Bacteriological inoculating loops and needles
Plastic inoculation loops are only designed for single, one-time use, and discarded in the biological waste bin afterwards. Metal loops are designed to be sterilized in the Bact-Cinerator between use, and may be used over and over again and sterilized countless times for reuse.
4 quadrant streak plate method for pure colony isolation
streak plate method of isolation:
As microbiologists, we streak plates for bacterial or fungal isolation. This is performed on a wide variety of different media. The goal is to obtain pure cultures for further testing. Individual microbial species in a mixed culture need to be isolated and cultivated as pure cultures before correct identification and further testing can be carried out. The most common and least expensive means of isolating organisms in a mixed culture is to use the streak plate method.
The streak plate method of isolation means to spread the microbes on plated agar media so that the individual cells or colony forming units (CFUs) can become isolated and grow into individual, pure colonies. The method most often used in the clinical laboratory is the quadrant method. This uses either the three-streak or four-streak methods shown above. The four-streak method is more common. It involves four individual streaks The best results are obtained when the loop is sterilized or flamed between streaks when using a stainless steel inoculating loop. The plate is turned between streaks. The first quadrant is a tight streak, and the most growth is here. Density decreases in the four-streak pattern. Isolation is first obtained in the third or fourth-streak most of the time. This is where the purest, youngest bacteria are found. Cells from the individual, pure colonies may be transferred to sterile media to start pure cultures. If it is a subculture, test the individual colonies in the last quadrant.
Streaking the agar plate for isolation is performed on selective media so that certain types of microbes are encouraged to grow, while others are inhibited. Some selective media contain color indicators or pH indicators to show the differences between organisms. These media types are both selective and differential because they "differentiate" between organisms.
The streak plate method of isolation means to spread the microbes on plated agar media so that the individual cells or colony forming units (CFUs) can become isolated and grow into individual, pure colonies. The method most often used in the clinical laboratory is the quadrant method. This uses either the three-streak or four-streak methods shown above. The four-streak method is more common. It involves four individual streaks The best results are obtained when the loop is sterilized or flamed between streaks when using a stainless steel inoculating loop. The plate is turned between streaks. The first quadrant is a tight streak, and the most growth is here. Density decreases in the four-streak pattern. Isolation is first obtained in the third or fourth-streak most of the time. This is where the purest, youngest bacteria are found. Cells from the individual, pure colonies may be transferred to sterile media to start pure cultures. If it is a subculture, test the individual colonies in the last quadrant.
Streaking the agar plate for isolation is performed on selective media so that certain types of microbes are encouraged to grow, while others are inhibited. Some selective media contain color indicators or pH indicators to show the differences between organisms. These media types are both selective and differential because they "differentiate" between organisms.
aseptic technique
The use of aseptic technique is so that contamination is reduced or eliminated when plating organisms. It insures that microbes are being handled in a safe manner to prevent contamination of the handler or others in the laboratory. And it means cleaning up after yourself so that no contamination remains after you have worked with bacteria or other microbes. It means being very careful with how you handle cultures, bacteria, and loops, treating the loops as sterile, and being careful not to touch anything with your loop prior to inoculation of the media, whether it be transferring a broth culture to a plate for streaking, or inoculating an isolated colony from a plate to another plate for purity and/or isolation. It may also mean inoculating many tubes of media and agar plates from a stock culture to identify a bacterium. It is crucial that you make sure that only your desired organism is successfully transferred during the inoculation process for successful and accurate identification.
Flaming the loop means to sterilize your loop or inoculating needle until is becomes bright red. The entire wire must be heated, but be careful not to insert the handle inside the incinerator or it might melt.
A pure culture is desired in order to isolate bacteria for identification and susceptibility studies. Methods for obtaining pure cultures are available by using aseptic technique and by using the streak plate method in order to dilute the bacterial cells in a sample to an end point where a single cell divides giving rise to a pure colony. One colony is referred to as a Colony Forming Unit (CFU). The colony is the identical progeny of the original cell and can be picked up and used for further study. The most isolated colony should be used when subculturing because any genetic mutations, such as antibiotic resistance, will have been gained by the most isolated colony (colonies).
Flaming the loop means to sterilize your loop or inoculating needle until is becomes bright red. The entire wire must be heated, but be careful not to insert the handle inside the incinerator or it might melt.
A pure culture is desired in order to isolate bacteria for identification and susceptibility studies. Methods for obtaining pure cultures are available by using aseptic technique and by using the streak plate method in order to dilute the bacterial cells in a sample to an end point where a single cell divides giving rise to a pure colony. One colony is referred to as a Colony Forming Unit (CFU). The colony is the identical progeny of the original cell and can be picked up and used for further study. The most isolated colony should be used when subculturing because any genetic mutations, such as antibiotic resistance, will have been gained by the most isolated colony (colonies).
- Disinfect your work area to kill any microbes that may be present. This process destroys vegetative cells and any viruses, but it may not destroy endospores. This is what the autoclave process is for. Cavicide wipes are used to wipe off countertop surfaces, along with bleach and 70% alcohol.
- Loops and needles need to be sterilized before transferring any culture when using metal loops or inoculating needles to burn and destroy any contaminating organisms that might be present. If using plastic loops, ensure that they have not touched any surfaces or your lab coat prior to inoculation, and that the bottom of the bag or case that the loops are in is properly sealed. If you accidentally drop a loop prior to inoculation, throw it away in the biological waste bin and do not use it to inoculate your media. Take out a new loop.
- Prior to inserting a cooled and sterilized loop or needle into a culture tube, once the cap is removed, flame the mouth of the tube. Inoculate your tube, reflame the moth, and recap it. Most tube media needs to have loosened caps prior to incubation.
- After the inoculation is complete, you need to resterilize your loop or needle to destroy the organisms you just inoculated into your media. Return the loop or needle to its storage place and never place it down on the countertop surface.
- Loops are used to inoculate or streak petri dish plates. The plate cover is raised and held at an angle over the plate to protect the surface from any contamination in the air, such as dust and bacterial particles. Never talk on the plates, as spit and organisms from your mouth may contaminate the cultures. Take care not to gouge or disturb the surface of the agar with the loop.
- When all work for the day is complete, treat your work area with disinfectant and empty the biological waste bins and autoclave any bags and sharps containers that are full and ready for autoclaving.
urine cultures and some sterile cultures: semiquantitative method
The semiquantitative method is a quick and easy way to semiquantitate the concentration of bacteria in a urine or sterile body fluid sample. Streak a known volume of urine on an agar plate, incubate, and perform a colony count. This is accomplished by using a volumetric inoculating loop calibrated to hold a specific volume of urine, preferably 0.001 mL or 0.01 mL. Carefully transfer a loopful to the agar plate by making a single streak down the plate. Turn the plate 90 degrees and streak across the original line to evenly disperse the bacteria across the agar. Incubate for 18-24 hours and check at regular intervals. Record your counts in CFU/mL.
OCD = CFU/loop volume
OCD = 150 CFU/0.001 mL
OCD = 1.5 x 10 to the fifth power CFU/mL
(1 colony = about 1,000 cells)
Approximately 150,000 CFU/mL
OCD = CFU/loop volume
OCD = 150 CFU/0.001 mL
OCD = 1.5 x 10 to the fifth power CFU/mL
(1 colony = about 1,000 cells)
Approximately 150,000 CFU/mL