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| 1.09 Inoculating Instruments Any of several different instruments may be used to transfer a microbial sample, the choice of which depends on the sample source, its destination, and any special requirements imposed by the specific protocol. Shown here are several examples of transfer instruments. From left to right: serological pipette, disposable transfer pipette, Pasteur pipette, inoculating loop, disposable inoculating loop/needle, cotton swab, and glass spreading rod. (Note: the glass rod is not an inoculation instrument, but it is used to spread an inoculum introduced to an agar plate by another instrument. As such, it is an instrument used in an inoculation process.) When transferring BSL-2 organisms, we advise using a sterile disposable loop or wooden stick (not pictured). Neither of these requires incineration after use and minimize the threat of aerosol production. |
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| 1.10 Label the Media To avoid consion after-the-fact, it is best to label sterile media prior to inoculating it. (A) Tubed media can be labeled with tape, paper, or directly on the glass with a marking pen. Labels must be removed when tubes are put in the autoclave bin for sterilization. (B) Plastic Petri dishes should be labeled on their base, not their lid, because the lid may get separated from its base during reading or rotated from its correct orientation. Because most labs use disposable Petri dishes, the labels do not have to be removed prior to autoclaving. |
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| 1.11 Microbiologist at Work Materials are neatly positioned and not in the way. To prevent spills, culture tubes are stored upright in a test tube rack. They are never laid on the table. The microbiologist is relaxed and ready to work. Notice he is holding the loop like a pencil, not gripping it like a dagger. |
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| 1.12 Hold the Loop Like a Pencil Holding the loop as shown puts the hand in a convenient position to hold tube caps by the "pinky" finger. |
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| 1.13 Bunsen Burner Flame When properly adjusted, a Bunsen burner produces a flame with two cones. Sterilization of inoculating instruments is done in the hottest part of the flame—the tip of the inner cone (red arrow). Heat-fixing bacterial smears on slides and incinerating the mouths of open glassware items may be done in the outer cone (white arrow). |
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| 1.14 Bacteriological Incinerator Bacterial incinerators use infrared heat and reach temperatures ober 800°C. A wire loop/needle is inserted into the incinerator and heated until the wire is red-hot (usually winin 5-7 seconds). (The handle may also get hot, so be careful.) The loop/needle is then removed and allowed to cool without touching anything. It may then be used to transfer microbes, or if this is done at the completion of a transfer, it may be set aside until needed again. |
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| 1.15 Procedural Diagram for an Aseptic Transfer from a Nutrient Broth Pure Culture to a Sterile Nutrient Broth Tube This is a summary of the procedure. Make every effor to keep your loop hand as still as possible throughout the transfer. Details can be found in the text. Make appropriate adjustments if transferring a BSL-2 organism. |
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| 1.16 Flaming Loop Incineration of an inoculating loop's wire is done by passing it through the tip of the flame's inner cone. Begin at the wire's base and continue to the end, making sure that all parts are heated to a uniform orange color. Allow the wire to cool before touching it or placing it on/in a culture. The former will burn you, the latter will cause aerosols of microorganisms. |
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| 1.17 Vortex Mixer Bacteria may be suspended in a broth using a vortex mixer. The switch on the bottom has three positions: "auto" (left), "off" (center), and "on" right. The rubber boot is activated when touched only if the "auto" position is used; "on" means the boot is constantly vibrating. Above the on/off/auto switch is a variable speed knob. The slowest speed that allows the vortex to reach the bottom of the tube is used. Caution must be used to prevent broth from getting into the cap or losing control of the tube and causing a spill (note the hand position around the tube, ready to grab it). Short bursts of vortexing can be used if the glassware is too full to allow vortexing to the bottom. |
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| 1.18 Mixing Broth by Hand A broth culture always should be mixed prior to transfer. Tapping the tube with your fingers gets the job done safely and without special equipment. |
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| 1.19 Removing the Tube Cap The loop is held in the dominant hand and the tube in the other hand. Remove th etube's cap with the little finger of your loop hand by pulling the tube away with the other hand; keep your loop hand still. Hold the cap in your little finger during the transfer. When replacing the cap, move the tube back to the cap to keep your loop hand still. The replaced cap doesn't have to be on firmly at this time—just enough to cover the tube. |
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| 1.20 Holding the Tube at an Angle The tube is held at an angle to minimize the chance that airborne microbes will drop into it. Notice that the tube's cap is held in the loop hand. |
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| 1.21 Flaming the Tube The tube's mouth is passed quickly through the flame a couple of times to sterilize the tube's lip and the surrounding air. Notice the tube's cap is held in the loop hand. |
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| 1.22 Move the Tube, Not the Loop The open tube is held at an angle to minimize airborne contamination of it. When placing a loop into a broth tube or removing it, keep the loop hand still and move the tube. Be careful not to catch the loop on the tube's lip when removing it. This produces aerosols that can be dangerous or produce contamination. |
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| 1.23 Do Not Catch the Loop on the Tube's Lip When Removing It Notice the film of broth in the loop (see inset). Be careful not to catch the loop on the lip of the tube when removing it. This would produce aerosols and droplets that can be dangerous or produce contamination. |
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| 1.24 Removing Excess Broth from Loop after Transferring Before removing it from the new culture tube, tap the face of the loop on the glass to remove the broth film. Failure to do so will result in splattering and aerosols when sterilizing the loop in a flame. |
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| 1.25 Procedural Diagram for an Aseptic Transfer from a Nutrient Agar Slant Pure Culture to a Sterile Nutrient Agar Slant This is a summary of the procedure. Make every effort to keep your loop hand as still as possible throughout the transfer. Details can be found in the text. Make appropriate adjustment if transferring a BSL-2 organism. |
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| 1.26 A Loop an an Agar Slant When placing a loop into a slant tube or removing it, the loop hand is kept still while the tube is moved. Hold the tube so the agar is facing upward. To pick up the inoculum, you only need to gently touch the growth on the agar surface. |
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| 1.27 Fishtail Inoculation of a Slant Begin at the base of the slant and gently move the loop back and forth as you withdraw the tube. Be careful not to cut the agar. After completing the transfer, sterilize the loop of dispose of it properly. |
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| 1.28 Procedural Diagram for an Aseptic Transfer form a Nutrient Agar Plate Pure Culture to a Sterile Tubed Medium This is a summary of the procedure. Make every effore to keep your loop hand as still as possible throughout the transfer. Details can be found in the text. Inoculation of a sterile broth is the same as in Figure 1.15, whereas inoculation of a sterile slant is the same as in Figure 1.25. Make appropriate adjustments in transferring a BSL-2 organism. |
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| 1.29 "Picking a Colony for Transfer Touch the tip of the loop to an isolated colony and get a small amount of growth. Use the lid as a shield from airborne contamination. |
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| 1.39 Spread Plate Set-up The spread plate technique requires a Bunsen burner, a screw-cap jar with alcohol, a glass spreading rod, and a plate. Position these components in your work area as shown: isopropyl alcohol, flame, and plate. This arrangement reduces the chance of accidentally catching the alcohol on fire. Notice the cotton in the jar's bottom to reduce the chance of breaking the glass rod. |
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| 1.40 Delivering the Inoculum Deposit the inoculum near one side of the agar surface. Use the lid as a shield and properly dispose of the pipette or pipette tip in a sharps or other biohazard container. |
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| 1.41 Flaming the Glass Rod Remove the glass spreading rod from the alcohol jar, tap off any excess alcohol, and then pass it through the flame away from the alcohol jar to ignite the alcohol on it. Allow the alcohol to burn off completely. Do not leave the rod in the flame; the combination of the alcohol and brief flaming are sufficient to sterilize it. Be careful not to drop any flaming alcohol on the work surface or back into the alcohol jar. If the alcohol catches on fire, smother the flame by replacing the cap on the jar. |
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| 1.42 Spreading the Inoculum After the flame has gone out on the rod, life the lid of the plate and use it as a shield from airborne contamination. Then, touch the rod to the agar surface away from the inoculum in order to cool it. To spread the inoculum, hold the plate lid with the base of your thumb and index finger, and use the tip of your thumb and middle finger to rotate the base. At the same time, move the rod in a back-and-forth motion across the agar surface. After a couple of turns, do one last turn with the rod next to the plate's edge. |
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| 3.121 Amphitrichous Flagella |
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| 3.122 Lophotrichous Flagella |
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| 3.123 Peritrichous Flagella of Proteus vulgaris |
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| 3.113 Central Elliptical Endospores Most are central, some are subterminal. |
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| 3.114 Terminal Elliptical Endospores Notice how the endospores have caused the ends of the cells to distend (swell). |
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| 3.115 Subterminal Elliptical Endospores Slightly distend the cell. |
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| 1.34A Beginning the Quadrant Streak Pattern Streak the mixed culture back and forth in one quadrant of the agar plate. Stay close to the plate's edge and make the streaks long. Do no cut the agar with the loop. Flame the loop, and then proceed. |
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| 1.34B Second Streak Rotate plate nearly 90° and touch the agar in an uninoculated region to cool the loop. Streak again, using the same wrist motion. Flame to loop afterward. (Note: In these illustrations, the plate is not rotated.) |
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| 1.34C Third Streak Rotate the plate nearly 90° and streak again, using the same wrist motion. Be sure to cool the loop prior to streaking and flame it afterward. |
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| 1.34D Fourth Streak Into the Center After cooling the loop, streak one last time into the center of the plate. Flame the loop, and incubate the plate in an inverted position for the assigned time at the appropriate temperature. |
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| 2.37A Uniform Fine Turbidity |
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