Zool 2232 Physiology Laboratory

 

MICROPIPETTING AND MEASUREMENT OF PLASMA GLUCOSE

 

I. Digital Micropipettors

 

A digital micropipettor is a precision pump fitted with a disposable tip. The volume of air space in the barrel is adjusted by screwing the plunger in or out of the piston, and the volume is displayed on a digital readout on the top upper portion of the pipette. Depressing the plunger displaces the specified volume of air from the piston; releasing the plunger creates a vacuum, which draws an equal volume of fluid into the tip. Depressing the plunger again then expels the withdrawn fluid.

 

There are different size micropipettors with various volume range: a small-volume (1-20 ul), a mid-range (20-100 ul), and a large-volume (100-1000 ul). Depending on the volume needed, appropriate size micropipette should be used to ascertain accuracy. Since these micropipettors are very delicate, hard to repair and very costly, please note the following rules in using a digital micropipettor.

 

• Never rotate the volume adjuster beyond the upper or lower range of the pipette as stated by the manufacturer.

• Never use the micropipettors without the tip in place; this could ruin the piston.

• Never invert or lay the micopipettor down with a filled tip; fluid could run back into the piston.

• Never let the plunger snap back after withdrawing or expelling fluid. This could damage the piston.

• Never immerse the barrel of the micropipettor in fluid.

• Never reuse a tip that has been used to measure a different reagent.

 

General use of digital micropipettors:

1. Rotate the volume adjustor to the desired setting. Be sure to locate the decimal point when reading the volume setting.

 

2. Firmly seat a proper-sized tip on the end of the micropipettor.

 

3. When withdrawing or expelling fluid, always hold the tube firmly between your thumb and forefinger. For best control, grasp the micropipettor in your palm and wrap your fingers around the barrel; work the plunger with your thumb.

 

4. The micropipettor plunger has two positions. Depress the plunger to the first stop and hold it in this position. Dip the tip into the solution to be pipetted and draw fluid into the tip by gradually releasing the plunger. Be sure the tip remains in the solution while you are releasing the plunger.

 

5. Slide the pipette tip along the inside wall of the reagent tube to dislodge any excess droplets adhering to the outside of the tip. Check and make sure there is no air space at the very tip of the tip.

 

6. To expel the sample into a reaction tube, slowly depress the plunger to the first stop to expel the sample. Depress to second stop to blow out the last bit of fluid. Hold the plunger in the depressed position and slide the tip out of the pipette along the inside wall of the reaction tube. This creates a capillary effect that helps draw the last bit of fluid out of the tip.

 

7. Eject the tip by depressing the tip-ejection button.

 

8. Use a fresh tip for each new reagent to be pipetted.

 

Practice with small-volume micropipettor

This exercise simulates setting up a reaction, using a micorpipettor with a range of 20-200 ul.

 

1.      Use a wax pencil to label three 2-ml tubes A, B, and C.

2.      Add solutions to these tubes using the following table:

 

Tube

Solution 1

Solution 2

Solution 3

Solution 4

A

10 ul

20 ul

20 ul

0 ul

B

30 ul

10 ul

0 ul

10 ul

C

10 ul

0 ul

20 u1

20 u1

 

3.  A total of 50 ul of reagents was added to each tube.  To check the accuracy of your measurements, set the pipette to 50ul and withdraw the solution from each tube.

a. Is the tip barely filled?

b. Does a small volume of fluid remain in the tube?  This indicates an over-measurement.

c. After redrawing all fluid, is a space left in the end of the tip?  This indicates an under-measurement.

d. If several measurements were inaccurate, repeat the exercise to obtain nearly perfect results.

 

II. Measurement of plasma glucose - enzymatic procedure

Measurement of blood glucose levels was among the first chemical procedures employed in clinical laboratory medicine.  Keilin and Jartree introduced glucose oxidase methodology in 1948.  Keston later reported use of the combined glucose oxidase-peroxidase reagent, followed by the Teller addition of chromogenic reagent to Keston's procedure.  

Glucose is oxidized in the presence of glucose oxidase (GO).  The hydrogen peroxide formed reacts, under the influence of peroxidase, with phenol and 4-aminophenazone to form a red-violet quinone complex.  The intensity of the color is proportional to glucose concentration.    

Glucose + O2 + H2O ------> gluconic acid + H2O2

H2O2 + 4-aminophenazone + phenol -------> quinone complex

Objective:

1.       Use one standard solution to make diluted solutions for standard curve.

2.       Explain the physiological significance of glucose in the blood and why abnormal levels (too high or too low) are clinically significant.  

3.       To determine how Beer’s law is used to determine the concentration of molecules in a solution.

4.       Use the graphic and formula method to determine the concentration of glucose in an unknown sample.

Materials:

Microfuge tubes (50)

Test tube racks (6)

Microliter pipette (6) and pipette tips.

Constant water bath set at 37oC.

Colorimeter (6)

Cuvettes (6sets of 7)

Glucose --250 ml @ 400mg/dl

Mixer-- vortex mixer

Unknown glucose solution (150 mg/dl-- 200 ml) and glucose reagent.

Procedure for using spectrophotometer

  1. Turn machine on—at lower back of machine
  2. Make sure cell holder is empty
  3. Warm the machine for 30 minutes before using
  4. Set wave length at 500 nm using I or L arrows
  5. Place glucose reagent on ice
  6. Set and label 5 3-ml microfuge tubes 1 through 5, and 7 glass tubes, and label them 1-7
  7. Place 2 ml of DI water in microfuge tube 1, and 2 ml of 400 mg/dl standard in tube 2.  The water is used for dilution.
  8. Place all tubes in a rack
  9. Prepare standard solutions using microfuge tubes —given a 400 mg/dl glucose solution
    1. Add 100 ul of 400 mg/dl in  tube 3 and add 100 ul of DI water, this will be a 200 mg/dl solution
    2. Add 100 ul of 200 mg/dl in  tube 4 and add 100 ul of DI water, this will be a 100 mg/dl solution
    3. Add 100 ul of 100 mg/dl in  tube 5 and add 100 ul of DI water, this will be a 50 mg/dl solution
  10. Dispense 2 ml of glucose reagent in each glass tube (1-7)
  11. Add 20 ul of DI water to tube 1 – this is the ‘blank’ to set Absorbance to 0
  12. Add 20 ul of 400 mg/dl standard to tube 2
  13. Add 20 ul of 200 mg/dl standard to tube 3
  14. Add 20 ul of 100 mg/dl standard to tube 4
  15. Add 20 ul of 50 mg/dl standard to tube 5
  16. Add 20 ul of unknown plasma to tube 6
  17. Add 20 ul of 100 mg/dl standard to tube 7 (This standard comes with the kit.)
  18. Incubate all glass tubes with the enzyme (1-7)  for 5 minutes at 37oC
  19. With a transfer pipette, transfer solutions from glass tubes to the spectrophotometer cuvettes 1-7.
  20. Press A/T/C to set to absorbance.
  21. Insert blank into cell holder, close door.
  22. Press 2nd button--setting absorbance to zero-- 0 ABS/100%T
  23. Remove blank, insert standard and sample and take reading.
  24. Press “Print” for a print-out of results or record data in your note book.
  25. Read the absorbance  values of each solution.
  26. Draw a linear standard curve (absorbance vs concentration).
  27. Even though your experimental values may deviate slightly from a straight line.

      The standard curve should intersect the origin (zero concentration = zero absorbance).

28.  Determine the concentration of the unknown using the standard curve.

29.  Locate on the y axis the absorbance of the unknown.

23.   Then, use a ruler to draw a horizontal line from this point to its intersection with the data line (standard curve). 

24.        Now extend a vertical line to x axis; this will give the concentration of the unknown.

 

Measurement of plasma glucose concentration

 Determine the concentration of the unknown (plasma) using the

                Beer's law calculation.

 

Values are derived by the following equation:

               Glucose (mg/dl) = Au/As*100,

               Where Au = absorbance value of unknown (tube 6)

                          As = absorbance of standard (tube 7)

                          and 100 = concentration of the standard (100 mg/dl)

                        Normal range: serum/plasma = 70-105 mg/dl

Reference:

1.  Fox, S.A.  Laboratory Guide- Human Physiology. 7th ed. Pp 28-32,1993.

      Wm. C. Brown Publishers, Dubuque, Iowa.

2.  Bloom,M.V., Freyer,G.A. and Micklos, D.A.  1996  Laboratory DNA Science.

     The Benjamin/Cummings Publishing Company, Inc.