Aseptic - “an setting or procedure the is cost-free of pollution by pathogens”

Bauman, Robert W. Microbiology mountain Francisco: Pearson education and learning Inc., 2004.

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Introduction

In the microbiology laboratory we usage aseptic an approach to:

Prevent contamination of the specific pathogen we space working with.Prevent pollution of the room and personnel with the pathogen we are working with.

Many of the microorganisms we will be working with in rap are well-known pathogens. Proper and also appropriate aseptic technique is vitally necessary for the safety of all lab personnel; the is also essential for the successful completion of the lab portion of this class. The an abilities and awareness you develop practicing aseptic technique will lug over to your career together a wellness professional. Plenty of of our previous students comment that this is the most essential thing lock learned in lab!

In this laboratory you will be finding out standard microbiological procedures proper for Biosafety Level (BSL) 1 and Biosafety Level (BSL) 2 precautions. We follow the security guidelines established by the facility for condition Control and Prevention (CDC). Complete documentation is accessible at the CDC website.

Part i - measures for exercise "Organisms"MaterialsUsing a Bunsen BurnerBroth to Broth Transfers utilizing BSL2 ProceduresBroth to Plate Transfers making use of BSL2 ProceduresPart II - procedures for BSL2 OrganismsMaterialsPlate come Plate Transfers making use of BSL2 ProceduresPlate to Broth Transfers utilizing BSL2 ProceduresDisposal Instructions

Overview

Aseptic technique involves emerging both manual dexterity in safely managing the microorganisms and mental dexterity in thinking ahead around what you are doing with the microorganism. In this laboratory you will certainly learn just how to:

You need to have your workspace fine organized. Rap bench an are is very limited. You need to have all your materials close to you. Do not stretch over the table for what friend need. Each of you have your own loop and tubes, but you will be share a Bunsen burner and other rap equipment.

Transferring Organisms

One of her main pertains to when working through microorganisms is come avoid producing aerosols the you deserve to breathe in and droplets that have the right to land on you, your lab partners, and also your rap equipment. Friend will spend a lot of time in lab delivering organisms from one tube to another, or to slides or to plates. The is imperative that you carry out this quickly and also safely. The longer your organism is exposed to the air, the an ext opportunities there are for the to obtain contaminated and/or to contaminate you, your lab partner or her equipment.

Part ns - steps for practice "Organisms"

After you have actually practiced these procedures several times your instructor or IA will certainly assess your proficiency. The is essential that you understand these skills before you proceed to working through actual microorganisms.

MaterialsBunsen burner and also strikerIncineratorInoculating loop1 selective media plate1 tube food dye colored water1 pipe sterile water1 marker pen or grease pencilUsing a Bunsen Burner

If you can not seem to acquire your burner lit, ask her instructor or IA if the main gas regulate is on.

Firmly affix the water tap to the tapered gas line. The burner has two adjustments. The knob under adjusts the quantity of gas going into the burner tube. The barrel of the burner transforms to adjust the amount of waiting going right into the burner. Before lighting a Bunsen burner, set the air and gas adjustments come a minimal open position. Revolve the air adjustment clockwise to decrease the waiting (a much more purple flame) and also counterclockwise to boost the wait (a an ext yellow flame). (Refer to figure 1.)
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Figure 1: Bunsen Burner


Look at the underneath of the striker. Over there is a roughened rod versus which a small flint rubs once you push the handle. If you are pushing firmly and also do not gain a spark, you probably need a new flint. Revolve the gas top top partway when originally lighting your burner.Hold the cup part of the striker in ~ an edge slightly above the opening of the burner and push the manage to generate a spark. You may need come repeat this several times prior to your burner lights. Alternatively, there may be butane lighters obtainable in lab come light her Bunsen burner.
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Figure 2: an excellent Flame


Modify the air and also gas adjustments till you acquire a hissing, small, blue fire within a taller lighter blue/violet flame. (Refer to number 2.) If your flame is just one violet flame you either have actually too lot air or gas. If your flame is yellow and blows in the waiting currents in the room, over there is too little air in your flame. (Refer to number 3.) Fiddle approximately with both of these adjustments until you acquire the appropriate flame. Over there are numerous other classes utilizing the exact same equipment. You will most most likely never have actually your burner the method you left it!
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Figure 3: bad Flame


The reminder of the tiny inside fire is the hottest component of the fire (1560°C). This is the area wherein you will desire to run the optimal of your tubes with to keep sterility.Never leaving a lit burner unattended!Using one Incinerator

Special precautions have to be bring away to avoid the formation of aerosols when working through BSL2 organisms. Rather of using a Bunsen burner to “flame” loops and other inoculating utensils, BSL2 measures require the use of one incinerator. An incinerator sterilizes inoculating utensils lot the same means as a Bunsen burner does other than the risk of aerosol production is reduced.


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Figure 4: Incinerator


Broth to Broth Transfers making use of BSL2 Procedures

If you are using screw cap tubes, be certain to ease them before you start this procedure.

Usually once you room working v a broth culture, the organisms will certainly have settled to the bottom.You need to very first re-suspend castle in the broth.Do not invert the tube! many of the tubes us use have actually slip caps with a space at the height to enable air right into the tubes. If friend invert the tube you will certainly pour bacteria every over. If you invert a screw lid tube, the liquid currently in the inside of the lid will certainly drip all over when you remove the lid.Hold the tube near the top and also flick the bottom the the tube with your other hand. Continue flicking until the organisms room re-suspended.Place both her colored practice tube and also your sterile water pipe in her left hand with the lids pointing up.Hold the tube closer come the bottom so that your hand will not it is in close come the flame when you sterilize the mouth the the tubes.Grab the inoculating loop far back on the take care of as if you to be going to write v it.Place her loop in the mouth the the incinerator briefly because that 2-4 secs to sterilize it.Do not leave her loop in the incinerator for more than 10 seconds, you will ruin the loop!Wait around 10 seconds for your loop to cool.While quiet holding the inoculating loop, usage the lower component of your hand to grab both of the slip caps and also pull them off. Save them tucked in her hand.Yes, this is together tricky as it sounds.Sterilize the mouth that the tube by happen them with the flame 2-3 times.This death organisms top top the opening of the tubes.Hold the tubes slightly tipped to minimization microorganisms in the waiting from falling right into the open up tubes.Insert the loop into the colored tube without touching the sides of the tube.As you pull it back out friend will notification a film across the loop, just like when you punch soap bubbles.Carefully withdraw the inoculating loop without emotional the sides of the tube and insert it into the sterile water tube without touching the political parties of the tube.Watch your droplet if you space transferring to see if the is still in the loop. If you are successful, you must see some color in her water tube. You have actually now transferred your organism to a new tube.Shaky hand or air currents have the right to make the drop autumn off ~ above the bench top, you, or your neighbor. It is in careful!When you retract your inoculating loop indigenous the second, freshly inoculated tube, be certain to touch it against the within of the pipe to knock off excess fluid.Flame the mouth of her tubes and replace the caps.Do not miss your caps and push the mouths that the warm tubes right into the palm of your hand.Place your loop in the mouth of the incinerator briefly because that 2-4 secs to sterilize it.Do not leave your loop in the incinerator for much more than 10 seconds, you will destroy the loop!Never lay her loop or various other inoculating tools on the respond to without an initial sterilizing it.Have your instructor observe and also record her technique.Broth to Plate Transfers utilizing BSL2 Procedures

This is a tiny easier because you only need to hold one tube in her hand.

Be certain you have actually your practice plate ~ above the rap bench prepared to use.Label the bottom through the date, the organism, your initials and lab section number.Write little and follow me the edges. Why?Follow the exact same procedure as above (steps 2-7) to get your sample indigenous the colored “broth” tube.While stop the loop v the practice "organism" (colored broth), fire the mouth that the tube and replace the lid. Return the pipe to the rack.Hopefully her loop still has its contents, if not wherein did the go?Open the lid of her dish through your left hand and also hold that ajar. Touch the surface of the loop come the agar surface. (Refer to figure 5.)Lightly traction your loop earlier and forth across the surface ar of the agar being cautious not to gouge the surface. Think that agar together really for sure Jell-O.The much more your drag the much more bacteria friend deposit. The basic idea is come decrease the bacter concentration with each swipe. 4 to 5 zigzags appears to occupational well. Experiment v your various plates. Be sure to keep track the what friend did on every plate.Sterilize your loop. Do not go earlier into the original broth tube.Wait about 10 secs for your loop to cool.Touch the agar surface against the far finish of your first streak.Repeat by dragging back and forth. Do not drag right into the facility of your plate. Friend should be able to see the pass out indentations of her streaking line on the agar surface.Sterilize her loop and also cool. Repeat the procedure on your 2nd streak.Sterilize her loop and cool. Repeat the procedure top top your third streak. Zigzag the last part into the center of the plate. You should finish up through isolated colonies somewhere in her last streak.This procedure is dubbed “streaking because that isolation”Replace the lid on your plate.The longer the plate is open up to the room air, the higher your chance of contamination.Sterilize your loop in the incinerator.

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Never lay her loop or other inoculating tools on the respond to without very first sterilizing it.Have your instructor observe and also record your technique.