Experimental principle Dilution plate measurement is a counting method designed according to the culture characteristics that a single colony formed on a solid medium under high-dilution conditions is a single cell, that is, one colony represents a single cell. When counting, first make the sample to be tested into a uniform breeding dilution, try to spread the microbial cells in the sample to make it exist as a single cell, otherwise a colony will not only represent a cell, but also take a certain dilution, certain The amount of the dilution was inoculated into the plate so that it was evenly distributed in the medium in the plate. After culture, colonies are grown by single cell growth, and the number of colonies in the sample can be calculated by counting the number of colonies. The number of bacteria counted by this counting method is the number of colonies growing on the medium, so it is also called the viable count. Generally used for certain product verification, such as product verification of rhizobium agent, biological product inspection, determination of soil bacteria content and inspection of pollution degree of food and water sources. Under natural conditions, microorganisms often exist in a community state, which is often a mixture of different types of microorganisms. In order to study the characteristics of a certain microorganism or to cultivate and use a certain microorganism in a large amount, it is necessary to obtain pure culture from these mixed microbial communities, and this method of obtaining pure culture is called separation and purification of microorganisms. In nature, soil is a good environment for microbial life. The number and types of microorganisms in life are extremely rich. Therefore, soil is an important base for human development and utilization of microbial resources. The number and type of microorganisms in the soil are related to soil fertility, and there are many fertile soils and few in poor soil. Its physiological group is related to other physical and chemical properties of the soil, such as aeration and pH. For example, in a well-ventilated vegetable garden soil, aerobic microorganisms have an absolute advantage. In this experiment, the aerobic bacteria in the soil were separated by the garden soil as the material, and the quantity was determined. When separating microorganisms, they are generally supplied with suitable living conditions according to the requirements of the microorganisms for nutrition, pH, oxygen, etc., or adding an inhibitor to cause an environment which is only beneficial to the growth of the strain and is not conducive to the growth of other strains. Thereby eliminating unwanted strains. Common methods for separating microorganisms include dilution plate separation method and scribing separation method. Different methods can be used according to different materials, and the final purpose is to present a single colony of the microorganism to be separated on the medium, and further to a single colony if necessary. Isolation and Purification. When the microorganism is separated by the dilution plate, the number of microorganisms to be separated can also be simultaneously determined. Actinomycetes and bacteria belong to the same prokaryotic microorganisms, and are important antibiotic-producing bacteria. The amount in the soil is second only to bacteria, especially in the neutral to slightly alkaline soil with abundant organic matter and good permeability. In this experiment, the actinomycetes in the garden soil were separated and counted using Gao's No. 1 agar medium. The number of fungi in the soil is inferior to bacteria and actinomycetes, and it is mainly in acidic soils with abundant organic matter and good permeability. It is not difficult to isolate the fungus in the soil, but because of its large colonies, it is easy to expand and the counting accuracy is low. In this experiment, the fungus in the garden soil was separated and counted using Martin's medium supplemented with chloramphenicol or gentamicin and bengal red. According to the general information, it is streptomycin, but this kind of antibiotic should be formulated into a certain concentration of solution, and should be added to the medium before the plate is inverted. On this medium, actinomycetes and bacteria are inhibited by chloramphenicol or gentamicin and bengal red, but most fungi survive and their colonies are less suppressed by Bengal red, thus avoiding certain The quantitative error caused by the spread of fungi. Experimental Materials 1. Living material: Bacillus thuringiensis fungicide. 2. Medium: beef paste peptone agar medium; Gao's No. 1 agar medium; Martin's agar medium supplemented with chloramphenicol (or gentamicin) and bengal red: added to 1000 mL of medium for injection Chloramphenicol 2 mL, Bengal Red 33.4 mg, were added before sterilization. 3. Material: Fertile tea garden soil. Laboratory supplies 90mL sterile water, 9mL sterile water, 9cm diameter sterile plate, 1mL sterile straw, balance, sample bottle, marker pen, glass spatula, sieved through 2mm soil sieve with 15-20 glass beads Vegetable garden, balance, test tube rack, sterile weighing paper, alcohol lamp, match, inoculating ring, sterile water. experimental method (1) Dilution plate measurement method 1. Preparation of sample diluent Accurately weigh 10g of the sample to be tested, put it into a 250mL flask containing 90mL of sterile water and put small glass beads, shake it by hand or shake on the shaker for 20min, disperse the microbial cells, let stand for 20~30s, Serve 10 -1 dilution; then use 1mL sterile pipette, draw 1mL of 10 -1 dilution, transfer to a test tube containing 9mL of sterile water, blow 3 times, let the bacteria mix evenly, then become a 10 -2 dilution Then change a sterile pipette, draw 1 mL of 10 -2 dilution, transfer it to a test tube containing 9 mL of sterile water, and also suck 3 times to form a 10 -3 dilution; and so on, serial dilution A series of diluted bacteria such as 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 . When counting with a dilution plate, the selection of the dilution of the bacteria to be tested should be determined according to the sample. When the number of bacteria to be tested contained in the sample is large, the dilution should be high, and vice versa. When measuring the bacterial count of bacteria, the dilution of 10 -7 , 10 -8 , and 10 -9 is used to determine the amount of soil bacteria. The actinomycetes are determined by 10 -4 , 10 -5 , and 10 -6 dilutions . For the quantity, the dilution of 10 -3 , 10 -4 , and 10 -5 is used to determine the number of fungi using 10 -2 , 10 -3 , and 10 -4 dilutions. Nano Gel Pad,Nano Adhesive Tape,Nano Gel Grip Tape,Washable Adhesive Tape Kunshan Jieyudeng Intelligent Technology Co., Ltd. , https://www.jerrytapes.com
Microbiology: separation and purification of microorganisms and dilution of plate colony counts