It is important during bacterial culture to avoid introducing additional foreign bacteria into the culture medium. Therefore, in addition to operations that must always be carried out in absolute aseptic conditions, all elements from the environment, containers, culture tools to necessary items must be disinfected before use. .
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Isolation is an important step in the study of bacterial culture.
The purpose of the isolation method is to isolate bacteria from the original population to form pure clones for investigation and identification. As bacteria grow and develop on the surface of solid media that have created colonies, the morphology of the colonies will be specific to each type of bacteria. Accurate characterization of isolated colonies can be of great help in bacteriological diagnosis. Microbiologists have standardized meaning when describing the shape, height, and margins of colonies.
It is important in the culture of bacteria to avoid introducing additional foreign microorganisms into the culture medium. Therefore, in addition to operations that must always be carried out in absolute aseptic conditions, all elements from the environment, containers, culture tools to necessary items must be disinfected before use. .
It is important to culture the bacteria to avoid introducing additional foreign microorganisms into the culture medium
2.1 Type of culture sample
The form of sample solution indicated for homogenized bacterial culture, culture solution or liquid medium containing the microorganism strain to be analyzed. Surface form of solid medium containing agar (from 1.5-2%) in inclined agar tubes or in petri dishes. Samples are deep in solid medium in test tubes Deep agar contains soft agar (0.5-0.7%).
Straight inoculation rod: Metallic rod with pointed tip, used for inoculating bacteria with bacteriocytosis. Hook inoculation rod: This is an inoculating rod with a perpendicular tip, used for inoculating bacteria with bacteriophage. Ring culture rod: Inoculation rod with metal ring tip, used to inoculate solids or liquids from solid or liquid media. Inoculation rod: A metal or glass rod, triangular tip, used to spread bacteria on Surface of solid agar. Glass straw: Used to transfer the required amount of bacteria to the surface of solid medium or into liquid medium. Sterile swab tip: Used to inoculate the culture from liquid medium to the surface of the lip. solid field.
3. Methods of isolating and culturing bacteria
3.1. Technique of growing whiskers on petri dishes
Dip a sterile loop into the sample solution to obtain the bacteria you want to isolate. Line the lines on a petri dish containing the appropriate agar. After each continuous stroke, sterilize the inocula and cool before proceeding. Turn the plate upside down and incubate at the appropriate temperature and time in the incubator.
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3.2. Page planting technique
Pipette 0.1 ml of the broth to the surface of the agar in a petri dish. Dip the tip of the wiper in alcohol and heat it over the flame to disinfect. Then, let the wiper tip cool in the sterile space of the flame. Open the petri dish, gently rub the wiper over the agar surface of the petri dish. Use the tip of the wiper to spread the bacterial solution evenly on the surface of the agar. To do this, rotate the plate a few times, about half the circumference of the plate each time to allow the wiper to spread the bacterial solution evenly over the surface of the medium. Turn the plate upside down and incubate at an appropriate temperature and time for thermostatic cabinet.
3.3. Inoculation from liquid medium to test tubes containing liquid medium
Heat the tip of the inoculum in a flame, then gently heat the handle, then hold the inoculating rod upright for even heating. The left hand holds the test tube and rotates slightly, the right hand holds the inoculating rod. Use the little finger of your right hand to open the cotton button. After opening the cotton stopper, rotate the mouth of the test tube over the flame. Insert the sterilized inoculum into the test tube and cool the inoculum (press the tip of the tube against the wall of the tube to cool). Immerse the rod in the liquid medium, pull the rod straight out to avoid sticking to the wall and mouth of the tube to collect biomass. Heat the mouth of the test tube and cover with a cotton stopper and place the test tube in the holder. The tip of the culture rod is kept in a sterile area of air near the lamp. Use your left hand to take the test tube containing the new medium, then open the cotton stopper and disinfect the mouth of the test tube. Next, insert the tip of the probe into the medium.Immerse and gently stir the rod in the medium to separate the biomass from the tip.Pull the tip of the rod straight out. Sterilize the mouth of the test tube and cover with a cotton stopper. Disinfect the implant immediately after inoculation.
3.4. Inoculation from liquid medium to inclined agar tubes
This method does the same thing as above, but with some differences:
Inoculate the culture on the inclined agar surface by gently placing the tip of the rod on the surface of the medium at the bottom of the tube. Then, inoculate in a zigzag pattern from the bottom of the test tube up to the tip on the slanted agar.
3.5. Inoculate from liquid medium with a removable pipette
Removable end pipettes allow precise handling of small capacity. In aseptic handling, the removable end pipette is very useful because it allows the easy transfer of bacterial aliquots onto the surface of solid media in a petri dish to form loose colonies or into a test tube or container of liquid medium for growth. born. Before use, the operator needs to know the basic requirements for growing bacteria with a removable pipette as follows:
Each pipette has a certain allowable working capacity limit: 0.1 ml, 1 – 20 ml, 20 – 20 ml, 0.2 – 1 ml, 1-5 ml, 1 – 10 ml. The removable end pipette should be selected with the appropriate capacity limit for the range of operation. The removable end pipette has two steps: Step 1 is equivalent to the volume selected to be used when aspirating the solution; Step 2 (beyond step 1) is used to pump the solution out of the tip of the removable end pipette. When using the removable end pipette to inoculate the inoculum, it should be carried out in the sterile flame space of the incubator. The right hand holds the removable tip pipette, the left hand opens the sterile tip container. Then, insert the pipette tip into the tip. Use your left hand to hold the test tube and the vial containing the microbiological fluid. Use the little finger, ring finger of the right hand holding the pipette to clamp and open the cotton stopper, then heat and disinfect the mouth of the test tube or container. Insert the sterile tip into the culture solution and aspirate the required volume. . Then, remove the tip from the mouth of the container, disinfect the mouth of the container and cover with the cotton stopper being held on the little finger and ring finger of the right hand. Tip containing microorganisms should be kept in a sterile area of air. near the lamp.Use your left hand to take a test tube or a container of new medium, use your pinky and ring fingers to clamp and open the cotton stopper, disinfect the mouth of the container.Insert the tip into the medium and then inject the inoculum into the medium. . Then, remove the tip from the mouth of the container, disinfect the mouth of the bottle and cover with a cotton stopper. The removable end pipette and the tip made of polymer require absolute sterilization of the pipette tip and tip by flame.
To ensure the growth of bacteria, after culturing the bacteria, attention must be paid to the conditions of the bacterial culture such as: Optimal temperature for the growth of each bacterial species and maintaining thermal stability. degree; humidity during incubation need to ensure enough water; Oxygen gas for aerobic microorganisms, the layer of culture medium has a moderate thickness so that atmospheric oxygen can penetrate.
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Topic: Mustache culture technique on petri dishes Microbiological diagnosis How to culture bacteria Indications for bacterial culture Bacterial culture