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Techniques Manual Table of Contents

EXERCISE IN DILUTION TECHNIQUE

Introduction

Abbreviations used:

A. Absorbance = Optical Density (O.D.) SRBC = Sheep Red Blood Cells
Conc. = Concentration PBS = Phosphate buffered saline
l (lambda) Wavelength TT = Test Tube
d= H2O Distilled water Hgb= Hemoglobin

In biology, it is frequently useful to prepare serial dilutions. For example, you may want to test the biological activity of a hormone over a range of concentrations and making a serial dilution is a quick, easy and accurate way to prepare these solutions. In this exercise you will test and practice your ability to pipette accurately by making a series of dilutions of a colored solution. The intensity of the color in each of the dilutions will serve as a measure of the precision of your pipetting. The source of the colored solution will be the hemoglobin contained in sheep red blood cells. The following suggestions will be helpful in developing good techniques:
1. Wipe the tip of the pipette with paper tissue to remove fluid adhering to the outside surface.
2. Pipettes should be held vertically and at eye level because pipettes are calibrated to be held vertically and read most accurately in this position.


Materials for dilutions using serological pipettes per table:

10% SRBC in PBS: 1+ mL per group d H2O: 50 ml
15 x 125 mm TT (16 ml capacity): 8 5 or 10 ml pipettes: 6
1 ml pipette: 6 Kimwipes
Parafilm to cover 15 x 125 mm TTs Scissors to cut parafilm
TT rack for 15 x 125 mm TT: 1 Container for dirty pipettes
Wax glass marking pencil 25 or 50 mL graduated cylinder
Unknown hemoglobin solution in PBS, somewhere between 0.3% - 10%: 5 ml Spectrophotometer (2 per lab)



Dilution procedure using serological pipettes.
The concentration of red blood cells (RBC) in a suspension can be accurately determined by photometrically measuring their hemoglobin content. To release the hemoglobin from the cells so that it can be measured, the cells are lysed by hypotonic shock (e.g., putting them into distilled water). The hemoglobin molecule has a characteristic red color in solution and absorbs visible radiation strongly at 541 nm. In order to assure that all RBCs lyse it is important that they be diluted in a large amount of hypotonic solution. We will use a 15x dilution of the SRBC (i.e., 1.0 ml of the 10% SRBC into 14.0 ml distilled H2O in tube 1. After mixing well and allowing a couple of minutes for SRBC to lyse, two-fold dilutions are made for tubes 2 through 7.
Before starting the exercise, practice your pipetting technique by seeing how accurately you can measure distilled water. Using the 5 mL pipet, transfer 13 mL of water to a graduated cylinderand then read the volume of water using the gradations marked on the cylinder. If you are off by more than 1/2 mL, you need more practice before beginning the exercise! All members of the group should become comfortable with the pipets as you will be required to use them in the future.

Procedure: Prepare the following solutions. Detailed instructions are below the table.

TT
1
2
3
4
5
6
7
Hgb (mL)
1.0
5.0
5.0
5.0
5.0
5.0
5.0
H20
14.0
5.0
5.0
5.0
5.0
5.0
5.0
Dilution
1/2
1/4
1/8
1/16
1/32
1/64
SRBC Conc.(%)
10
5
2.5
1.25
0.63
0.32
0.16


1. Pipet 14 ml dH2O in TT #1 and add 1.0 ml of 10% SRBC to this. Cover with parafilm and mix well by inverting 5 times. (In TT #1 hemoglobin is considered undiluted because all subsequent calculations are made relative to that sample.)
2. Pipet 5.0 ml of dH2O into each of 6 TTs.
3. Transfer 5 ml of #1 to TT #2 containing 5.0 ml of dH2O. Mix as before (= 1/2 dilution).
4. Repeat the above process to obtain 1/4, 1/8, 1/16, 1/32, and 1/64 dilutions.
5. Place solutions in a cuvette, starting from 1/64 dilution, and read absorbance (O.D.) values at 541 nm.
6. You will obtain a hemoglobin solution of unknown concentration from your instructor.
Determine its O.D. at 541 nm.
Results:
a. Prepare a graph showing O.D.541 vs. relative hemoglobin concentration.
b. Determine the relative hemoglobin concentration of the unknown sample.

Question:
Why not measure the darker colored solutions before the lighter colored ones?



Some Notes on Graphing
We will be doing a significant amount of graphingin this laboratory and it is very important that you do it correctly. It is even more important that you be able to look at graphs in the literature and really understand what they mean. Having some practice with making your own graphs will make interpretation of literature data much simpler. Here are some rule to follow while preparing your graphs. Most important rule of all. All graphs must be done neatly, with the X and Y axis clearly labeled using the proper units.
Other important rules.
1. Always graph absorbance on the Y axis, concentration on the X axis.
2. Make sure you position the concentration numbers to be linear on your graph.
3. Always have the lowest concentration on the left side of the graph. Frequently, but not always, the origin will be zero. Likewise, when graphing O.D., start with the smallest number at the bottom of the graph.
4. Use as much of the graph paper as possible while still keeping the graph accurate. Tiny graphs using just a small corner of the paper are unacceptable. Your instructors eyes are getting old and need all the help they can get.
5. It is often very useful to generate a standard curve to be used when calculating the concentration of an unknown. In a standard curve, the absorbance of a series of solutions with known concentrations are determined The absorbance values are plotted on a graph and the standard curve drawn. When you are making a standard curve using absorbance values, always draw your best fit straight line between the data points. DO NOT connect all the data points if they are not directly on this line. Your best fit line is an average of all the data points, and thus is a more accurate representation of absorbance vs. concentration than any single data point.
Determining the concentration of unknown solutions. We commonly use graphs to determine the concentration of unknowns. In these types of problems, you will be given a standard and will make serial dilutions. Reading the absorbance of these standard solutions will allow you to make your standard curve (using the instructions above) and then determine the concentration of your unknown. We will use the plot and drop method with the following steps:
1. Determine the absorbance of the unknown solution.
2. Draw a straight line from the absorbance value on the graph until it intersects with the standard curve. This is your plot.
3. Drop a straight line from the point of intersection to the X axis and read the concentration off the graph.
4. When determining the actual concentration of the unknown, remember to multiply by any dilution factors used while preparing the unknown.



Serial Dilution Problems.

Work these problems BEFORE leaving class today. Be forewarned: you will see this kind of problem again!


We commonly use multiple dilutions of a single sample in procedures for estimating concentrations of serum proteins. Frequently, these will be very high dilutions, making it necessary to use serial dilutions for minimizing the use of excess reagents and in order to work with appropriate volumes. Also, we may need to run experiments on each of the dilutions to see which has a desired effect or to make a standard curve like we did in today’s exercise. Often, we will do these dilutions as serial dilutions, which are a series of equal small dilutions used to obtain a large final dilution. For example, we might want to do 5 serial two-fold dilutions. We could do this by adding 1 mL of solute to 1 mL of solvent, mixing, transferring 1 mL of this mixture to another mL of solvent, mixing, transferring to 1 mL of solvent and so on. The dilution would be 1/2 in the first tube, 1/4 in the second, then 1/8, 1/16 and finally 1/32 in the final tube. This final tube would have a dilution factor (the inverse of the ratio) of 32. Likewise, we could perform serial 5-fold dilutions by transferring 1 mL of solute into 4 mL of solvent . The dilutions for 5 of these steps would be 1/5, 1/25, 1/125, 1/625 and 1/3125, for a dilution factor of 3,125. You can see the savings in solvent clearly in the latter example as only 25 mL of solvent was necessary to prepare this 3,000+-fold dilution.


1. Starting with 1 ml of an undiluted solution, perform the serial dilutions shown below. Calculate the dilution at each step. What is the final dilution factor?


2. Perform a series of 6, 5-fold dilutions. What is the final dilution? You may find it helpful to actually sketch the test tubes as was done for you in problem 1.


3. If you performed four 2-fold dilutions and the final concentration was 1.5%, what was the initial concentration? 1 point


4. What is the final dilution of the following?

 


5. If you begin with a 1/10 dilution and do 5 more 2-fold dilutions, what is the final dilution?