can be initially considered that there is no contamination of the seed liquid 3.3 flat-panel examination from the nutritional agar flat medium observed the form of the colonies is round, neat edges, smooth surface, translucent or milky white, colonies have small protrusions, this is a typical E coli colonies, no other forms of colonies observed, so it can be initially considered that the seed liquid is not contaminated with bacteria 3.4 Broth to detect whether the medium is thoroughly sterilized phenolred broth in the medium, phenol red in the acidic environment is yellow, alkaline environment is red If the inoculated fluid in the broth medium is present, the bacteria metabolism will make the medium acidic and show yellow After culture, the broth medium is still red, did not turn yellow, and clarified, not cloudy, indicating that there is no bacteria in the liquid to be measured, therefore, the measured sterilized seed medium is not contaminated with bacteria 4, experimental discussion 4.1 to activate the seed to obtain active seeds before the expansion of seed culture, if the bacteria has been activated can omit this step When the seed is expanded, if the bacteria of low temperature preservation contaminates the hybrid bacteria, it should be discarded or purified with flat-plate medium and then used to activate and expand the culture 4.2 using the microscope method, if from the field of view found with different forms of bacteria than the target strain can be considered to be contaminated bacteria, the method is simple, fast, can be timely to detect the bacteria But there are its shortcomings: (1) the sample containing few eras is not easy to draw the correct conclusion, so should check a few more horizons; 4.3 using the flat-panel inspection method, if the emergence of bacteria with different forms from the target strain, it indicates that it may be contaminated with bacteria, to further confirm, can be observed with microscope form, if the individual form and colonic form are different from the target bacteria, can confirm the contamination of the bacteria This method is suitable for the detection of fermentation fluid with more solids, the image is intuitive, the naked eye can be discernable, and no instrument is required However, sterile operation techniques need to be strictly enforced, which take longer and cannot distinguish between bacteria similar in form to the target bacteria In the initial stage of pollution, the target bacteria accounted for the vast majority, the number of contaminated bacteria is very small, so to do more parallel tests to detect the pollution bacteria 4.4 Brod culture inspection method can only be used to detect whether the sterilization of the medium is thorough, not applicable to the fermentation liquid inspection 4.5 can be used to determine the fermentation parameters to detect the presence of contamination of the bacteria That is, in the fermentation process, the fermentation time is horizontal coordinates, to dissolve oxygen or exhaust carbon dioxide content or the pH of the fermentation liquid as the vertical coordinates, as a curve, if the process unchanged under the circumstances of these characteristic curves change, it is likely to contaminate the bacteria However, fermentation parameter stoice is difficult to detect early contaminated bacteria 5, the experimental conclusion this experiment we understand the method of seed preparation and the harm of fermentation pollution, know that the contamination of bacteria not only waste a lot of raw materials, causing serious economic losses, but also pollution of the environment, so, in the seed activation culture and expansion of culture every step of the need for a hybrid-free inspection Microscope direct examination method and flat-panel indirect examination method can be used to detect bacteria pollution, the method is simpler and visual, but, to carry out more accurate detection, it is best to do more parallel experiments The broth culture check method is an effective method for detecting the thoroughness of media sterilization