Lab classes #1-4
Page 1: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013)
Assigned reading, problems, quizzes, graphing exercises and schedule. (Lab classes #1-4) (1) Lab Notes p. 1-9 (2) Lab Notes p. 16 Overview (3) Resource Manual p. 28-31 Graphs for print publication. (4) Resource Manual p. 34-42 Ionic product of water, pH and buffers (solve problems)* (5) Resource Manual p. 42-43 Light (6) Resource Manual p. 49-51 Pipetting (7) Resource Manual p. 57-60 Spectrophotometry (try calculations on p.60)* (8) Resource Manual p. 90-91 Lab Notebook and Protocols (9) Resource Manual p. 6 – 9 Philosophy and ground rules (10) Resource Manual p. 67-68 Units of Measure & Exponents (solve problems)* (11) Resource Manual p. 43-45 Light reactions & Light quantity (12) Resource Manual p. 20 Chlorophyll (13) Resource Manual p. 61-64 Suspensions, solutions, concentrations (solve problems on pp. 63-64)* (Lab classes #5-6) (14) Lab Notes p. 10 - 15 (15) Lab Notes p. 17 Overview (16) Resource Manual p. 17-19 Cell counting (17) Resource Manual p. 19-20 Centrifugation (18) Resource Manual p. 45-49 Microscopy (19) Resource Manual p. 64-67 Dilutions (solve problems on pp.66 - 67)* * Solutions to problems in the Resource Manual are on p.54-57 of the Resource Manual. Lab class #1
Lab class #2 Graph assignment #1 set
Lab class #3
Lab class #5
Graph assignment #1 due
Graph assignment #2 due
Lab class #4 Lab Quiz #1: 4 questions on (7), (10) and (13). Graph assignment #2 set
Lab class #6 Hand in lab notebook at the start of class. Lab Quiz #2: 5 questions on (4) & (19)
Grading for this unit. A total of 14% of the course mark will be available for the following: Graph assignment #1 1% Graph assignment #2 2% Lab Quiz #1 4% Lab Quiz #2 5% Lab Book 2% Total 14%
Page 2: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013)
General Introduction Read “The Laboratory Notebook” in the Resource Manual (p. 90-91). Follow these guidelines for keeping your lab notebook. It should be clear what each graph, table or piece of information represents. Information should be clearly titled and recorded on numbered and dated pages. Experimental Spectrophotometry OBJECTIVES: At the end of the 6 lab classes in this unit you should be able to: (1) draw a diagram of the optical system of the Spectronic 20 and explain how the instrument works. (2) describe what is meant by transmittance (T) and absorbance (A) and be able to convert transmittance values to absorbance values. Use the equation A = Ecl to calculate A, c or E. (3) use the spectrophotometer with confidence. (4) state Lambert’s Law and Beer’s Law in words. (5) make an appropriate reference blank to zero the spectrophotometer in an experimental situation. (6) give an example of each of the principal uses of the spectrophotometer (Lab Notes, p.16). (7) calculate reaction rates using spectrophotometric data (A/time). (8) describe the meaning and units of photosynthetic photon flux (Resource Manual, p. 45). (9) explain the meaning of resolution in microscopy and the theoretical limit to resolution by reference to Abbe’s Equation (Resource Manual, p. 47-49). (10) use S.I. units of measure and exponents (scientific notation) with confidence. (11) calculate molar concentrations and dilutions. (12) remember and use the following equations: [H+][OH-] = 1 x 10-14, pH = - log[H+], base . [H+] = 10-pH, pOH = - log[OH-], [OH-] = 10-pOH, pH + pOH = 14, pH = pKa + log acid (13) make a buffer solution and explain how it minimizes changes in pH. (14) be aware of factors to consider when selecting a buffer solution for a specific experimental context (Resource Manual, p. 41-42). (15) know how to determine the number of cells/mL using a haemacytometer and a spectrophotometer. (16) design an experiment, with appropriate controls, to investigate the rate of electron flow from photosystem II in isolated, illuminated chloroplasts. Lab Class #1: Introduction to spectrophotometry (Resource Manual, p. 57–60). (1) An image of the optical system of the Spectronic 20 (Resource Manual. p. 59) will be displayed and a dismantled model is available for inspection. (2) In your lab book, make a table with the following headings: Transmittance (T)
%T
Log %T
Absorbance (A)
1.0 0.9 etc.
100 90 etc.
2.0 1.95 etc.
0 0.046 etc.
(Suggested values for T = 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, and 0.1) Use the equation A = log
1 to calculate the values for A. T
Page 3: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013)
(a) Use the data in the table to make a graph of A vs. T. (b) Use the data in the table to make a graph of A vs. Log % T. (All graphs must have a descriptive title). [GRAPHS REQUIRED]. (3) Absorption Spectra. Spectrophotometry can be used to identify or characterize compounds in solution by producing an absorption spectrum. An absorption spectrum is a graph of the absorbance values of a chemical compound in solution measured at a series of different wavelengths. We will make an absorption spectrum for a food colouring dye and a solution of 2, 6- dichlorophenol indophenol (DCPIP). Protocol Set up the spectrophotometer by following the instructions on page 60 in the Resource Manual. Solutions of a red food dye at a concentration of 0.06% (v/v) (Resource Manual, p. 61) and DCPIP at a concentration of 2 x 10-5 moles/litre will be provided. Record the absorbance value for each solution at 20 nm increments over the range of 340 to 600 nm. YOU MUST ZERO THE INSTRUMENT EACH TIME THAT YOU CHANGE THE WAVELENGTH. Plot all of the data on one graph and note the wavelength at which absorbance is maximal (the max) for each solution. [GRAPH REQUIRED]. (4) Standard Curve Protocol Use the stock DCPIP solution (1 x 10-4 mol/L) to make a dilution series according to the following table: 1 x 10-4 M Deionised DCPIP Absorbance DCPIP (mL) water (mL) concentration at max (= nm) 1 2.0 3.0 4 x 10-5 M 2 1.5 3.5 3 1.0 4.0 4 0.5 4.5 5 0 5.0 Read the absorbance (A) of each solution the wavelength of maximum (max ) absorbance for DCPIP. Tube #
Make a graph of absorbance vs. DCPIP concentration. The line should pass through the origin because at zero DCPIP concentration the absorbance will be zero. This graph, a standard curve, exemplifies Beer’s Law (i.e. up to a concentration limit, absorption is linearly related to the concentration of the absorbing compound in solution). [GRAPH REQUIRED]. The spectrophotometer can therefore be used to determine an unknown solution concentration if you first prepare a standard curve of known concentrations of that substance vs. absorbance. The absorption coefficient (E) for DCPIP in deionized water can be calculated from the slope of the standard curve: If l = I cm, then A = Ec E = A/c Calculate the value for E (the absorption coefficient). You will use this derived E value to analyse data obtained in the next lab class.
Page 4: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013)
Lab Class #2: DICHLOROPHENOL INDOPHENOL (DCPIP) We will be using the dye 2,6-dichlorophenolindophenol (DCPIP) as an artificial electron acceptor in our experimental work on the rate of electron flow from photosystem II in isolated chloroplasts. In the oxidized state, DCPIP is blue. When it is reduced by gaining electrons, DCPIP becomes colourless. The reduction of DCPIP can therefore be monitored with a spectrophotometer by measuring the decrease in absorbance as it is decolourized. To optimize the data obtained from using DCPIP as an electron acceptor, we need to determine two things:(i) The wavelength of light that is maximally absorbed by DCPIP. (You determined the max for DCPIP from its absorption spectrum in lab class #1). (ii) The range of DCPIP concentration over which a plot of concentration vs. absorbance is linear. This is determined by producing a standard curve of DCPIP concentration vs. absorbance at the wavelength at which DCPIP shows maximum absorbance (λmax). (also done in lab class #1). So far you have used the spectrophotometer to (i) produce characteristic absorption spectra for compounds in solution and (ii) to determine an unknown concentration for a specific compound in solution by preparing a standard curve of solute concentration vs. absorbance. Another major use of this instrument (Lab Notes, p. 13) is to determine the rate of a (bio)chemical reaction. 1. Experiment to determine the rate of reduction of DCPIP by different concentrations of the reducing agent, sodium hydrosulphite Protocol. Prepare 7 tubes according to the following table: Tube # ______ 1 2 3 4 5 6 CONTROL 7 BLANK
Room temperature =
1 x 10-4 M DCPIP (mL)
Deionized water (mL)
Deionized water (L)
1.5 1.5 1.5 1.5 1.5 1.5 0
3.5 3.5 3.5 3.5 3.5 3.5 5.0
0 10 20 30 40 -
C
505 mM sodium hydrosulphite (L) 50 40 30 20 10 * -
[Best practice: Instead of adding 1.5 mL of dye and 3.5 mL of D.I. water individually to each of tubes 1 to 6, make a bulk solution by mixing 10.5 mL of stock DCPIP and 24.5 mL of D.I. water in a beaker. Then transfer 5 mL aliquots to each of tubes 1 – 6. This is more efficient and all of the components in tubes 1 – 6 will be at exactly the same concentration]. Read the initial absorbance (zero time) of the solution in tube #1 tube just before you add the reducing agent. Then start timing when you add the 50 L of 5.05 mM sodium hydrosulphite and invert the tube three times to mix the contents. When 30 seconds have elapsed from the time that you added the reducing agent, take the absorbance reading. Then take absorbance readings at 60, 90 and 120 seconds from the time you added the reducing agent. Reactions in tubes 2 – 5 are started by adding 40, 30, 20 and 10 L of reducing agent respectively. * The control is started by adding 50 L of deionized water.
Page 5: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013)
Draw a single graph to show the A (change in absorbance) vs. time for reactions in tubes 1 – 5 to illustrate the kinetics of these reactions. [GRAPH REQUIRED]. Make a second graph to the rate of DCPIP reduction (moles/minute) vs. the initial concentration of the reducing agent present in each reaction. [GRAPH REQUIRED]. 2. Calculating the average rate of reaction during the first minute of each reaction using A values and the absorption coefficient (E) for DCPIP derived from the slope of the standard curve.
A600
In the example standard curve for DCPIP shown below, the slope of the line (the absorption coefficient or E) = 16,400 litres/mole/cm. As an example, if the absorbance of a reaction mixture is 0.500 at the start of the reaction and declines to 0.336 in the first minute of the reaction, then the change in absorbance during the first minute (the A/min) is 0.500 – 0.336 = 0.164/min. 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0
0.5 1 1.5 2 DCPIP concentration (m oles/L x 10-5)
2.5
The change in the DCPIP concentration during the first minute (c/min) can be calculated using the absorption coefficient (E).
0.164 A / min = 1 x 10-5 moles/litre of DCPIP reduced/minute = E 16,400 Since you are using 5.0 mL of DCPIP solution plus 50 L of reducing agent, the actual amount of 5.05ml DCPIP reduced in 1 minute = x (1 x 10-5 M) = 5.05 x 10-8 moles of DCPIP 1000ml / L reduced/minute (average rate). c/min =
Preparation of a buffer solution. In the next class you will be measuring the rate of reduction of DCPIP by illuminated chloroplasts isolated from spinach leaves. Like most biological reactions, this reaction is very sensitive to changes in pH. Consequently, the reactions will be run in the presence of a buffer solution to minimize changes in pH during the course of the reactions. In preparation for the next class, an instructor will show your group how to make this buffer solution. Instructions for preparing this buffer are on pages 40 – 41 in the Resource Manual.
Page 6: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013)
Lab Class #3: Experimental determination of rates of DCPIP photoreduction by a suspension of chloroplasts isolated from spinach leaves
The experience you have gained in using the Spectronic 20 to measure reaction rates will be used to study a biological phenomenon: namely, the rate at which electrons flow from photosystem II in illuminated, isolated spinach chloroplasts. A brief description of this phenomenon will be given in class (you should already have reviewed the notes on light harvesting and electron transport in photosynthesis, p. 43-45, Resource Manual). In summary, photons of light are absorbed by pigment molecules in the antenna complexes surrounding photosystem II in the thylakoid membranes. Some of the absorbed energy is funneled towards a pair of chlorophyll a molecules (the reaction centre) in photosystem II which respond by emitting electrons that sequentially pass through other electron acceptor molecules to photosystem I. The electrons lost from photosystem II are replaced by electrons released in the oxidation of water. (See Resource Manual, p. 43-45). Oxidized DCPIP can be used to intercept the electrons somewhere between photosystem II and photosystem I and as it does so, it becomes reduced and loses its blue colour. The rate at which DCPIP loses colour, detected by a decrease in its A600 with time, is proportional to the number of electrons that the dye has accepted from photosystem II. (1) Protocol for Chloroplast Isolation (Performed by your instructor). (i) Weigh out 25 grams of deribbed spinach leaves. (ii) Chop leaves into small pieces with scissors and place them in a chilled Waring blender with 100 mL cold isolation buffer (the recipe for this buffer is given in qu. 9 on p. 63, Resource Manual) to which 0.2L/mL of -mercaptoethanol has been added. (iii) Blend the leaves with 3 x 5 second bursts at full speed in the blender. (iv) Filter the resulting suspension through 4 layers of cheesecloth into a chilled beaker (Volume = approximately 75 mL). (v) Centrifuge the filtrate at 1300 x g. (see Resource Manual, p. 19-20) for 5 minutes. (vi) Discard the supernatant and add isolation buffer to the pellet to a total volume equal to ~0.5 mL/g spinach leaves used. Resuspend the pellet with a paint brush. This suspension is the chloroplast preparation. Determination of Chlorophyll concentration in the chloroplast suspension. (See Resource Manual p. 20). Briefly stated, 50 L of the suspended chloroplast preparation is added to 5 mL of 80% (v/v) aqueous acetone and the mixture is centrifuged to remove debris. The A652 of the resulting solution = _______. The concentration of chlorophyll in the chloroplast suspension is calculated as follows: mg of chlorophyll/mL = A652 ___________ x 100 34.5 mL/mgcm = _________mg/mL = _________ g/L the volume of chloroplast suspension that contain 20 g of chlorophyll: = 20 g ________ g/L = __________ L of the chloroplast suspension.
Page 7: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013)
(2) Determination of the rate of photreduction of DCPIP by an isolated chloroplast suspension Protocol (1) Prepare 10 tubes according to the following table but do not add the chloroplast suspension yet: Tube # ____________ 1 (blank) 2 3 4 5 6 7 8 9 10 (control)
1 x 10-4 M Reaction Deionized Chloroplast buffer (mL) water (mL) suspension (L*) DCPIP (mL) 0 1.0 4.0 * 1.5 1.0 2.5 * 1.5 1.0 2.5 * 1.5 1.0 2.5 * 1.5 1.0 2.5 * 1.5 1.0 2.5 * 1.5 1.0 2.5 * 1.5 1.0 2.5 * 1.5 1.0 2.5 * 1.5 1.0 2.5 * * a volume of chloroplast suspension containing 20 g of chlorophyll.
(2) There is a piece of masking tape marked in 5 cm increments from 0 up to 40 cm on the bench. Place the lamp over the 0 cm ruling. Reaction tubes are supported in a 100 mL beaker. The 100 mL beaker can be placed astride any of the ruled lines on the masking tape to vary the amount of light falling on the reaction mixtures (see diagram below)
(3) Add a volume of chloroplast suspension containing 20 g of chlorophyll to the blank (tube #1) and invert three times to mix. Use the blank solution to set the spectrophotometer to zero absorbance. (4) Working quickly, add a volume of chloroplast suspension containing 20 g of chlorophyll to tube #2, invert to mix and record the absorbance of the reaction mixture at 600 nm (= A600 at 0 minutes). (5) Place the reaction mixture in a beaker astride the line at 5 cm from the lamp and start timing when you turn on the lamp. (6) After 55 seconds have elapsed, put the tube into the spectrophotometer and record the 1 minute absorbance reading (do not turn off the lamp and do not stop timing). (7) Immediately return the tube to the beaker at 5 cm from the lamp and repeat the procedure at 55 seconds into the second minute and 55 seconds into the third minute.
Page 8: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013)
(8) Repeat steps 4 – 7 with the remaining reaction mixtures at 10, 15, 20, 25, 30 35 or 40 cm away from the lamp respectively. (9) The control (tube #10) is treated identically except that it is not illuminated and is wrapped in aluminum foil between A600 readings. Rate calculations. (a) Subtract the A600/ 2 minutes for the control (tube #10) from the A600/ 2 minutes for each of the other reactions to derive the corrected A600/ 2 minutes for each reaction. (b) Divide each corrected A600/ 2 minutes by 2 to derive the corrected A600/ minute value for each reaction. (c) Divide each corrected A600/minute by the slope (the absorption coefficient or E) of the new standard curve at pH 7.5 (see 4, below) to determine rates in terms of moles/litre of DCPIP photoreduced/2 minutes. (d) Correct for the volume of the reaction mixture by multiplying each corrected A600/minute by the total volume of the reaction mixture (5.0 mL plus the volume of the chloroplast suspension[?]) expressed as a fraction of a litre, i.e: corrected A600/minute x 5.0? mL = moles of DCPIP photoreduced/minute. 1000 mL/L (e) Divide by the number of g of chlorophyll in the volume of chloroplast suspension used (typically 20 g) = moles of DCPIP photoreduced/minute/g of chlorophyll.
The rate of photoreduction in each tube should be plotted against the photon fluence rate measured at each experimental distance from the lamp (see 3, below). [GRAPH REQUIRED] (3) Measuring the photon fluence rate with a quantum sensor. (See Resource Manual, p. 45. Light Ouantity: fluence measurements). Use the Li-Cor quantum sensor to measure the photon fluence rate, in moles photons/m2/sec, at each distance from the lamp that you ran the reactions. How does the photon fluence rate change with distance from the lamp? Make a graph of photon fluence rate vs. distance from the lamp. [GRAPH REQUIRED]. (4) Make a new standard curve for DCPIP at pH 7.5. (see table below). [GRAPH REQUIRED] Tube # 1 x 10-4M DCPIP A600 1 2.0 mL 2 1.5 mL 3 1.0 mL 4 0.5 mL 5 Blank 0
5x reaction buffer
Water
[DCPIP]
1.0 1.0 1.0 1.0 1.0
2.0 2.5 3.0 3.5 4.0
4 x 10-5M
0
Page 9: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013)
Lab class #4: The effect of different concentrations of dichlorophenyl dimethyl urea (DCMU) on the rate of photoreduction of DCPIP by isolated chloroplasts In this class you will again measure the rate of photoreduction of DCPIP but, this time, in the presence of different concentrations of dichlorophenyl dimethyl urea (DCMU). DCMU is an agricultural herbicide marketed as Diuron™. When working with reagents that affect the rates of biological reactions, two questions are commonly asked: (i) Is the relationship between the reaction rate and the reagent concentration linear or is there another relationship between these two variables? (ii) If the reagent inhibits the reaction, what concentration of the reagent reduces the reaction rate to 50% of the rate in the absence of the inhibitor (the control rate)?
Protocol It is not necessary to repeat the protocol for the basic method. Just refer to the lab notebook page number on which the protocol from the previous class is recorded. You should, however, include the following information and the table below: Room temperature = C. The photon fluence rate used = moles of photons/m2/second. The volume of chloroplast suspension that contains 20 g of chlorophyll = L. Tube # 1 Blank 2 Control-dark 3 Control-no DCMU 4 5 6 7 8
1 x 10-4M DCPIP(mL) 0 1.5 1.5 1.5 1.5 1.5 1.5 1.5
5 x reaction Buffer(mL) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Deionized Water (mL) 4.0 2.5 2.5 2.5 2.5 2.5 2.5 2.5
Methanol (L) 50 50 50 40 30 20 10 0
30.3 M DCMU(L) 0 0 0 10 20 30 40 50
[Best practice: Instead of adding 1.5 mL of dye, 1.0 mL of buffer and 2.5 mL of D.I. water individually to each of tubes 2 to 8, make a bulk solution by mixing 12.0 mL of stock DCPIP, 8.0 mL of buffer solution and 20. mL of D.I. water in a beaker. Then transfer 5 mL aliquots to each of tubes 2 – 8]. [BAR GRAPH REQUIRED]. At the start of lab class #4, the instructor will describe the components of the microscopes and show you how to set them up for optimal optical performance. The instructor will also explain the logic of using a haemacytometer (Resource Manual, p. 17 – 19) to determine the number of cells/mL in liquid suspension cultures of cells.
Page 10: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013)
Lab class #5: In this class you will use a microscope, a haemacytometer and a spectrophotometer to determine the number of cell/mL in a liquid suspension cell culture. These methods will be used to start a simple experiment that will be completed in lab class #6.
Cells and cell culture medium Each group will be provided with 5 mL of a suspension of yeast cells (Saccharomyces cerevisiae). The cell cultures will have been incubated for 48 hours at room temperature, on a rotary shaker, in a sterile culture medium called yeast carbon base (YCB). YCB contains all of the nutrients needed to sustain the cells except for a source of nitrogen. To provide the necessary nitrogen source, the cultures are supplemented with 0.05% (w/v) of the amino acid asparagine. Because YCB absorbs light to some extent, it is useful to separate the cells from the culture medium and resuspend them in deionized water. This allows us to use deionized water as the reference blank when working with the spectrophotometer. We can separate the cells from the growth medium using a centrifuge (Resource Manual, p. 19 – 20). Centrifugation (a) Place tubes in the swinging buckets in the centrifuge. Make sure that the rotor is balanced. Close the lid. (b) Turn on the centrifuge at full speed (# 7) and wait for 30 seconds before turning off the power. (c) Wait for the rotor to stop completely before removing the tubes. (d) Carefully discard the supernatant (growth medium) and add enough deionized water to bring the total volume to 5 mL. (e) Thoroughly resuspend the cells in the deionized water by aspiration with a Pasteur pipette. Care of the haemacytometer After each use, wash the haemacytometer chamber and its cover with warm water containing a small quantity of detergent. Rinse with deionized water and place the chamber and cover on a paper towel on a flat surface. Gently pat them dry with a Kimwipe™. Protocol: Determination of the number of cells/mL using a haemacytometer and preparation of a standard curve of the apparent absorbance at 400 nm (‘A400’) vs. the number of cells/mL (#cells/mL)
(1) Pellet cells in centrifuge (see Centrifugation – above), discard supernatant (growth medium), resuspend cells up to 5 ml in deionized water. (2) Make a 10-fold dilution of the resuspended cells (1.5 mL of cell suspension + 13.5 mL of D.I. water). (3) Load a sample of the 10-fold diluted cell suspension on to the haemacytometer. (4) Count the number of cells you see in 10 of the 0.04 mm2 squares (as explained in class): First grid #cells/square Second grid #cells/square Top left
___
Top left
___
Top right
___
Top right
___
Middle
___
Middle
___
Bottom left
___
Bottom left
___
Bottom right
___
Bottom right
___
(5) Calculate the average # cells in the 10 squares =
cells/square.
Page 11: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013) (6) Multiply the average #cells/square x 250,000 = cells/mL. (Place this value next to the * in the following table and use this value to calculate the #cells/mL in tubes # 2, 3 and 4). (7) Read the “A400” for tubes #1 – 4 and make a standard curve to show the relationship between the “A400” and the #cells/mL. Calculate the slope of the graph. Slope = Tube # 1 2 3 4 5 Blank
10-fold diluted suspension (mL) 5.0 3.0 2.0 1.0 0
Deionized water (mL) 0
# cells/mL
‘A400’
*
2.0 3.0 4.0 5.0
* # cells/ml from your first haemacytometer count. Experiment to determine the yield of Saccharomyces cerevisiae cells grown for 48 hours in media containing different concentrations of nitrogen (i.e.asparagine). Protocol:
Three duplicate pairs of cultures containing the following media will each be inoculated with 3.0 ml of a two day old culture of S. cerevisiae cells on the first day of the experiment. (a) 1A and 2A: 65.0 ml of 1.17% (w/v) yeast carbon base medium (no asparagine). (b) 1B and 2B: 65.0 ml of 1.17% (w/v) yeast carbon base medium + 0.01% (w/v) of asparagine. (c) 1C and 2C: 65.0 ml of 1.17% (w/v) yeast carbon base medium + 0.05% (w/v) of asparagine. On day zero (lab class #5), the initial number of cells/ml in one of each pair of cultures will be determined using both the haemacytometer and the spectrophotometer. The remaining 3 duplicate cultures will be maintained at room temperature for 48 hours. In the final lab you will estimate the number of cells/ml in these cultures using the haemacytometer and the spectrophotometer to determine the increase in the number of cells/ml in each treatment during the 48 hour incubation period. Final #cells/ml Yield = Original #cells/ml At the end of the experiment, produce a bar chart for the yield of cells against asparagine concentration. At each concentration, place two bars: one to show the yield as determined by using the haemacytometer, and one to show the yield as determined by spectrophotometry.
Page 12: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013) (a) Pellet the cells in 1A, 1B and 1C in the centrifuge, discard supernatant (growth medium), resuspend cells up to 5 ml in deionized water. (b) In turn, read the “A400” for 1A, 1B and 1C: “A400” for 1A =
slope of standard curve =
cells/mL
“A400” for 1B =
slope of standard curve =
cells/mL
“A400” for 1C =
slope of standard curve =
cells/mL
(c) In turn, load samples of resuspended 1A, 1B and 1C on to the haemacytometer. In each sample, count the number of cells you see in 10 of the 0.04 mm2 squares: Counts for 1A. First grid #cells/square
Second grid #cells/square
Top left
___
Top left
___
Top right
___
Top right
___
Middle
___
Middle
___
Bottom left
___
Bottom left
___
Bottom right
___
Bottom right
___
Calculate the average # cells in the 10 squares =
cells/square.
Multiply the average #cells/square x 250,000 =
original #cells/mL for 1A.
Counts for 1B. First grid #cells/square
Second grid #cells/square
Top left
___
Top left
___
Top right
___
Top right
___
Middle
___
Middle
___
Bottom left
___
Bottom left
___
Bottom right
___
Bottom right
___
Calculate the average # cells in the 10 squares =
cells/square.
Multiply the average #cells/square x 250,000 =
original #cells/mL for 1B.
Page 13: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013) Counts for 1C. First grid #cells/square
Second grid #cells/square
Top left
___
Top left
___
Top right
___
Top right
___
Middle
___
Middle
___
Bottom left
___
Bottom left
___
Bottom right
___
Bottom right
___
Calculate the average # cells in the 10 squares =
cells/square.
Multiply the average #cells/square x 250,000 =
original #cells/mL for 1C.
Day 0: Summary: original # cells/mL # of cells/mL in culture
Method
1a
1b
1c
Spectrophotometer Haemacytometer
Lab class #6: Your laboratory notebook should be handed in at the beginning of lab class #6. It should be complete up to and including the work for labs 1 – 5. After the second lab quiz, the instructor will explain the meaning of resolution in microscopy and the use of Abbe’s equation to calculate the theoretical limit to resolution in microscopy. We will then complete the experimental work started in lab class #5.
Resolution in microscopy.(Resource Manual, p.47–49) Calculation of the theoretical resolution of your microscope.
0.612 n sin to calculate d, the theoretical resolution of your microscope for both the x10 and x 40 objective lenses. Use a wavelength of 550 nm and give the answer in m. The numerical aperture (N.A.) value (n sin ) is stamped on each objective lens. N.A. for x 10 objective lens = 0.25 and for the x 40 objective lens = 0.66. Use the equation d =
Page 14: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013) Protocol to complete the experimental work. (a) Pellet the cells in 2A, 2B and 2C in the centrifuge, discard supernatant (growth medium), resuspend cells up to 5 ml in deionized water. (b) Dilute cell suspensions as necessary: Dilution factor for 2A =
-fold.
Dilution factor for 2B =
-fold.
Dilution factor for 2C =
-fold.
(c) In turn, read the “A400” for undiluted 2A,
-fold diluted 2B and
-fold diluted 2C:
“A400” for 2A =
slope of standard curve =
x (d.f.)
=
cells/mL
“A400” for 2B =
slope of standard curve =
x (d.f.)
=
cells/mL
“A400” for 2C =
slope of standard curve =
x (d.f.)
=
cells/mL
(d) In turn, load samples of resuspended undiluted 2A, -fold diluted 2B and -fold diluted 2C on to the haemacytometer. In each sample, count the number of cells you see in 10 of the 0.04 mm2 squares: Counts for 2A. First grid #cells/square
Second grid #cells/square
Top left
___
Top left
___
Top right
___
Top right
___
Middle
___
Middle
___
Bottom left
___
Bottom left
___
Bottom right
___
Bottom right
___
Calculate the average # cells in the 10 squares =
cells/square.
Multiply the average #cells/square x 250,000 =
final #cells/mL for 2A.
Page 15: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013) Counts for 2B. First grid #cells/square
Second grid #cells/square
Top left
___
Top left
___
Top right
___
Top right
___
Middle
___
Middle
___
Bottom left
___
Bottom left
___
Bottom right
___
Bottom right
___
Calculate the average # cells in the 10 squares =
cells/square.
Multiply the average #cells/square x 250,000 =
x d.f.
Counts for 2C. First grid #cells/square
=
final #cells/mL for 2B.
Second grid #cells/square
Top left
___
Top left
___
Top right
___
Top right
___
Middle
___
Middle
___
Bottom left
___
Bottom left
___
Bottom right
___
Bottom right
___
Calculate the average # cells in the 10 squares = Multiply the average #cells/square x 250,000 =
cells/square. x d.f.
=
Yield Calculations
By haemacytometer
By Spectrophotometer
(i)
2A 1A
(iv) 2A 1A
(ii) 2B 1B
(v) 2B 1B
(iii) 2C 1C
(vi) 2C 1C
final #cells/mL for 2C.
Page 16: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013)
OVERVIEW – EXPERIMENTAL SPECTROPHOTOMETRY Introduction –
Direct observation is not always possible in biological research: the biologist frequently has to use specialized equipment to follow the course of a biological process. An understanding of how the equipment works, and what it can and cannot do, is often important for producing reliable, reproducible information. In other words, many machines can generate numbers but these will only be meaningful if the instrument is correctly set up, properly operated and measuring in the range for which it is designed.
Spectrophotometry –
In this rotation, you will ultimately use a spectrophotometer to study the rate of electron flow from photosystem II in isolated chloroplasts.
However, you will first spend some time on seeing how the spectrophotometer is constructed and in understanding the theoretical aspects that are important in its use. You will then gain practical experience using the instrument in exercises designed to demonstrate its major uses: (1)
to produce absorption spectra that can be used to characterize or identify compounds in solution.
(2)
to make quantitative determinations of the concentration of materials in solution by reference to your own standard curves.
(3)
to study the rate at which (bio)chemical reactions proceed.
Having worked through the exercises, you should have the experience and skill required to use this technology in a more independent investigation. With the assistance of instructors and colleagues, you will carry out experiments to investigate factors affecting the rate of electron flow in isolated chloroplasts.
Important technical and background information can be found in the Spectrophotometry section of the Resource Manual and your first year biology textbook. Review your first year biology notes and the relevant section in the textbook to refresh your memory on the mechanism of the light-dependent stage of photosynthesis.
Page 17: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013)
Overview: Estimation of cell numbers. Introduction When working with cells in culture media, it is frequently necessary to know how many cells are present. Direct counts of the number of cells in a sample of culture medium can be made using a particular kind of microscope slide, the haemacytometer. On the upper mirrored surface of the haemacytometer are precisely etched lines that intersect to form squares of known area. With an appropriate type of cover in place, a known volume of fluid is enclosed above each square. Therefore, if the number of cells contained in the liquid above each square is counted, the number of cells in a given volume of the culture from which the sample was taken can be calculated.
Rapid estimates of cell numbers per unit volume of culture can be made with a spectrophotometer. A beam of light passing through a suspension of cells is scattered in proportion to the turbidity of the suspension. The turbidity of the suspension, over a limited range of cell concentration, is proportional to the number of cells per unit volume of suspension. To use this method, it is first necessary to make a standard curve of absorbance at a given wavelength versus the number of cells per millilitre. The number of cells per ml must be determined by some other method such as haemacytometry. In this class you will participate in an experiment to determine the yield of cells over a given time period as a function of the nutritional quality of the medium or other experimental variables.
Assigned reading, problems, quizzes, graphing exercises and schedule. (Lab classes #1-4) (1) Lab Notes p. 1-9 (2) Lab Notes p. 16 Overview (3) Resource Manual p. 28-31 Graphs for print publication. (4) Resource Manual p. 34-42 Ionic product of water, pH and buffers (solve problems)* (5) Resource Manual p. 42-43 Light (6) Resource Manual p. 49-51 Pipetting (7) Resource Manual p. 57-60 Spectrophotometry (try calculations on p.60)* (8) Resource Manual p. 90-91 Lab Notebook and Protocols (9) Resource Manual p. 6 – 9 Philosophy and ground rules (10) Resource Manual p. 67-68 Units of Measure & Exponents (solve problems)* (11) Resource Manual p. 43-45 Light reactions & Light quantity (12) Resource Manual p. 20 Chlorophyll (13) Resource Manual p. 61-64 Suspensions, solutions, concentrations (solve problems on pp. 63-64)* (Lab classes #5-6) (14) Lab Notes p. 10 - 15 (15) Lab Notes p. 17 Overview (16) Resource Manual p. 17-19 Cell counting (17) Resource Manual p. 19-20 Centrifugation (18) Resource Manual p. 45-49 Microscopy (19) Resource Manual p. 64-67 Dilutions (solve problems on pp.66 - 67)* * Solutions to problems in the Resource Manual are on p.54-57 of the Resource Manual. Lab class #1
Lab class #2 Graph assignment #1 set
Lab class #3
Lab class #5
Graph assignment #1 due
Graph assignment #2 due
Lab class #4 Lab Quiz #1: 4 questions on (7), (10) and (13). Graph assignment #2 set
Lab class #6 Hand in lab notebook at the start of class. Lab Quiz #2: 5 questions on (4) & (19)
Grading for this unit. A total of 14% of the course mark will be available for the following: Graph assignment #1 1% Graph assignment #2 2% Lab Quiz #1 4% Lab Quiz #2 5% Lab Book 2% Total 14%
Page 2: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013)
General Introduction Read “The Laboratory Notebook” in the Resource Manual (p. 90-91). Follow these guidelines for keeping your lab notebook. It should be clear what each graph, table or piece of information represents. Information should be clearly titled and recorded on numbered and dated pages. Experimental Spectrophotometry OBJECTIVES: At the end of the 6 lab classes in this unit you should be able to: (1) draw a diagram of the optical system of the Spectronic 20 and explain how the instrument works. (2) describe what is meant by transmittance (T) and absorbance (A) and be able to convert transmittance values to absorbance values. Use the equation A = Ecl to calculate A, c or E. (3) use the spectrophotometer with confidence. (4) state Lambert’s Law and Beer’s Law in words. (5) make an appropriate reference blank to zero the spectrophotometer in an experimental situation. (6) give an example of each of the principal uses of the spectrophotometer (Lab Notes, p.16). (7) calculate reaction rates using spectrophotometric data (A/time). (8) describe the meaning and units of photosynthetic photon flux (Resource Manual, p. 45). (9) explain the meaning of resolution in microscopy and the theoretical limit to resolution by reference to Abbe’s Equation (Resource Manual, p. 47-49). (10) use S.I. units of measure and exponents (scientific notation) with confidence. (11) calculate molar concentrations and dilutions. (12) remember and use the following equations: [H+][OH-] = 1 x 10-14, pH = - log[H+], base . [H+] = 10-pH, pOH = - log[OH-], [OH-] = 10-pOH, pH + pOH = 14, pH = pKa + log acid (13) make a buffer solution and explain how it minimizes changes in pH. (14) be aware of factors to consider when selecting a buffer solution for a specific experimental context (Resource Manual, p. 41-42). (15) know how to determine the number of cells/mL using a haemacytometer and a spectrophotometer. (16) design an experiment, with appropriate controls, to investigate the rate of electron flow from photosystem II in isolated, illuminated chloroplasts. Lab Class #1: Introduction to spectrophotometry (Resource Manual, p. 57–60). (1) An image of the optical system of the Spectronic 20 (Resource Manual. p. 59) will be displayed and a dismantled model is available for inspection. (2) In your lab book, make a table with the following headings: Transmittance (T)
%T
Log %T
Absorbance (A)
1.0 0.9 etc.
100 90 etc.
2.0 1.95 etc.
0 0.046 etc.
(Suggested values for T = 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, and 0.1) Use the equation A = log
1 to calculate the values for A. T
Page 3: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013)
(a) Use the data in the table to make a graph of A vs. T. (b) Use the data in the table to make a graph of A vs. Log % T. (All graphs must have a descriptive title). [GRAPHS REQUIRED]. (3) Absorption Spectra. Spectrophotometry can be used to identify or characterize compounds in solution by producing an absorption spectrum. An absorption spectrum is a graph of the absorbance values of a chemical compound in solution measured at a series of different wavelengths. We will make an absorption spectrum for a food colouring dye and a solution of 2, 6- dichlorophenol indophenol (DCPIP). Protocol Set up the spectrophotometer by following the instructions on page 60 in the Resource Manual. Solutions of a red food dye at a concentration of 0.06% (v/v) (Resource Manual, p. 61) and DCPIP at a concentration of 2 x 10-5 moles/litre will be provided. Record the absorbance value for each solution at 20 nm increments over the range of 340 to 600 nm. YOU MUST ZERO THE INSTRUMENT EACH TIME THAT YOU CHANGE THE WAVELENGTH. Plot all of the data on one graph and note the wavelength at which absorbance is maximal (the max) for each solution. [GRAPH REQUIRED]. (4) Standard Curve Protocol Use the stock DCPIP solution (1 x 10-4 mol/L) to make a dilution series according to the following table: 1 x 10-4 M Deionised DCPIP Absorbance DCPIP (mL) water (mL) concentration at max (= nm) 1 2.0 3.0 4 x 10-5 M 2 1.5 3.5 3 1.0 4.0 4 0.5 4.5 5 0 5.0 Read the absorbance (A) of each solution the wavelength of maximum (max ) absorbance for DCPIP. Tube #
Make a graph of absorbance vs. DCPIP concentration. The line should pass through the origin because at zero DCPIP concentration the absorbance will be zero. This graph, a standard curve, exemplifies Beer’s Law (i.e. up to a concentration limit, absorption is linearly related to the concentration of the absorbing compound in solution). [GRAPH REQUIRED]. The spectrophotometer can therefore be used to determine an unknown solution concentration if you first prepare a standard curve of known concentrations of that substance vs. absorbance. The absorption coefficient (E) for DCPIP in deionized water can be calculated from the slope of the standard curve: If l = I cm, then A = Ec E = A/c Calculate the value for E (the absorption coefficient). You will use this derived E value to analyse data obtained in the next lab class.
Page 4: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013)
Lab Class #2: DICHLOROPHENOL INDOPHENOL (DCPIP) We will be using the dye 2,6-dichlorophenolindophenol (DCPIP) as an artificial electron acceptor in our experimental work on the rate of electron flow from photosystem II in isolated chloroplasts. In the oxidized state, DCPIP is blue. When it is reduced by gaining electrons, DCPIP becomes colourless. The reduction of DCPIP can therefore be monitored with a spectrophotometer by measuring the decrease in absorbance as it is decolourized. To optimize the data obtained from using DCPIP as an electron acceptor, we need to determine two things:(i) The wavelength of light that is maximally absorbed by DCPIP. (You determined the max for DCPIP from its absorption spectrum in lab class #1). (ii) The range of DCPIP concentration over which a plot of concentration vs. absorbance is linear. This is determined by producing a standard curve of DCPIP concentration vs. absorbance at the wavelength at which DCPIP shows maximum absorbance (λmax). (also done in lab class #1). So far you have used the spectrophotometer to (i) produce characteristic absorption spectra for compounds in solution and (ii) to determine an unknown concentration for a specific compound in solution by preparing a standard curve of solute concentration vs. absorbance. Another major use of this instrument (Lab Notes, p. 13) is to determine the rate of a (bio)chemical reaction. 1. Experiment to determine the rate of reduction of DCPIP by different concentrations of the reducing agent, sodium hydrosulphite Protocol. Prepare 7 tubes according to the following table: Tube # ______ 1 2 3 4 5 6 CONTROL 7 BLANK
Room temperature =
1 x 10-4 M DCPIP (mL)
Deionized water (mL)
Deionized water (L)
1.5 1.5 1.5 1.5 1.5 1.5 0
3.5 3.5 3.5 3.5 3.5 3.5 5.0
0 10 20 30 40 -
C
505 mM sodium hydrosulphite (L) 50 40 30 20 10 * -
[Best practice: Instead of adding 1.5 mL of dye and 3.5 mL of D.I. water individually to each of tubes 1 to 6, make a bulk solution by mixing 10.5 mL of stock DCPIP and 24.5 mL of D.I. water in a beaker. Then transfer 5 mL aliquots to each of tubes 1 – 6. This is more efficient and all of the components in tubes 1 – 6 will be at exactly the same concentration]. Read the initial absorbance (zero time) of the solution in tube #1 tube just before you add the reducing agent. Then start timing when you add the 50 L of 5.05 mM sodium hydrosulphite and invert the tube three times to mix the contents. When 30 seconds have elapsed from the time that you added the reducing agent, take the absorbance reading. Then take absorbance readings at 60, 90 and 120 seconds from the time you added the reducing agent. Reactions in tubes 2 – 5 are started by adding 40, 30, 20 and 10 L of reducing agent respectively. * The control is started by adding 50 L of deionized water.
Page 5: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013)
Draw a single graph to show the A (change in absorbance) vs. time for reactions in tubes 1 – 5 to illustrate the kinetics of these reactions. [GRAPH REQUIRED]. Make a second graph to the rate of DCPIP reduction (moles/minute) vs. the initial concentration of the reducing agent present in each reaction. [GRAPH REQUIRED]. 2. Calculating the average rate of reaction during the first minute of each reaction using A values and the absorption coefficient (E) for DCPIP derived from the slope of the standard curve.
A600
In the example standard curve for DCPIP shown below, the slope of the line (the absorption coefficient or E) = 16,400 litres/mole/cm. As an example, if the absorbance of a reaction mixture is 0.500 at the start of the reaction and declines to 0.336 in the first minute of the reaction, then the change in absorbance during the first minute (the A/min) is 0.500 – 0.336 = 0.164/min. 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0
0.5 1 1.5 2 DCPIP concentration (m oles/L x 10-5)
2.5
The change in the DCPIP concentration during the first minute (c/min) can be calculated using the absorption coefficient (E).
0.164 A / min = 1 x 10-5 moles/litre of DCPIP reduced/minute = E 16,400 Since you are using 5.0 mL of DCPIP solution plus 50 L of reducing agent, the actual amount of 5.05ml DCPIP reduced in 1 minute = x (1 x 10-5 M) = 5.05 x 10-8 moles of DCPIP 1000ml / L reduced/minute (average rate). c/min =
Preparation of a buffer solution. In the next class you will be measuring the rate of reduction of DCPIP by illuminated chloroplasts isolated from spinach leaves. Like most biological reactions, this reaction is very sensitive to changes in pH. Consequently, the reactions will be run in the presence of a buffer solution to minimize changes in pH during the course of the reactions. In preparation for the next class, an instructor will show your group how to make this buffer solution. Instructions for preparing this buffer are on pages 40 – 41 in the Resource Manual.
Page 6: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013)
Lab Class #3: Experimental determination of rates of DCPIP photoreduction by a suspension of chloroplasts isolated from spinach leaves
The experience you have gained in using the Spectronic 20 to measure reaction rates will be used to study a biological phenomenon: namely, the rate at which electrons flow from photosystem II in illuminated, isolated spinach chloroplasts. A brief description of this phenomenon will be given in class (you should already have reviewed the notes on light harvesting and electron transport in photosynthesis, p. 43-45, Resource Manual). In summary, photons of light are absorbed by pigment molecules in the antenna complexes surrounding photosystem II in the thylakoid membranes. Some of the absorbed energy is funneled towards a pair of chlorophyll a molecules (the reaction centre) in photosystem II which respond by emitting electrons that sequentially pass through other electron acceptor molecules to photosystem I. The electrons lost from photosystem II are replaced by electrons released in the oxidation of water. (See Resource Manual, p. 43-45). Oxidized DCPIP can be used to intercept the electrons somewhere between photosystem II and photosystem I and as it does so, it becomes reduced and loses its blue colour. The rate at which DCPIP loses colour, detected by a decrease in its A600 with time, is proportional to the number of electrons that the dye has accepted from photosystem II. (1) Protocol for Chloroplast Isolation (Performed by your instructor). (i) Weigh out 25 grams of deribbed spinach leaves. (ii) Chop leaves into small pieces with scissors and place them in a chilled Waring blender with 100 mL cold isolation buffer (the recipe for this buffer is given in qu. 9 on p. 63, Resource Manual) to which 0.2L/mL of -mercaptoethanol has been added. (iii) Blend the leaves with 3 x 5 second bursts at full speed in the blender. (iv) Filter the resulting suspension through 4 layers of cheesecloth into a chilled beaker (Volume = approximately 75 mL). (v) Centrifuge the filtrate at 1300 x g. (see Resource Manual, p. 19-20) for 5 minutes. (vi) Discard the supernatant and add isolation buffer to the pellet to a total volume equal to ~0.5 mL/g spinach leaves used. Resuspend the pellet with a paint brush. This suspension is the chloroplast preparation. Determination of Chlorophyll concentration in the chloroplast suspension. (See Resource Manual p. 20). Briefly stated, 50 L of the suspended chloroplast preparation is added to 5 mL of 80% (v/v) aqueous acetone and the mixture is centrifuged to remove debris. The A652 of the resulting solution = _______. The concentration of chlorophyll in the chloroplast suspension is calculated as follows: mg of chlorophyll/mL = A652 ___________ x 100 34.5 mL/mgcm = _________mg/mL = _________ g/L the volume of chloroplast suspension that contain 20 g of chlorophyll: = 20 g ________ g/L = __________ L of the chloroplast suspension.
Page 7: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013)
(2) Determination of the rate of photreduction of DCPIP by an isolated chloroplast suspension Protocol (1) Prepare 10 tubes according to the following table but do not add the chloroplast suspension yet: Tube # ____________ 1 (blank) 2 3 4 5 6 7 8 9 10 (control)
1 x 10-4 M Reaction Deionized Chloroplast buffer (mL) water (mL) suspension (L*) DCPIP (mL) 0 1.0 4.0 * 1.5 1.0 2.5 * 1.5 1.0 2.5 * 1.5 1.0 2.5 * 1.5 1.0 2.5 * 1.5 1.0 2.5 * 1.5 1.0 2.5 * 1.5 1.0 2.5 * 1.5 1.0 2.5 * 1.5 1.0 2.5 * * a volume of chloroplast suspension containing 20 g of chlorophyll.
(2) There is a piece of masking tape marked in 5 cm increments from 0 up to 40 cm on the bench. Place the lamp over the 0 cm ruling. Reaction tubes are supported in a 100 mL beaker. The 100 mL beaker can be placed astride any of the ruled lines on the masking tape to vary the amount of light falling on the reaction mixtures (see diagram below)
(3) Add a volume of chloroplast suspension containing 20 g of chlorophyll to the blank (tube #1) and invert three times to mix. Use the blank solution to set the spectrophotometer to zero absorbance. (4) Working quickly, add a volume of chloroplast suspension containing 20 g of chlorophyll to tube #2, invert to mix and record the absorbance of the reaction mixture at 600 nm (= A600 at 0 minutes). (5) Place the reaction mixture in a beaker astride the line at 5 cm from the lamp and start timing when you turn on the lamp. (6) After 55 seconds have elapsed, put the tube into the spectrophotometer and record the 1 minute absorbance reading (do not turn off the lamp and do not stop timing). (7) Immediately return the tube to the beaker at 5 cm from the lamp and repeat the procedure at 55 seconds into the second minute and 55 seconds into the third minute.
Page 8: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013)
(8) Repeat steps 4 – 7 with the remaining reaction mixtures at 10, 15, 20, 25, 30 35 or 40 cm away from the lamp respectively. (9) The control (tube #10) is treated identically except that it is not illuminated and is wrapped in aluminum foil between A600 readings. Rate calculations. (a) Subtract the A600/ 2 minutes for the control (tube #10) from the A600/ 2 minutes for each of the other reactions to derive the corrected A600/ 2 minutes for each reaction. (b) Divide each corrected A600/ 2 minutes by 2 to derive the corrected A600/ minute value for each reaction. (c) Divide each corrected A600/minute by the slope (the absorption coefficient or E) of the new standard curve at pH 7.5 (see 4, below) to determine rates in terms of moles/litre of DCPIP photoreduced/2 minutes. (d) Correct for the volume of the reaction mixture by multiplying each corrected A600/minute by the total volume of the reaction mixture (5.0 mL plus the volume of the chloroplast suspension[?]) expressed as a fraction of a litre, i.e: corrected A600/minute x 5.0? mL = moles of DCPIP photoreduced/minute. 1000 mL/L (e) Divide by the number of g of chlorophyll in the volume of chloroplast suspension used (typically 20 g) = moles of DCPIP photoreduced/minute/g of chlorophyll.
The rate of photoreduction in each tube should be plotted against the photon fluence rate measured at each experimental distance from the lamp (see 3, below). [GRAPH REQUIRED] (3) Measuring the photon fluence rate with a quantum sensor. (See Resource Manual, p. 45. Light Ouantity: fluence measurements). Use the Li-Cor quantum sensor to measure the photon fluence rate, in moles photons/m2/sec, at each distance from the lamp that you ran the reactions. How does the photon fluence rate change with distance from the lamp? Make a graph of photon fluence rate vs. distance from the lamp. [GRAPH REQUIRED]. (4) Make a new standard curve for DCPIP at pH 7.5. (see table below). [GRAPH REQUIRED] Tube # 1 x 10-4M DCPIP A600 1 2.0 mL 2 1.5 mL 3 1.0 mL 4 0.5 mL 5 Blank 0
5x reaction buffer
Water
[DCPIP]
1.0 1.0 1.0 1.0 1.0
2.0 2.5 3.0 3.5 4.0
4 x 10-5M
0
Page 9: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013)
Lab class #4: The effect of different concentrations of dichlorophenyl dimethyl urea (DCMU) on the rate of photoreduction of DCPIP by isolated chloroplasts In this class you will again measure the rate of photoreduction of DCPIP but, this time, in the presence of different concentrations of dichlorophenyl dimethyl urea (DCMU). DCMU is an agricultural herbicide marketed as Diuron™. When working with reagents that affect the rates of biological reactions, two questions are commonly asked: (i) Is the relationship between the reaction rate and the reagent concentration linear or is there another relationship between these two variables? (ii) If the reagent inhibits the reaction, what concentration of the reagent reduces the reaction rate to 50% of the rate in the absence of the inhibitor (the control rate)?
Protocol It is not necessary to repeat the protocol for the basic method. Just refer to the lab notebook page number on which the protocol from the previous class is recorded. You should, however, include the following information and the table below: Room temperature = C. The photon fluence rate used = moles of photons/m2/second. The volume of chloroplast suspension that contains 20 g of chlorophyll = L. Tube # 1 Blank 2 Control-dark 3 Control-no DCMU 4 5 6 7 8
1 x 10-4M DCPIP(mL) 0 1.5 1.5 1.5 1.5 1.5 1.5 1.5
5 x reaction Buffer(mL) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Deionized Water (mL) 4.0 2.5 2.5 2.5 2.5 2.5 2.5 2.5
Methanol (L) 50 50 50 40 30 20 10 0
30.3 M DCMU(L) 0 0 0 10 20 30 40 50
[Best practice: Instead of adding 1.5 mL of dye, 1.0 mL of buffer and 2.5 mL of D.I. water individually to each of tubes 2 to 8, make a bulk solution by mixing 12.0 mL of stock DCPIP, 8.0 mL of buffer solution and 20. mL of D.I. water in a beaker. Then transfer 5 mL aliquots to each of tubes 2 – 8]. [BAR GRAPH REQUIRED]. At the start of lab class #4, the instructor will describe the components of the microscopes and show you how to set them up for optimal optical performance. The instructor will also explain the logic of using a haemacytometer (Resource Manual, p. 17 – 19) to determine the number of cells/mL in liquid suspension cultures of cells.
Page 10: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013)
Lab class #5: In this class you will use a microscope, a haemacytometer and a spectrophotometer to determine the number of cell/mL in a liquid suspension cell culture. These methods will be used to start a simple experiment that will be completed in lab class #6.
Cells and cell culture medium Each group will be provided with 5 mL of a suspension of yeast cells (Saccharomyces cerevisiae). The cell cultures will have been incubated for 48 hours at room temperature, on a rotary shaker, in a sterile culture medium called yeast carbon base (YCB). YCB contains all of the nutrients needed to sustain the cells except for a source of nitrogen. To provide the necessary nitrogen source, the cultures are supplemented with 0.05% (w/v) of the amino acid asparagine. Because YCB absorbs light to some extent, it is useful to separate the cells from the culture medium and resuspend them in deionized water. This allows us to use deionized water as the reference blank when working with the spectrophotometer. We can separate the cells from the growth medium using a centrifuge (Resource Manual, p. 19 – 20). Centrifugation (a) Place tubes in the swinging buckets in the centrifuge. Make sure that the rotor is balanced. Close the lid. (b) Turn on the centrifuge at full speed (# 7) and wait for 30 seconds before turning off the power. (c) Wait for the rotor to stop completely before removing the tubes. (d) Carefully discard the supernatant (growth medium) and add enough deionized water to bring the total volume to 5 mL. (e) Thoroughly resuspend the cells in the deionized water by aspiration with a Pasteur pipette. Care of the haemacytometer After each use, wash the haemacytometer chamber and its cover with warm water containing a small quantity of detergent. Rinse with deionized water and place the chamber and cover on a paper towel on a flat surface. Gently pat them dry with a Kimwipe™. Protocol: Determination of the number of cells/mL using a haemacytometer and preparation of a standard curve of the apparent absorbance at 400 nm (‘A400’) vs. the number of cells/mL (#cells/mL)
(1) Pellet cells in centrifuge (see Centrifugation – above), discard supernatant (growth medium), resuspend cells up to 5 ml in deionized water. (2) Make a 10-fold dilution of the resuspended cells (1.5 mL of cell suspension + 13.5 mL of D.I. water). (3) Load a sample of the 10-fold diluted cell suspension on to the haemacytometer. (4) Count the number of cells you see in 10 of the 0.04 mm2 squares (as explained in class): First grid #cells/square Second grid #cells/square Top left
___
Top left
___
Top right
___
Top right
___
Middle
___
Middle
___
Bottom left
___
Bottom left
___
Bottom right
___
Bottom right
___
(5) Calculate the average # cells in the 10 squares =
cells/square.
Page 11: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013) (6) Multiply the average #cells/square x 250,000 = cells/mL. (Place this value next to the * in the following table and use this value to calculate the #cells/mL in tubes # 2, 3 and 4). (7) Read the “A400” for tubes #1 – 4 and make a standard curve to show the relationship between the “A400” and the #cells/mL. Calculate the slope of the graph. Slope = Tube # 1 2 3 4 5 Blank
10-fold diluted suspension (mL) 5.0 3.0 2.0 1.0 0
Deionized water (mL) 0
# cells/mL
‘A400’
*
2.0 3.0 4.0 5.0
* # cells/ml from your first haemacytometer count. Experiment to determine the yield of Saccharomyces cerevisiae cells grown for 48 hours in media containing different concentrations of nitrogen (i.e.asparagine). Protocol:
Three duplicate pairs of cultures containing the following media will each be inoculated with 3.0 ml of a two day old culture of S. cerevisiae cells on the first day of the experiment. (a) 1A and 2A: 65.0 ml of 1.17% (w/v) yeast carbon base medium (no asparagine). (b) 1B and 2B: 65.0 ml of 1.17% (w/v) yeast carbon base medium + 0.01% (w/v) of asparagine. (c) 1C and 2C: 65.0 ml of 1.17% (w/v) yeast carbon base medium + 0.05% (w/v) of asparagine. On day zero (lab class #5), the initial number of cells/ml in one of each pair of cultures will be determined using both the haemacytometer and the spectrophotometer. The remaining 3 duplicate cultures will be maintained at room temperature for 48 hours. In the final lab you will estimate the number of cells/ml in these cultures using the haemacytometer and the spectrophotometer to determine the increase in the number of cells/ml in each treatment during the 48 hour incubation period. Final #cells/ml Yield = Original #cells/ml At the end of the experiment, produce a bar chart for the yield of cells against asparagine concentration. At each concentration, place two bars: one to show the yield as determined by using the haemacytometer, and one to show the yield as determined by spectrophotometry.
Page 12: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013) (a) Pellet the cells in 1A, 1B and 1C in the centrifuge, discard supernatant (growth medium), resuspend cells up to 5 ml in deionized water. (b) In turn, read the “A400” for 1A, 1B and 1C: “A400” for 1A =
slope of standard curve =
cells/mL
“A400” for 1B =
slope of standard curve =
cells/mL
“A400” for 1C =
slope of standard curve =
cells/mL
(c) In turn, load samples of resuspended 1A, 1B and 1C on to the haemacytometer. In each sample, count the number of cells you see in 10 of the 0.04 mm2 squares: Counts for 1A. First grid #cells/square
Second grid #cells/square
Top left
___
Top left
___
Top right
___
Top right
___
Middle
___
Middle
___
Bottom left
___
Bottom left
___
Bottom right
___
Bottom right
___
Calculate the average # cells in the 10 squares =
cells/square.
Multiply the average #cells/square x 250,000 =
original #cells/mL for 1A.
Counts for 1B. First grid #cells/square
Second grid #cells/square
Top left
___
Top left
___
Top right
___
Top right
___
Middle
___
Middle
___
Bottom left
___
Bottom left
___
Bottom right
___
Bottom right
___
Calculate the average # cells in the 10 squares =
cells/square.
Multiply the average #cells/square x 250,000 =
original #cells/mL for 1B.
Page 13: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013) Counts for 1C. First grid #cells/square
Second grid #cells/square
Top left
___
Top left
___
Top right
___
Top right
___
Middle
___
Middle
___
Bottom left
___
Bottom left
___
Bottom right
___
Bottom right
___
Calculate the average # cells in the 10 squares =
cells/square.
Multiply the average #cells/square x 250,000 =
original #cells/mL for 1C.
Day 0: Summary: original # cells/mL # of cells/mL in culture
Method
1a
1b
1c
Spectrophotometer Haemacytometer
Lab class #6: Your laboratory notebook should be handed in at the beginning of lab class #6. It should be complete up to and including the work for labs 1 – 5. After the second lab quiz, the instructor will explain the meaning of resolution in microscopy and the use of Abbe’s equation to calculate the theoretical limit to resolution in microscopy. We will then complete the experimental work started in lab class #5.
Resolution in microscopy.(Resource Manual, p.47–49) Calculation of the theoretical resolution of your microscope.
0.612 n sin to calculate d, the theoretical resolution of your microscope for both the x10 and x 40 objective lenses. Use a wavelength of 550 nm and give the answer in m. The numerical aperture (N.A.) value (n sin ) is stamped on each objective lens. N.A. for x 10 objective lens = 0.25 and for the x 40 objective lens = 0.66. Use the equation d =
Page 14: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013) Protocol to complete the experimental work. (a) Pellet the cells in 2A, 2B and 2C in the centrifuge, discard supernatant (growth medium), resuspend cells up to 5 ml in deionized water. (b) Dilute cell suspensions as necessary: Dilution factor for 2A =
-fold.
Dilution factor for 2B =
-fold.
Dilution factor for 2C =
-fold.
(c) In turn, read the “A400” for undiluted 2A,
-fold diluted 2B and
-fold diluted 2C:
“A400” for 2A =
slope of standard curve =
x (d.f.)
=
cells/mL
“A400” for 2B =
slope of standard curve =
x (d.f.)
=
cells/mL
“A400” for 2C =
slope of standard curve =
x (d.f.)
=
cells/mL
(d) In turn, load samples of resuspended undiluted 2A, -fold diluted 2B and -fold diluted 2C on to the haemacytometer. In each sample, count the number of cells you see in 10 of the 0.04 mm2 squares: Counts for 2A. First grid #cells/square
Second grid #cells/square
Top left
___
Top left
___
Top right
___
Top right
___
Middle
___
Middle
___
Bottom left
___
Bottom left
___
Bottom right
___
Bottom right
___
Calculate the average # cells in the 10 squares =
cells/square.
Multiply the average #cells/square x 250,000 =
final #cells/mL for 2A.
Page 15: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013) Counts for 2B. First grid #cells/square
Second grid #cells/square
Top left
___
Top left
___
Top right
___
Top right
___
Middle
___
Middle
___
Bottom left
___
Bottom left
___
Bottom right
___
Bottom right
___
Calculate the average # cells in the 10 squares =
cells/square.
Multiply the average #cells/square x 250,000 =
x d.f.
Counts for 2C. First grid #cells/square
=
final #cells/mL for 2B.
Second grid #cells/square
Top left
___
Top left
___
Top right
___
Top right
___
Middle
___
Middle
___
Bottom left
___
Bottom left
___
Bottom right
___
Bottom right
___
Calculate the average # cells in the 10 squares = Multiply the average #cells/square x 250,000 =
cells/square. x d.f.
=
Yield Calculations
By haemacytometer
By Spectrophotometer
(i)
2A 1A
(iv) 2A 1A
(ii) 2B 1B
(v) 2B 1B
(iii) 2C 1C
(vi) 2C 1C
final #cells/mL for 2C.
Page 16: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013)
OVERVIEW – EXPERIMENTAL SPECTROPHOTOMETRY Introduction –
Direct observation is not always possible in biological research: the biologist frequently has to use specialized equipment to follow the course of a biological process. An understanding of how the equipment works, and what it can and cannot do, is often important for producing reliable, reproducible information. In other words, many machines can generate numbers but these will only be meaningful if the instrument is correctly set up, properly operated and measuring in the range for which it is designed.
Spectrophotometry –
In this rotation, you will ultimately use a spectrophotometer to study the rate of electron flow from photosystem II in isolated chloroplasts.
However, you will first spend some time on seeing how the spectrophotometer is constructed and in understanding the theoretical aspects that are important in its use. You will then gain practical experience using the instrument in exercises designed to demonstrate its major uses: (1)
to produce absorption spectra that can be used to characterize or identify compounds in solution.
(2)
to make quantitative determinations of the concentration of materials in solution by reference to your own standard curves.
(3)
to study the rate at which (bio)chemical reactions proceed.
Having worked through the exercises, you should have the experience and skill required to use this technology in a more independent investigation. With the assistance of instructors and colleagues, you will carry out experiments to investigate factors affecting the rate of electron flow in isolated chloroplasts.
Important technical and background information can be found in the Spectrophotometry section of the Resource Manual and your first year biology textbook. Review your first year biology notes and the relevant section in the textbook to refresh your memory on the mechanism of the light-dependent stage of photosynthesis.
Page 17: Biology 2290F Lab Notes (Dean Unit, North Campus Building, Room 325) (Fall 2013)
Overview: Estimation of cell numbers. Introduction When working with cells in culture media, it is frequently necessary to know how many cells are present. Direct counts of the number of cells in a sample of culture medium can be made using a particular kind of microscope slide, the haemacytometer. On the upper mirrored surface of the haemacytometer are precisely etched lines that intersect to form squares of known area. With an appropriate type of cover in place, a known volume of fluid is enclosed above each square. Therefore, if the number of cells contained in the liquid above each square is counted, the number of cells in a given volume of the culture from which the sample was taken can be calculated.
Rapid estimates of cell numbers per unit volume of culture can be made with a spectrophotometer. A beam of light passing through a suspension of cells is scattered in proportion to the turbidity of the suspension. The turbidity of the suspension, over a limited range of cell concentration, is proportional to the number of cells per unit volume of suspension. To use this method, it is first necessary to make a standard curve of absorbance at a given wavelength versus the number of cells per millilitre. The number of cells per ml must be determined by some other method such as haemacytometry. In this class you will participate in an experiment to determine the yield of cells over a given time period as a function of the nutritional quality of the medium or other experimental variables.
