In the field of water Q treatment and sewage treatment, E. coli is used in a large number of feces, and it is commonly used as a marker for checking whether a water source is contaminated with faeces, and its standard coliform group index is measured. In addition, E. coli is harmless in most cases and does not "escape" from the laboratory and harm humans. The use of E. coli as an indicator of fecal contamination may also lead to misleading conclusions, as E. coli can also be present in large quantities in other environments such as paper mills. Cable Try Roll Forming Machine Cable Try Roll Forming Machine,Roll Forming Machine Botou Golden Integrity Roll Forming Machine Co.,Ltd , http://www.jcxsteelrollformer.com
1. E. coli Bacteria are single-cell prokaryotes. The structure of bacterial cells has cell walls, cell membranes, and cytoplasm. Bacteria have no shaped nuclei and the cell wall consists of peptidoglycans. Due to the different bacterial cell wall structure, bacteria can be divided into Gram-positive bacteria and Gram-negative bacteria. Gram-positive bacteria have a thick cell wall, no decidua, and produce exotoxins; gram-negative bacteria have a thin cell wall, decidua, and endotoxin production. Gram-positive bacteria are more sensitive to penicillin.
Escherichia coli is a Gram-negative, heterotrophic, anaerobic enterobacteriaceae. In the intestine it is generally harmless to humans, but any E. coli entering the human urinary system can cause harm to the human body. E. coli is widely used in genetic engineering technology. Its plasmid is the most commonly used carrier, and it is also a commonly used recipient cell in genetic engineering.
Second, the medium configuration
The compounds needed during the microbial life activities include carbon sources, nitrogen sources, growth factors, inorganic salts, and water. Some compounds are both carbon and nitrogen sources and energy sources. Growth factors are indispensable trace organic substances for microbial growth, but they do not necessarily need external supplements. Some microorganisms can synthesize themselves. On the basis of providing the above-mentioned major nutrients, the culture medium also needs to meet the requirements for the growth of microorganisms on pH, special nutrients, and oxygen.
We generally use LB liquid culture medium to expand E. coli, after culture, can be streaked on LB solid medium. The following are the media configuration steps for this experiment:
1. Weighing: accurately weigh each component. 0.5 g of peptone, 0.25 g of yeast extract, 0.5 g of sodium chloride, and 50 ml of water. Add 1g agar to LB solid medium.
2. Dissolve: Heat to melt and bring to 50 mL with distilled water. When LB solid medium is used, it is necessary to add agar, and the whole process is constantly stirred with a glass rod in order to prevent the bottom of the agar and cause the beaker to burst.
3. Adjust the pH: Adjust the pH to 1K alkaline with 1mol/L NaOH solution.
4. Sterilization: Into two 250 ml flasks were respectively filled with 50 ml LB liquid medium and 50 ml LB solid medium, plus tampon. The culture dish was wrapped in kraft paper and placed in an autoclave, and 1 kg was pressure-sterilized for 15 minutes.
5. Pour the plate: After sterilization, the solid medium is cooled to about 60°C and operated near the flame of the alcohol lamp. The process is:
1 Place the sterilized petri dish next to the flame, hold the flask with the medium in the right hand, and pull out the tampon with the left hand;
2 Hold the flask in the right hand so that the bottle mouth of the flask quickly passes through the flame;
3 Open the Petri dish with a left hand to a gap slightly larger than the mouth of the triangle, and pour the medium into the Petri dish with the right hand, and immediately close the lid;
4 After the plate cools and solidifies, place the plate upside down.
The purpose of placing it upside down is to prevent the water droplets formed during cooling of the medium from falling onto the surface of the medium. When transferring sterilized media to a Petri dish, do not stick the media to the wall. Otherwise, bacteria in the air will multiply on these adherent media and contaminate the culture medium in the dishes.
Third, sterilization and disinfection
1. Aseptic technique:
1 Clean and disinfect the test operating space, the operator’s clothing and hands;
2 Sterilize culture vessels, inoculation equipment, and culture media;
3 In order to avoid the surrounding microbial contamination, the experiment should be carried out next to the flame of the alcohol lamp;
4 Avoid contact with surrounding materials by sterilized material appliances.
2. Disinfection method:
1 Boiling sterilization is commonly used in daily life;
2 For some liquids that are not resistant to high temperatures, use pasteurization;
3 Carcinogens, inoculation boxes or clean benches are first sprayed with a solution of carbolic acid or phenolic soap to enhance the disinfection effect, and then use ultraviolet light for physical disinfection;
4 The experiment operator's hands use alcohol to disinfect.
3. Sterilization method:
1 Inoculation loop, inoculation needle, test tube mouth, etc.
2 medium, sterile water, etc. use high pressure steam sterilization method, the equipment used is high pressure steam sterilization pot;
3 Ultraviolet sterilization is used for surface sterilization and air sterilization. The instrument used is an ultraviolet lamp.
4. Comparative disinfection and sterilization:
Compare the strength of physical and chemical factors to eliminate the number of microorganisms Spores and spores can be eliminated
Disinfection of microorganisms that are relatively mild in some living conditions cannot
Sterilization Strong All Microbes Energy
5. Precautions:
1 cotton used in the experiment can not use absorbent cotton, because absorbent cotton is easy to absorb water, easy to cause pollution after absorbing water. After sterilization, the sterilized moisture is usually removed in an oven at 60-80°C;
2 After the article is loaded into the autoclave, it is necessary to first open the exhaust valve to boil and remove the cold air from the pan. The purpose is to facilitate the temperature rise in the pan and then close the row.
The air valve continues to heat. After the heat is cut off and the heat source is cut off, the temperature must be lowered naturally. When the air pressure must fall to zero, the lid is opened. The purpose is to prevent the liquid in the container from boiling.
3 When transferring with any utensils, the stopper and the sealing film can only be clamped on the hand, and should not be placed on the table. The inoculation should be done by the flame of the alcohol lamp;
4 The culture medium must not be stained on the mouth of the triangle, the mouth of the tube and the wall of the culture dish, otherwise it is easy to be contaminated;
5 Inoculation must be bold and careful, and the action is fast, which is the key to reduce pollution;
6 After inoculation, the Petri dish must be placed upside down (covered below) in a constant temperature incubator, and if it is placed, the water vapor is condensed on the lid and dropped onto the surface of the inoculated medium.
Diffusion of the water causes the colony to spread, making it difficult to form a single colony.
Fourth, the separation of bacteria
1. Cross-separation method: After inoculated with Cyclic Bacteria liquid, the streak plate containing the solid medium is streaked, and the bacterial liquid gradually decreases during the streak process, and the bacteria gradually decreases. To the end, the distance between bacteria can be increased. After 10 to 20 hours in culture, a single colony can be produced by one bacterium and the colonies do not overlap. If each colony is inoculated separately onto the bevel of the test tube containing the solid medium and streaked on the slope, the flora of each bevel is the offspring produced by one bacterium.
The procedure is as follows: The bottom of the dish is fixed with the thumb and the ring finger in an inclined state, and the petri dish is slightly opened beside the flame. At the same time, a small amount of bacterial suspension was placed next to the flame with a loop inoculation needle and rapidly transferred to a Petri dish. On the side of the plate medium, the first parallel line was streaked with 3 to 5 pieces, and the Petri dish was rotated at about 70°. In the corners, use the inoculated needles that have been cooled and cooled, pass the second parallel line through the first scribe section, and then in the same way, pass the second parallel scribe line for the third parallel scribe and pass the third The sub-parallel lined portion makes a fourth parallel line. When marking, the inoculation needle should be at a 30° angle to the surface of the plate. Do not allow the inoculation needle to hit the edge of the dish and do not scratch the culture medium. After the marking is completed, the lid is covered and placed in a constant temperature box.
The purpose of the sterilizing and inoculating ring before the bacteria seeding is to destroy the microorganisms on the inoculation loop; in addition to the first marking, the purpose of burning the inoculation loop before the other markings is to eliminate the residual bacteria on the inoculation loop; Both before and after marking require the loop to be cooled and the purpose is to prevent high temperatures from killing the bacteria; the purpose of burning the loop is to prevent bacteria from contaminating the environment and the operator.
2. Coating separation method: first dilute the culture broth, usually diluted to 10-5 ~ 10-7, and then take 0.1ml diluted dilutions of different dilutions on the culture medium in a solid medium, using a glass squeegee The culture is applied on the plane of the culture and, at the appropriate dilution, colonies separated from each other can be generated. Usually, a single colony within 20 dishes per dish is most suitable. Each colony was inoculated on the slant, cultured, and then functional experiments were performed.
The separation method is simple and the method is simple. The colony separation method makes single colonies easier to separate, but the operation is more complicated. The two separation methods of bacteria have their own advantages and can be used.
V. Identification of colonies and species
Individual bacteria are invisible to the naked eye. However, when a single or a small number of bacteria multiply on a solid medium, they form a group of subcells visible to the naked eye and having a certain morphological structure called colonies. Colonies formed by different types of bacteria have certain characteristics in terms of size, shape, color, glossiness, transparency, and the like.
Bacterial colony surface is generally smooth and moist, viscous, most transparent or translucent, but there are bacterial colonies surface is dry, wrinkled; actinomycetes due to the fine hyphae and can produce spores, so the colony The surface is tightly velvety, solid and wrinkly. The long spores become powdery. Since the mycelium and spores have multiple pigments, they have different colors at the bottom of the colony medium and on the surface of the colony, and the colony is not easy to irritate with the inoculation ring. Yeast colonies are similar to bacterial colonies and have a smooth and moist surface that is viscous but mostly milky white and a few red.
If colonies cannot be identified, they can only be observed with the help of a microscope. Under the microscope, the bacteria are generally rod-shaped, spherical and arc-shaped, and their diameters are all around 1um. Actinomycetes mycelium is a branching filament without space, the apical mycelium splits into conidial spores, and the spores have straight lines. Shaped, spiral, curved or round, etc.; yeast has oval or filamentous, but oval cells larger than bacteria, diameter of about 5 ~ 7um.