Spectral Analysis of Photosynthetic Pigments

Chlorophyll and accessory pigments are used by leaves to collect energy from light and transduce it to chemical energy be used to synthesize sugar. These pigments can be separated by chromatography in which a solvent system travels through paper by capillary action, carrying the pigments with it. Because each pigment has a different solubility and/or affinity for paper, they move at different rates, and separate along the paper. Pigments separated in the way in a previous lab have varying absorption spectra which may be demonstrated using the technique for determining the absorption spectra.

Alternatively, simple analysis of the composition of plants may be performed by drying, extracting with various organic solvents, and determining the absorbtion spectra of each extract. In this experiment, we will determine percent water, and relative pigment concentrations in a variety of plant materials, notably salad greens.

NOTE:

It is required that you know how to use a spectrophotometer and correctly plot data on a graph to complete this lab. Ask the instructor for previous protocols if you lack this knowledge.

Questions:
Which solvent is most effective extracting chlorophyll?
How does the chlorophyll content compare among salad greens?
What differences do you note when comparing spectra of various greens?
How does the absorption spectrum of plant extract compare with known dyes?
Might the concentration of photosynthetic pigments correlate with the nutritional quality of these salad greens?

Supplies:

  • Variety of salad greens, fresh and unfaded
  • Organic solvents:
    • 95% ethyl alcohol
    • petroleum ether
    • acetone, etc.
  • Whatman #1 filter paper
  • Diluted standard microbiological stains:
  • Hucker’s: 1.0 uL into 10 mL dH2O
  • Safranin O: 3.0 uL into 3 mL dH2O
  • Methylene Blue: 2.0 uL into 3 mL dH2O

Equipment:

  • Large strainers with feet
  • Drying oven, 80°C
  • Balance
  • Mortar and pestle
  • 16 x 150 mm test tubes with corks
  • Filter funnel
  • Spectrophotometer
  • 5 cuvettes in plastic rack
  • Lens paper

Protocol:
Continued from the extraction protocol…

8. Dilute filtered extract 1:10: 5.0 mL EtOH + 0.555 mL extract (we are ignoring effect of diluting alternate solvents into EtOH.).

9. Prepare cuvettes: Rinse five cuvettes with 95% EtOH. Fill each with 3.00 mL EtOH, polish outside, read A350. If the difference is >than 0.005, clean and polish again. Mark the cuvette with the lowest A350 as “B” (blank), the others S1, S2, etc (S = sample).
10. Read absorbencies at 350 nm
11. Dilute the extract to adjust absorbency so that A350 ~ 0.800 to 1.000

12. Read absorption spectra for all group samples every 25 nm from 350-800 nm. (Read all samples at a given wavelength, then re zero and reblank for the next wavelength and read all samples at that wavelength, etc). Rotate reading and recording roles. Enter into the computer as instructed.

13. Graph the data: Graph all data on the same graph, plotting wavelength (on the X axis) versus absorbance (on the Y axis), noting maxima for each of the samples. Follow graphing protocol previously distributed. Note maxima for each of the solutions tested. Which of the original questions can you answer? What conclusions do you draw?

Bacterial Growth Curve

Bacterial Growth Curve

Escherichia coli Grown on Minimal Salts Versus Complex Media

Bacteria display a characteristic four-phase pattern of growth in liquid culture. The initial Lag phase is a period of slow growth during which the bacteria are adapting to the conditions in the fresh medium. This is followed by a Log Phase during which growth is exponential, doubling every replication cycle. Stationary Phase occurs when the nutrients become limiting, and the rate of multiplication equals the rate of death. Logarithmic Decline Phase occurs when cells die faster than they are replaced. (This latter occurs over a much longer period of time that the previous three.)

We will study the patterns of aerated growth in minimal salts medium (either Cold Spring Harbor A + 0.1% glucose (CSHA), or Vogel’s E + 0.1% glucose (VE)) compared to growth in a complex medium (Tryptic Soy Broth = TSB). Bacterial population in the culture will be estimated by measuring its turbidity, (to which it is proportional) a using spectrophotometer. Turbidity is classically measured as the absorbance at 660 nanometers wavelength. You should record the collected data and make two graphs of it in your notebook: one on a linear scale, and one on a semi-log scale. Labeled the phases and determine the doubling times for each medium.

EQUIPMENT
compressed air source or air pump
humidification flask
manifold with spaghetti tubing (be sure the tubing is in good repair
sterile 16 x 150 bubbler tubes
37C hot block, to accept 16 mm tubes
Timex “Triathlon”® watch
sterile pipettes (5 & 1 mL)
two spectrophotometers (one each for CSHA & TSB)

SUPPLIES
Nutrient Broth and/or Tryptic Soy Broth
Cold Spring Harbor A minimal medium with 0.1% glucose
10x Cold Spring Harbor A Medium
(NH4)2SO4 10 g
K 2HPO4 105 g
KH2PO4 45 g
MgSO4•H2O 1g
dissolve in dH2O, q.s. to 1 L
E. coli stationary overnight culture

PROCEDURE

growth_curve_setup_16mm_tubes_P7130008

Set up and illustrate:
GROWTH CURVE APPARATUS (here is a labeled image):
air pump bubbles air through a humidification flask, the humidified air is piped to a valved manifold, which is connected by spaghetti tubing to bubbler tubes, set in the holes of a pre-heated 37C hot block.
To simplify readings, use two spectrophotometers, one blanked to the minimal salts medium, the other blanked to the TSB.

Inoculate 0.02 mL of an overnight shaken culture (left image) of Escherichia coli into 5.0 mL Tryptic Soy Broth (right image) and into 5.0 mL of minimal salts medium (for instance, Vogel’s E or Cold Spring Harbor A) + 0.1% glucose.

Mix and determine the resulting A660 of the inoculated cultures, (it shold be between 0.010 and 0.020. This A660 is the time zero reading. Insert the bubbler apparatus, place in 37 C hot block with aeration adjusted, and start the 30 minute count down repeat watch.

07_Triathelon_watch_P7114806

Read the A660 of the two cultures every 30 minutes. The intervals may be conveniently timed using a Timex Triathlon in its “CDR” mode (Count Down Repeat) set to repeat a signal at 30 minute intervals.

Data_Table

Collect the growth data for at least three hours (five if possible). Use a computer for ease and ability to manipulate the data at the end of the experiment.

Bact_Growth_Linear

Make two graphs of the A660 of the cultures versus time in minutes:
First, a standard linear-linear graph of a time-course experiment, with minutes incubation on the X axis, and A660 along the Y axis. I have my students construct a graph by hand to develop their graphing skills. If they then wold like to use a spreadsheet program to construct a graph, that is fine.

Bact_Growth_SemiLog

Second, produce a semi-log graph using three cycle semi-log graph paper. Take care to correctly label the Y axis values. (First label 0.010, 0.100 and 1.000 lines, then fill in values between.)
DOUBLING TIME: Use a ruler to draw aline through the linear phases of these curves. This represents their exponential or log phases. Pick an A660 value on the lower end of this straight line, note its time. Then find the point at which the A660 has doubled and note that time. The time elapsed between the two points is the doubling time.
Label the lag and log phases, and determine the generation time. Mount these in your notebook

Graph Construction by Hand

Graph Construction by Hand

While computer programs can conveniently construct graphs from data, hand construction of a graph remains an important means of analyzing and appreciating the value and patterns present in data.  Follow the following rules in order to produce a properly sized and accurately plotted graph.

1.  DETERMINE THE RANGE LIMITS OF THE X-AXIS AND THE Y-AXIS

Examine the data set and note the minimum and maximum values:
X-axis:     ordinate (independent or known variable):       time, added concentration, etc.
Y-axis:     abscissa (dependent or unknown variable):     what was measured: weight, A660 etc

(If the zero value of X or Y is important for your graph, it should be included in the limits.)

2.  DETERMINE THE LIMITS OF YOUR GRAPH PAPER

Count the number of squares available for the X and Y axes, leaving at least 3 square at the bottom and sides, and 9 squares at the top.  Graph-lined composition notebooks with 5 X 5 quad ruling allow for a graph of no more than 35 squares wide and 40 squares tall.

3.  ASSIGN VALUES TO AXES WHICH INCLUDE THE RANGE LIMITS

Assign values to the coordinates which meet the following requirements:

a.  They include the limits determined in step 1.
b.  They make an adequately large graph as large as the available space will accommodate.
c.  They do not exceed the space available on the page.

Divide the value of the range by the number of squares available along the given axis.  Round up so that the first significant figure of the result equals 1, 2, 5 or 10 units per square. For example, if you have Y axis range from 0.000 to 1.212, divide the 1.212 by the 40 squares available, which equals 0.0303/square.  This would be rounded up to 0.05/square.  Memorize the 1, 2, 5 and 10 values per square.  Other values will make plotting the data difficult, and it will cost you points when graded.  The quantity zero should often be the space most to the left and/or bottom.

Here is a page from a notebook illustrating the parameters for sizing a graph to a page.

4.  CONSTRUCT AXES, MARK WITH REGULAR VALUES

Draw lines for the X-axis below and the Y-axis to the left of the selected open area on the graph paper.  Label each axis.  Mark off the selected regular values (often every 5 or 10 squares) with a small line corresponding to units/square selected in step 2.  Label each small line with its corresponding value.  (Do not label every square.)   Be certain to maintain linearity: all spaces must have equal value.

5.  ENTER DATA POINTS

For the first point, locate the appropriate value along the X axis and then follow that line up until the appropriate value of Y is reached.  Double check that you have not shifted from the desired location, and make a small dot at the point.  Draw a small circle around the point, making it easier to see, but preserving the integrity of the point.  Repeat until all data have been entered.  Did you include the zero point if it is a significant data point?  Use squares to indicate a second data set, triangles the third, etc.

6.  CONNECT THE CIRCLES

If the function you are graphing is linear, carefully connect the circles by lining a ruler up with the points and drawing a line between them.  (Do not violate the interior of the circles so that the value of the point will remain clear.)  Alternatively, if the function is non linear, you may either connect the circles or approximate the curve plots with a “best fit” curve.

7. TITLE THE GRAPH AND AXES

Create a title which is meaningful and explicitly reflects the value of the experimental data you have graphed.  Place it in CAPITAL LETTERS as the title of the page.  Below the title, indicate from where the original data came with a cross reference.  Be certain that the axes are correctly labeled.  Label any significant break points or phases in the curve, briefly indicate their meaning, if known.