Laboratory 1 Lab Techniques (Revised Aug 8, 06)

Transcription

1 Laboratory 1 Lab Techniques (Revised Aug 8, 06) Pipetting: Micropipettors: In this experiment you will learn how to pipette solutions accurately and how to use the "spectrophotometer". Virtually any area of science that you enter will require that you master the skills of pipetting and many biological assays require the use of the spectrophotometer. Many clinical diagnostic tests use spectrophotometry to determine the levels of substances in a patient s blood. There are many devices that laboratory workers use to pipette solutions. Accuracy in pipetting is very important, as delivering the precise amount of solution in biological and chemical assays is critical, particularly when constructing "standard curves" with which the "unknown or experimental samples" are to be compared. Your laboratory instructor will demonstrate the use of various pipetting aids that are frequently used. You will be working with pipettes of various sizes, 10 ml; 5 ml; 1 ml and micropipettors that dispense smaller volumes 0.1 ml (100 μl) down to.001 ml (1 μl). To refresh your memory the following table may be helpful: 1ml = 1000 μl 0.1ml = 100 μl 0.01 ml = 10 μl 0.001ml= 1 μl First you will practice with the larger pipettes. You have two colors of pipette aides the green one is for 5 and 10 ml pipettes and the blue one is for 1 and 2 ml pipettes. The pipette is placed in the rubber gasket carefully and the "wheel" at the top is rotated down to bring up the solution. To dispense the solution the wheel is rotated up. When you look at the solution in the pipette, it is not quite "flat" in the pipette but is a bit higher on the sides of the pipette, making the surface of the solution slightly concave. The bottom of this surface is called the "meniscus". The way to accurately pipette is to line the mark (say 10 ml) with the bottom of the meniscus. When you release the fluid entirely you have dispensed 10 ml. Practice this. Now practice pipetting 8 ml; 5 ml; 2 ml. Now try the 1 ml pipette with the appropriate pipette aid (blue). This is somewhat more difficult to control because small hand movements will cause the volume in the pipette to change rapidly. Practice pipetting 1 ml; 0.8 ml;.5ml and 0.2 ml. There are various types of micropipettors which dispense smaller volumes of solutions generally from as high as 1 ml down to 1µl or even less. The pipetting devices that we will be using are Hamilton Pipettors. There are three sizes of pipettors with which you will be working. The largest one pipettes up to 1000 μl (1ml). This has a blue plunger and is most accurate for pipetting from 200 μl up to 1000 μl (1ml). This has the largest barrel. The second, which has a red plunger, pipettes up to 300 μl and is most accurate from 30 μl up to 300 μl (0.3ml). The third pipettor, with a green plunger, pipettes up to 25 μl and is accurate from about 2 μl up to 25 μl (.025ml). All of these devices use plastic pipette tips to take up the liquid. The largest tips (blue) are for the 1000 μl (1ml) Hamilton pipettor. The smaller pipette tips are for both the 300 μl pipettor and the 25 μl pipettor. There are other pipettors which can pipette quantities under 1 μl, which would take even smaller tips. The plungers are connected to a shaft. If you push the plunger down you will find that there are two stops. Your lab instructor will demonstrate this. To pipette a solution, you push down to the first stop and then carefully take up the solution from a beaker. Make sure that the end of the tip is in the solution before you start letting the plunger up, or you will take in air bubbles. When the solution is in the pipette tip, push the plunger down until the shaft goes all the way down to the second stop, releasing the entire amount of fluid. Try this with the 1ml (1000 μl pipette set at 1 ml. The way to set this is to have the top number (which is black) set at 1 and the lower two numbers set at 0. This is the setting for 1 ml. To adjust the volume on all pipettors, turn the center wheel. Do not set the pipettor over its limit (in this case 1ml) or it can break!!. Try setting the 1000 μl pipettor to 500 μl (0.5ml) by turning the center wheel down to 500. This is done by setting the top number to 0 and the lower two numbers to 50. Try pipetting 500 μl. Next try 300 and 200

2 Dilutions: μl, respectively. Take great care that the solution does not get up into the barrel of the pipette!! Make sure that the pipette tip is down in the solution before you take up the liquid and this will help avoid this problem. Now try the 300 μl pipettor. It has a red plunger. Set it to pipette 200 μl and then 100 μl. For 200 μl the top number should read 2 and the lower numbers 00. All numbers are in black for this pipettor. Try the 25 μl pipettor and set it up to pipette 20 μl. To set this up, the top number (black) should read 2, the next number (black) should read 0 and the bottom number (red) should read 0. For 2.5 μl, the top number would be 0 and the middle number 2 and the bottom number (red) would be set at 5. Learn to recognize the different pipettors, tips and how to set the appropriate volumes for the amount of solution that you want to pipette. Testing your pipetting skills: To test accuracy of the pipettor, you will weigh the amount of water pipetted into a plastic weigh boat on a balance. Take the 1 ml (blue plunger) pipettor and set it for 1ml (1000 μl). Put a weighing boat on the scale and tare the scale so the weight reads zero. Then, pipette the 1 ml from the micropipettor into the weighing boat. What does the scale read? Try pipetting 200 μl, then 100 μl, then 50 μl. Why does this work to test the accuracy of the pipettor? What is the density of water? To test your pipetting skills, perform the following operation? Obtain a piece of the filter paper provided. Take the 25 μl pipetttor and set it for 1 μl. Pipette 1 μl of the colored solution onto the filter paper. Be sure you know how to take up the solution (from the first stop on the pipettor) and release it (going down to the second stop). Then reset the pipette to 2 μl. Apply the solution to the filter paper near the previous spot. Then reset the pipette to 5 μl and, again, apply the solution to another area of the filter paper. Then, in sequence, apply 10 μl, 20 μl, 30 μl, 40 μl, 50 μl and 75 μl - USING THE APPROPRIATE PIPETTOR. Note that you will have to change pipettors after 20 μl. Observe the spots that were generated. Can you tell that when you pipette twice as much, say comparing 10 μl to 20 μl, that the spot on the filter paper is about twice as big? Measure the diameter of the spots. Plot this to see what kind of relationship you observe. Very often in biological assays it is necessary to make dilutions of various solutions. For example, to make a tenfold dilution,you would pipette 1 ml of a stock colored solution and add 9 ml of distilled water (or whatever diluent you require for your assay). Try this, using the colored solution that has been provided and diluting with water. Observe what the color looks like. Note that it would also work to take 100 µl of the stock plus 900 µl of water (0.1 ml + 0.9ml). Try this, using the pipettor. The color should look the same as your previous dilution but just a smaller volume. To make a 5-fold dilution you would pipette 1 ml of stock plus 4 ml of water. For a 2-fold dilution (or actually diluting in half) you would pipette 1 ml of stock plus 1 ml of water or any combination which would dilute the stock in half i.e. 2 ml stock + 2 ml water. To prepare your standard curve, take the stock colored solution and pipette 8 ml into a test tube and make successive 2-fold dilutions as follows: Take 4 ml of the stock + 4 ml of water into a test tube. This is a 1 in 2 dilution. Take 4 ml of that solution into another test tube and add 4 ml of water. This is now a 1 in 4 dilution. Now take 4 ml of this solution and put it in a fourth test tube and add 4 ml of water. This is now a 1 in 8 dilution. Now, take 4 ml of this solution and 4 ml of water. This is a 1 in 16 dilution. Then do a final dilution of 4 ml from the last tube, containing 1/16 dilution, and add 4 ml of water. This is a 1/32 dilution. You can see that as you made successive serial dilutions the solution got lighter and lighter. We can accurately measure this in a device called a spectrophotometer. Spectrophotometry: A substance in solution may absorb some wavelengths of light while freely transmitting others. For example, a solution of chlorophyll appears green because it transmits a combination of wavelengths that we perceive as the color green. A spectrophotometer in its simplest form consists of a white light source that is focused on a prism to separate the white light into bands of differing wavelengths. When a given wavelength band is focused on a sample,

3 some fraction of the incident light is transmitted through the sample to a photocell. The ratio of radiant light transmitted (I) to radiant light emitted from the source (incident light) (I0 ) is called transmittance (T ) (T= I/ I0). Transmittance varies from 0 to 1.0 and is often called per cent transmittance. This is the light that you actually see coming through the solution. The darker the solution the less light is transmitted. Absorbance (A), also called optical density (O.D), is more commonly used in biological studies than is transmittance. Absorbance is the amount of light that is absorbed by the solution. It is defined as log 1/T or -log T. Absorbance may be partial or complete and thus A varies from 0 to infinity. The darker the solution the more light is absorbed and the higher the optical density. This often indicates a higher concentration of material in the solution, as in a biological assay where a colored product is formed to indicate concentration of a biological substance. An absolutely clear solution would absorb little or no light and would have a high percent transmittance and a low absorbance or O.D. We will be using a Spectronic 20 to measure our dilutions. It consists of a white light source that is focused on a prism to separate the white light into bands of differing wavelengths. A given wavelength band is focused through a narrow slit and then through the sample. Some fraction of the incident light I0 is transmitted through the sample to a photocell. The ratio of radiant light transmitted (I) to radiant light emitted from the light source (I0) is called the Transmittance (T) as we discussed above. This is read on the per cent transmittance scale on the spectrophotometer, or more often on the Absorbance scale. This exercise contains a diagram of the Spectronic 20 and some tips for its use. We will see how much light is absorbed by our various dilutions of the colored solution which we prepared above. First, we have to set the appropriate wavelength (lambda) for the color that we are working with. Different colors have different absorption maxima (different colors will have higher O.D. at specific wavelengths) and we can find the peak absorption maxima for the color of our solution. To determine the wavelength of maximum absorption for the colored solution you are working with, you need only use one of the tubes from the serial dilutions which you prepared- generally the darkest one. When you do the readings you will set the spectrophotometer at various wavelengths (say from 400nm up to 750nm) and determine the absorption (O.D.) of the solution you are working with at each specific wavelength. Remember to reblank the machine each time you change the wavelength. Your instructor will show you how to blank the spectrophotometer. Then, having determined the wavelength of maximum absorption, you will (staying at this wavelength) determine the O.D. of each of the tubes in the dilution series.

4 1. Set the wavelength scale to the wavelength for maximum absorption of the test material, if this is known. If it is not known, determine as directed below (Item 8). 2. Turn on the instrument by rotating the amplifier control (left knob) clockwise. After 10-15minutes warmup, adjust this control until the meter needle indicates 0% transmittance ( on the Optical Density scale). 3. Place a cuvette (special test tube used for spectrophotometers) containing the blank solution, usually water, in the sample holder and close the lid. 4. Rotate the light control (right knob) until the needle indicates 100% transmittance (O optical density). You have now blanked in or zeroed the spectrophotometer 5. Replace the blank with the test solution and read directly the % Transmittance (top scale) or the O.D. (bottom scale). For this experiment we will mainly be reading the O.D. 6. Repeat with the other samples at the same wavelength of absorption. Occasionally, reset 100% T with the blank in the sample holder to make sure that the spectrophotometer is reading accurately. 7. For samples of different wavelengths of absorption (different colors) reset the wavelength knob, and restandardize the instrument by resetting the light control with the appropriate blank in the sample holder. 8. To determine the absorption characteristics (within the range of mµ) of an unknown solution or suspension: for each reading, reset both the wavelength and the light control (right knob), with the blank in place, before substituting the sample. In other words, every time you change the wavelength you must re-blank the machine. Plot the Optical density vs. wavelength. The wavelength selector then is set at the wavelength of maximum absorption for measurements of unknown concentrations of solution. 9. Turn off the amplifier control (left knob) at the end of the series of tests and rinse the cuvettes and test tubes.. Notify the instructor or consult the Operations Manual in case the instrument does not function properly. The spectrophotometers need to warm up for approximately 20 minutes to yield accurate readings. The spectrophotometer is turned on using the knob on the left side. If it is already on the red light will be glowing. Take just one of your tubes to determine the appropriate wavelength at which to read the solution. Start at 400 nm and go up to at least 750 nm in increments of 25 nm. To read the O.D. of a solution you first have to blank in the machine. In this case, since our diluent was distilled water (and the stock solution was prepared in water) we will blank the machine with water. Put some distilled water into one of the cuvettes which are special tubes made from glass of a particular uniformity. Before you put the cuvette in the machine adjust the knob on the left hand side of the Spec 20 such that the per cent Transmittance reads 0 (Absorbance scale reads infinity). Then put the tube into the cuvette holder in the spectrophotometer (gently too forcefully and the cuvette may break!) Now adjust the right hand knob such that the per cent transmittance reads 100 (0 % Absorbance). You have now blanked in the machine so that water is reading 100 % Transmittance (0% Absorbance). Now put your colored solution in a cuvette and read its per cent transmittance and optical density. Move on to the next wavelength you are going to use to determine the maximum absorbance for the colored solution. YOU WILL NEED TO REBLANK THE SPECTROPHOTOMETER AT EACH DIFFERENT WAVELENGTH. To go further than 650 nm you must change the toggle switch from the left to the right, activating a different lamp. Once you have found where the maximum absorption is for your solution using one of your dilutions, you can then read the rest of the tubes at that same wavelength.

5 Data for determining the wavelength at which the solution should be read:

6 Wavelength Optical Density Graph the Optical density vs the wavelength for the dilution which you picked Data for determining Optical Density of the Serial dilutions that we made. Using the wavelength that we previously determined gave the highest O.D. for the color we are working with, measure all of the dilutions that we prepared from our stock colored solution. Note- if you read the least concentrated first and then proceed to the highest concentration, you do not need to wash the cuvette out in between. Sample Optical Density 1. Stock solution (neat) 2. 2 fold dilution (1/2) 3. 4 fold dilution (1/4) 4. 8 fold dilution (1/8) fold dilution (1/16) fold dilution (1/32) Graph the Optical density vs the sample dilution. Do you observe a pattern? Serial dilutions are often used in biological assays and can be done in a variety of ways: The following is an example of some different types of serial dilutions:

7 Questions: 1. I have a stock solution and I need to make a 1/10,000 dilution. How would I go about this? Are there other ways I could prepare this? 2. I am doing a biological assay where a red product is formed. One of the samples is from a patient. A high optical density means that the patient is ill a low O.D. means that the sample is normal. How would I go about determining this? What would I need for quantization and standardization of this assay? 3. By mistake you read the per cent transmittance scale off the spectrophotometer but your TA wants data expressed as optical density. The per cent transmittance which I read for a particular sample was 50. What is my OD. 4. What is a standard curve? How is this used to determine an unknown sample?