Lab8 Pipette Exercise 2021 FOR BSCI223 PDF

Preview

Summary

Lab description for pipette exercise used in bsci223 course...

Description

1

Lab 8: Use of a Micropipette INTRODUCTION Pipettes Microbiology and molecular genetics protocols often require accurately dispensing very small amounts of culture or reagents. Pipettes are the tool of choice for aseptic measuring and transferring of sterile liquids and bacterial cultures. Understanding how to use micropipettes is crucial for success in any modern biology lab. Using pipettes requires care to ensure accuracy of measurements and sterility. Micropipettes are used with tips, which are disposable portions of the pipette that can be sterilized. Micropipettes and appropriate tips are specifically calibrated for dispensing particular volumes. P200 micropipettes use yellow tips and dispense 20-200 µl. P1000 micropipettes use blue tips and dispense 200-1000 µl. Review the following conversions. 1 l = 1000 ml = 106 µl 0.001 l = 1 ml = 1000 µl 10-6 l = 0.001ml = 1 µl As a convention, any number with more than three digits is expressed using scientific notation. In this class, any number bigger than 1000 or smaller than 0.001 should be expressed using scientific notation. Each pipette has a window that tells you the volume it is set to. However, these boxes represent different numbers places, so even if the windows on a P200 and P1000 read the same number, it is NOT the same volume. A. P200 0 Hundred s 9 Tens 0 Ones

B. P1000 0 Thousand s 9 Hundreds 0 Tens

Figure 1. The window on (A) P200 and (B) P1000 pipettes. The volume the P200 will draw up is 90 µL (0.090 ml) while the volume the P1000 will draw up is 900 µL (0.900 ml). For sterile transfer of liquids, researchers use sterile tips, which are kept in sterile boxes. Aseptic technique must be practiced when using tips. Any time a pipette tip comes into contact with anything in the environment, it should be considered contaminated. The

2

golden rule of sterile technique is to change your tip whenever in doubt. If or when you suspect contamination, discard the pipette tip and start again with a fresh one. In this exercise, you will view two activities:  Pipette solutions of acids, bases, pH indicators and water as an exercise of accuracy and consistency.  Determine the number of viable cells per ml of a given suspension of bacterial culture. Concentrations of cells in suspension are expressed as viable cells/ml or colony forming units/ml (CFU/ml). For many cultures, each colony arises from one cell (cells/ml), but in cultures that grow in groups or chains, it is very likely that each colony starts from more than one cell (colony forming units/ml). A typical E. coli culture grown in lab in a rich media can have a concentration of 108 cells/ml or 100 million cells/ml, disqualifying Figure 3. A 1:10 dilution hemocytometer direct counting and accurate spectrophotometer reading. Putting even small volumes of this suspension onto agar media will result in a lawn of bacterial growth with no distinct colonies. It must be diluted to get the cell number down to a number that can be realistically counted on a plate. Typically, dilutions are done in ten-fold increments, each dilution reducing the concentration by a factor of 10. Taking 1 ml out of the original culture (Figure 3) and putting that milliliter into 9 ml diluent is a 1:10 dilution. (A diluent is an isotonic solution like saline that allows the cells to survive but not grow.) Now the concentration of E. coli is 1/10 of the Figure 4. Two 1:10 dilutions, resulting in original culture or 10 million cells/ml. This a 1:100 dilution concentration is still too high to count directly so more dilutions are needed. Since dilutions are multiplicative, each dilution results in another 10-fold reduction in concentration. Therefore, taking 1 ml out of the 1:10 dilution and adding it to 9 ml diluent is by itself a 1:10 dilution, but the concentration of this second dilution tube is 1/100 of the original culture or 1 million cells/ml (Figure 4). Another 1:10 dilution results in a 1:1000 dilution of the original culture. By repeating these steps, the culture is diluted 1:10000 or 10-4, then to 10-5, 10-6, and 10-7 (Figure 5). The number of colonies people can count without worrying that one colony was started by more than one cell is around 300 colonies per plate; under 30 colonies per plate is when colony counts are too variable to be trusted statistically. A

3

serial dilution of a culture will result in plates whose colony count falls in this thirty to three hundred colonies per plate range.

Figure 5. A series of seven 1:10 dilutions, resulting in a total dilution of 10 -7. If the original suspension started at 108 CFU/ml, what is the CFU/ml each tube? The dilutions are spread on plates, but the plates must be able to completely absorb all the culture spread on them. Since small volumes would absorb faster than larger ones, 0.1 ml (100 µl) is plated because it absorbs in a reasonable amount of time. The units for cell concentration are CFU/ ml, but we are only plating 0.1 ml, which is 1/10th of 1 ml, and therefore acts as one more 1:10 dilution (Figure 6). After colonies grow, count and record the number of colonies on each plate. Find the plates that are between 30 and 300 colonies to calculate the CFU/ml from the original suspension. Use this formula to determine the CFU/mL of the original culture. CFU/mL = (Colony #)/(Total Dilution)

Figure 6. Four dilutions, each has 100 µl sampled for plating. The final units are CFU/ml, but only 1/10th of 1 ml was sampled. Therefore, the plate counts will reflect the number of colony forming units in 0.1ml, with the sampling acting as an additional 1:10 dilution.

The colony number is what you count on your plate containing 30-300 colonies. The total dilution is the dilution you plate. (The dilution of the tube with the plating factor accounted for, see figure 6) Remember that the original CFU/mL is probably going to be a very big number, like 108 CFU/ml in the example E. coli culture. The exponent should be a positive number when calculating out CFU/ml. If it isn’t, ask for help to check your math. Dilutions are usually small numbers with negative exponents. If your dilutions don’t have negative exponents, ask for help to check your math.

4

To Do in Session 1 1. Become comfortable using pipettes (Protocol 1-4). 2. Perform a serial dilution and plate dilutions on plates (Protocol 5, Steps 1-7).

To Do in Session 2 1. Count colonies and determine CFU/mL of original culture (Protocol 5, Step 8). Protocol 1: Setting Up Micropipettes MATERIALS  Micropipettes – P200 and P1000  Blue and yellow tips 1. Before using a pipette, review these rules:  Never use the pipette without a tip in place, as it would ruin the precision piston that measures the volume of fluid.  Never lay down a pipette that has a filled tip because fluid could run back into the piston.  Never let the control button “snap back” after withdrawing or ejecting fluid. (Always draw up liquid slowly)  Never rotate the volume adjustment beyond the highest value for the micropipette, which is 200 µL for a P200 and 1000 µL for a P1000, or below the lowest value for a micropipette, which is 20-50 µL for a P200 and 200 µL for a P1000. Doing so will destroy the pipette. To determine the range for a pipette, look at either the top of the plunger or the side of the pipette. 2. Examine the P200 pipette, especially the window that displays the volume. Using the knurled knob at the top of the pipette, set the P200 at 200 microliters. 2 0 0

Hundreds Place Tens Place Ones Place

3. Use the knurled knob at the top to reset the pipette to dispense 100 microliters. 4. The P1000 pipette has a maximum volume of 1000 L and uses blue disposable tips. Examine the P1000 pipette, especially the window that displays the volume. 5. Using the knurled knob at the top of the pipette, set the P1000 at 1000 microliters. 1 0 0

Thousands Place Hundreds Place Tens Place

6. Use the knurled knob at the top to reset the pipette to dispense 900 microliters.

5

Protocol 2: Using Traditional Model Micropipettes 1. Choose the appropriate pipette for the desired volume. Set the volume using the rotating knob. Rotate to the desired volume. 2. Push the pipette end firmly into the proper size tip. Use a fresh tip for each new reagent or each new concentration of culture. Pull up pipette and close tip box. 3. Hold culture or reagent container at eye level. 4. Hold the pipette almost vertically. Depress plunger to the first stop and hold it there. 5. Lower tip into the reagent/culture. Slowly release plunger to draw the fluid into the tip. 6. Slide tip out of reagent/culture tube with tip gliding along the inside wall of the reagent tube. This will “knock off” excess liquid adhering to the outside of the tip. 7. Touch the pipette tip to the inside wall of the receiving tube. 8. Slowly depress the button to the first stop to expel fluid. Wait for a second or two. Press down to the second stop to eject remaining fluid. Hold button down in second position. 9. Slide the pipette out of the receiving tube with button depressed to second stop to avoid sucking any liquid back into the tip. 10. Eject tip into a discard bucket using the tip ejector button. Protocol 3: Using Ergonomically Corrected Micropipettes 1. Rotate the control button dial or adjusting column to the desired volume. 2. Push the pipette end firmly into the proper-size tip. Use a fresh tip for each new reagent or each new concentration of culture. Pull up pipette and close tip box. 3. While withdrawing or expelling fluid, always hold the vessel at nearly eye level. 4. Hold the pipette almost vertically while filling it. 5. Depress the top button to the first stop until it clicks and release. Dip tip into the fluid to be pipetted and press on the release button to draw the fluid into the tip. If the speed of fluid withdraw is too fast or too slow, adjust the control lever on side of pipette as appropriate. 6. Slide tip out along the inside wall of the reagent tube to knock off any excess fluid adhering to the outside of the tip. 7. Touch the pipette tip to the inside wall of the receiving tube. 8. To expel the sample, slowly depress the top button to the first stop. Wait for a second or two. Press down to the second stop to eject remaining fluid. Hold button down in second position. 9. Slide the pipette out of the reagent tube with button depressed to second stop to avoid sucking any liquid back into the tip. 10. Eject tip into discard beaker using the tip ejector button.

6

Protocol 4: Micropipette Exercise MATERIALS P200 and P1000 pipettes Blue and yellow tips 3 dilution tubes/pair Solution I: Thymol Blue (0.2 mg/ml in ethanol) Solution II: distilled water Solution III: 0.1 N NaOH Solution IV: 0.1 N HCl

      

1. Label six dilution tubes A through F. 2. Make tubes as described below: Solution Tube A Tube B Tube C

Tube D

Tube E

Tube F

1 2 3 4

80 µL 100 µL 20 µL 0 µL

80 µL 100 µL 0 µL 20 µL

80 µL 80 µL 20 µL 20 µL

400 µL 500 µL 100 µL 0 µL

400 µL 500 µL 0 µL 100 µL

400 µL 400 µL 100 µL 100 µL

Total

200 µL

200 µL

200 µL

1000 µL

1000 µL

1000 µL

3. Cap tubes A-C and briefly vortex to mix solutions. Record observations. 4. Set your pipette to 200 µl and very carefully pipet out the solution from each tube to see if your volume just fills the tip. Protocol 5: Dilution Plate Exercise MATERIALS      

E. coli broth culture Saline Blue and yellow tips P1000 set to 900 (0.9ml) P200 set to 100 (0.1ml) 4 TSA plates/pair

1. Label tubes 10-1 – 10-7. 2. Add 0.9 mL saline to each tube. 3. Pipet 0.1 mL E. coli culture into 10-1 tube. Mix using the vortex mixer. Discard tip.

7

4. Using new tip, transfer 0.1 mL from the 10 -1 tube to the 10-2 tube. Mix. Discard tip. Repeat as shown by figure below. 5. Label four TSA plates with name, group ID, date, species, and dilution. 6. Plate 0.1 mL of the last four dilutions on separate TSA plates. 7. Incubate at 37 oC for 24 hours. 8. Count colonies and calculate CFU/mL from plate that has between 30-300 colonies and record results in table.

Results Record colony numbers and CFU/mL calculations. Use the formula below to calculate CFU/mL from colony number. CFU/mL = (Colony #)/(Total Dilution) Table 1. Dilution Plating Colony Counts and CFU/mL Calculations. Total Dilution

Colonies Counted

-5

10 10-6 10-7 10-8 The CFU/mL for this culture is __________________....