In order to correctly treat an infection or identify a contaminant microbiologists must first isolate the target microbe from a sample or specimen that often contains other microorganisms. Streaking agar plates is an essential technique used in the laboratory to isolate pure individual colonies from a larger sample source. Before beginning it is important to remember individual microbial cells are not visible to the naked eye. Avoid the tendency to over-inoculate the plate a common mistake that will cause the plate to be overloaded with colonies and result in a confluence of growth. This is particularly critical for certain specimen types such as stool samples where isolation of single colonies is extremely important. Also agar is similar in consistency and firmness to jello, and can be prone to breaking the gel matrix. The goal when inoculating plates is to streak the surface gently and not to cut, tear, or stab the gel matrix with the inoculating loop. To prepare ensure the bench top is clean, disinfected, and organized. Make sure the tools such as loops and labeling pens are within easy reach. Laboratory personnel should use good hand hygiene, aseptic technique, and wear appropriate clothing such as a lab coat and gloves. Ensure clothing is tight fitting near open flames or sterilizing equipment such as a Bunsen burner or incinerator. Allow the plate to reach room temperature prior to inoculation to avoid temperature shock to cells which may affect recovery. Always label plates on the bottom of the dish prior to use. This is essential to identify the sample after incubation and to ensure the plate can be identified in the event the lid is lost, dropped, or accidentally switched to another plate. The purpose of the streak plate technique is to reduce the number of cells from an area of high concentration from the original sample and where the streak begins to an area of low concentration in the final streak. This is accomplished by using a series of loop passes over the agar called the four quadrant method. By the final streak the number of cells on the loop should be diluted and low enough that single cells are dispersed and isolated on the agar surface. After incubation these cells will grow into individual pure isolated colonies. Thus one viable cell will give rise to one single colony-forming unit known as a CFU. To streak plates three types of tools can be used a non-sterile metal loop which can be repeatedly sterilized by heat between uses, a pre-sterilized disposable plastic loop, or a sterile specimen swab. These loops come in different sizes and provide a calibrated volume making it easier for the microbiologist to determine the number of cells per milliliter in a liquid sample. Loops are generally calibrated to deliver either 10 microliters or 1 microliter. Depending upon the testing site or sample either a standard lab bench or biological safety cabinet which protects the microbiologist may be used. To sterilize a metal loop begin by flaming or incinerating the loop. Once it grows red hot and is sterilized allow the loop to cool in ambient air. Do not blow on or wave the loop to speed up the cooling process and do not touch the loop to any contaminated surface prior to sampling. Cool the hot loop by touching it to a sterile area of the Petri plate. Depending upon the sample type use the loop to collect a small number of cells from confluent growth, obtain a loop full of liquid sample, or touch the edge of a single colony from a source plate. Remove the plate lid and place it face down on the lab bench. Pick up the plate in one hand and the metal loop in the other hand. Streak the sample using a sweeping motion from side to side across quadrant 1 approximately a quarter of the agar surface. To prevent contamination avoid streaking too close to the perimeter of the agar the outside perimeter is where airborne contaminants are most likely to be located. Re-sterilize the loop and remember to let the loop cool before continuing to the next quadrant. You can quickly do this by stabbing the loop into the agar. Be sure to avoid the streak lines. Rotate the plate 90 degrees using a similar sweeping motion, streak the loop into the quadrant 1 streak lines two to three times, and then proceed to streak the remainder throughout quadrant 2 using about a quarter of the agar surface. If you used a specimen swab for quadrant 1 now switch to the metal loop for quadrants 2, 3, and 4. Only overlap quadrant 1 two or three passes and no more. The idea is to continue to reduce the number of cells spread across each quadrant so don't over saturate the loop. Re-sterilize and cool the loop in the agar before continuing. Rotate the plate another 90 degrees and spread the cells from quadrant 2 into quadrant 3 using a similar reduction strategy and a quarter of the agar surface. Re-sterilize and cool the loop one last time before continuing. Turn the plate a final 90 degrees and streak the cells from quadrant 3 into quadrant 4. This time sweep only once from quadrant 3 into quadrant 4 and continue the streak over the final quarter of the agar surface to disperse the cells to achieve isolated colonies. Once completed place the lid back on the plate and place the plate in the incubator. The plate should be placed media side up that is the media on the top and the lid on the bottom to prevent contamination from water condensation. An alternative to a metal loop is a plastic loop these loops are pre-sterilized and disposable. They are a good alternative if it's not feasible for a Bunsen burner or incinerating device at the plating location. Aseptically remove a sterile loop and touch a single colony from the source plate. Never lay the loop down on the bench top or touch it to a non-sterile surface. Proceed to streak the plate to the four quadrants. Use the same technique as the metal loop only instead of heating the loop between streaks just flip the plastic loop over to the clean side of the loop between streaking quadrants 1 and 2. Thank you for watching our four quadrant streak demonstration. Hardy Diagnostics is your complete microbiology supplier. Check out our full lineup of inoculating loops by clicking the link below. You can also see our complete catalog in the link below. We are 100% employee owned and have been serving microbiologists for over 40 years. To learn more about Hardy Diagnostics and the products we offer visit us at HardyDiagnostics.com