✦When reading volumes, use the base of the meniscus. The volume in the center pipette is read at exactly 3.0 mL because the meniscus is rest- ing on the line. The left pipette is read as 2.9 mL and the right pipette is read as 3.1 mL (0 is always at the top of the pipette).
C-5 GETTING THESTERILEPIPETTE✦Pipettes of the same size are autoclaved in canisters, which then are opened and placed flat on the table. Pipettes are removed as needed.
10 What you do next depends on the medium to which you are transferring the liquid. Please continue with the appropriate inoculation section.
Inoculation of Broth Tubes with a Pipette
Pipettes are often used to inoculate a known volume of culture into a known volume of diluent during serial dilutions.
1 While keeping the pipette hand still, bring the broth culture tube toward it. Use your little finger to re- move and hold its cap.
2 Flame the tube by quickly passing it through the Bunsen burner flame two or three times. Keep your pipette hand still.
3 Hold the open tube on an angle to minimize air- borne contamination. Keep your pipette hand still.
4 Insert the pipette tip and dispense the correct volume of inoculum.
5 Withdraw the tube from over the pipette. Before completely removing it, touch the pipette tip to the glass to remove any excess broth.
6 Completely remove the pipette, but avoid waving it around. This can create aerosols.
7 Flame the tube lip as before. Keep your pipette hand still.
8 Keeping the pipette hand still, move the tube to replace its cap.
9 The pipette is contaminated with microbes and must be disposed of correctly. Each lab has its own specific procedures, and your instructor will advise you what to do. Glass pipettes typically are placed in a pipette disposal container containing a small amount of disinfectant until they are autoclaved and reused (Figure C-8). Disposable pipettes must be placed in an appropriate biohazard container. In either case, be careful when removing the pipette from the me- chanical pipettor. There is danger of culture dripping from the pipette or of breaking the glass or plastic.
C-6 ASSEMBLING THEPIPETTE✦Carefully inser t the pipette into a mechanical pipettor. Notice that the pipette is held near the end with the finger tips. For safety, the hand is out of the way in case the pipette breaks.
C-7 FILLING THEPIPETTE✦Carefully draw the fluid into the pipette. Briefly bring it to a ver tical position and read the volume. Notice how the tube’s cap is being held by the pinky of the pipettor hand.
Inoculation of Agar Plates with a Pipette
Pipettes also can be used to dispense a known volume of inoculum to an agar plate.
1 Lift the lid of the plate and use it as a shield to protect it from airborne contamination.
2 Hold the pipette over the agar and dispense the correct volume (often 0.1 mL) onto the center of the agar surface (Figure C-9). From this point, the remainder of steps should be completed within about 15 seconds to prevent the inoculum from soaking into the agar.
3 The pipette is contaminated with microbes and must be disposed of correctly. Each lab has its own specific procedures, and your instructor will advise you what to do. Glass pipettes typically are placed in a pipette disposal container containing a small amount of disinfectant until they are autoclaved and reused (Figure C-8). Disposable pipettes must be placed in an appropriate biohazard container. In either case, be careful when removing the pipette from the mechanical pipettor. There is danger of culture dripping from the pipette or of breaking the glass or plastic.
4 Continue with the spread plate technique (Exercise 1-5).
C-9 INOCULATING THEPLATE WITH APIPETTE✦Open the lid and dispense the inoculum onto the sur face of the agar near the middle. Notice how the plate’s lid is held to prevent aerial contamination.
C-8 DISPOSE OF THE
PIPETTE✦The pipette may be contaminated with microbes and must be disposed of correctly. Each lab has its own specific procedures, and your instructor will advise you what to do. Shown here is a canister used for glass pipettes. Disposable pipettes must be placed in an appropriate biohazard container. In either case, be careful when removing the pipette from the me- chanical pipettor. There is danger of culture dripping from the pipette or of breaking the glass or plastic.
Modern molecular biology procedures often involve transferring ex tremely small volumes of liquid with great precision and accuracy. This has led to the development of digital pipettors (Figure D-1) that can be set up to dispense microliter volumes through milliliter volumes (recall that 1 mL equals 1000 àL). Common digital pipettors are calibrated to dispense volumes of 1 to 10 àL, 10 to 100 àL, 100 to 1000 àL, or 1 to 5 mL.
Filling a Digital Pipettor
Many manufacturers make digital pipettors, but they all work basically the same way. The following instructions are for Eppendorf Series 2100 models.
1 Growth may be suspended in the broth with a vortex mixer (Figure 1-15) or by agitating with your fingers (Figure 1-16). Be careful not to splash the broth into the cap or lose control of the tube.
2 Determine which digital pipettor should be used to dispense the desired volume.
3 Turn the setting ring to set the desired volume (Figure D-2). If you turn the dial past the volume range of the pipettor, you will damage it.
4 Hold the digital pipettor in your dominant hand.
5 Open the rack of appropriate pipette tips for your pipettor (these are often color-coded and match the pipettor) and push the pipettor into a sterile tip (Figure D-3). Close the rack.
Never touch the pipette tip with your hands or leave the rack open. Never use a digital pipettor without a tip.
6 Remove the cap from the culture tube with the little finger of your dominant hand, and flame the tube.
7 Press down the control button with your thumb to the first stop. This is the measuring stroke.
Transfer from a Broth Culture Using a Digital Pipette
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D-1 DIGITALPIPETTOR✦This is a digital pipettor to be used for dispensing volumes betwen 10 àL and 100 àL. Never set the pipettor to volumes above or below the intended volumes as damage may occur. Also, always use a digital pipettor with the appropriate tip. As shown here, tips and pipettors are often color-coded.
8 Insert the tip into the broth approximately 3 mm while holding it vertically (Figure D-4).
9 Slowly release pressure with your thumb to draw fluid into the tip. Be careful not to pull any air into the tip.
10 Remove the pipettor and hold it still as you flame the tube as before.
11 Keeping the pipettor hand still, move the tube to replace its cap.
12 What you do next depends on the medium to which you are transferring the growth. Please continue with the appropriate inoculation section.
Inoculation of Broth Tubes with a Digital Pipettor
1 Remove the cap of the sterile medium with the little finger of your pipettor hand, and hold it there.
2 Flame the tube by quickly passing it through the Bunsen burner flame a couple of times. Keep your pipettor hand still.
3 Insert the pipette tip into the tube. Hold it at an angle against the inside of the glass (Figure D-5).
4 Depress the control button slowly with your thumb to the first stop and pause until no more liquid is dispensed. Then continue pressing to the second stop to deliver the remaining volume. This is the blow-out stroke.
5 While keeping pressure on the control button, care- fully remove the pipettor from the tube by sliding it along the glass. Once it is out of the tube, slowly release pressure on the control button.
D-3 DIGITALPIPETTORTIPS✦Digital pipettors must be fitted with a sterile tip of appropriate size (often color-coded to match the pipettor). Open the case and press the pipettor into a tip, then close the case to maintain sterility. Do not touch the pipettor tip.
D-4 FILLING THEPIPETTE✦Depress the control button with your thumb to the first stop (measuring stroke). Holding the tube and pipettor in a ver tical position, inser t the tip into the fluid to a depth of 3 mm (see inset). Slowly release pressure with your thumb to fill the pipettor.
Control button
D-2 SETTING THEVOLUME✦Rotate the adjustment knob to set the volume on a digital pipettor. This pipettor has been set at 90.0 àL (see inset—the horizontal line between the third and four th numerals is a decimal point). Never rotate the adjustment knob beyond the volume limits of the pipettor.
Setting ring
6 Flame the tube lip as before. Keep your pipette hand still.
7 Keeping the pipette hand still, move the tube to replace its cap.
8 The pipettor tip is contaminated with microbes and must be disposed of correctly. Use the ejector button to remove the tip into an appropriate biohazard container (Figure D-6). Each lab has its own specific pro cedures, and your instructor will advise you what to do.
Inoculation of Agar Plates with a Pipettor
1 Lift the lid of the plate and use it as a shield to protect from airborne contamination.
2 Place the pipette tip over the agar surface. Be sure to hold the pipettor in a vertical position (Figure D-7).
3 Depress the control button slowly with your thumb to the first stop, and pause until no more liquid is dispensed. Then continue pressing to the second stop to deliver the appropriate volume. This is the
D-5 DISPENSING TO ALIQUID✦Hold the pipettor at an angle with the tip against the glass. Press the control button to the first stop. When no more fluid comes out, continue pressing to the second stop. Remove the pipettor, then slowly re lease pressure on the control button, and eject the tip into a biohazard container. Finally, replace the cap on the tube.