SUMMARY

For this experiment, we placed 400 mL of Tetraselmis sp. in plastic bags and exposed them to sunlight.  We measured a baseline concentration of chlorophyll a and b and did a baseline cell count before exposure.  The bags were placed in boxes that were in a water bath.  The boxes were covered with various filters and one had no filter.  Samples were taken on a regular basis.
 
 
 

MATERIALS NEEDED
 

Tetraselmis,sp.	2-2000 mL flasks
Enriched sea water	100 mL graduated cylinder
1000 mL graduated cylinder	pipettes
3 opaque boxes with lids	Whirl Pack bags or Ziploc bags
Kiddie Swimming Pool	A piece of fine mesh
Cuvettes	Lugol’s solution
Hemocytometer	Waterproof black marker
Spectrophotometer	Centrifuge with centrifuging tubes
Plexiglass lids for boxes	Microscope
Mylar UV-A filter for box lid	UV-A+UV-B filter for box lid

Note about materials: Lugol's solution is made by mixing 70 mL water, 5 g sodium acetate, 5 g iodine, and 10 g sodium iodide.  Enriched sea water can be purchased under the name of "Instant Ocean."  Also pet stores may have products similar to ocean water.

EXPERIMENTAL PROCEDURE

1.  You will need to obtain the algae, Tetraselmis sp.  Inoculate two 2000 mL flasks of sea water with the algae.  For the High Light culture, use 50 mL of algae and for the Low Light culture, use 200 mL.  Expose the High Light culture to bright sunlight and wrap the Low Light culture in fine mesh to reduce the amount of light.  Culture for one week.  For more information about the care and treatment of algae, see http://www.flinnsci.com/homepage/bindex.html.

2. You will need also to prepare the exposure boxes.  Set up three opaque boxes in a water bath.  You can use a “Kiddie Pool” filled with water.  Place the pool in a sunny spot outdoors.  Cover one box with a lid with filtered glass which is transparent only to visible light but blocks UV-A and UV-B rays.  This will be known as “PAR.”  On the second box, place a lid that is covered with filtered glass and a strip of mylar.  Mylar blocks UV-B rays.  This will be known as “PAR + UV-A.”  On the third box, place a cover with unfiltered glass.  This will be known as “PAR + UV-A + UV-B.”

3.  Prepare culture samples of 200 mL of HL mixed with 800 mL enriched sea water.  Pour 400 mL of this mixture into a Whirl Pack Thio (Plastic) bag labeled with HL or LL, amount in bag, date, and type of light to which it will be exposed.  For example, "HL, 400 mL, 07/07/00, PAR".  Take a baseline of the concentration of chlorophyll and the cell count (see step 5 and 6 below) and record data.

4. How to find measurement of absorbence of chlorophyll a using a spectrophotometer:

• Give the spectrophotometer a 20 minute warm up time after turning it on.
• Pour 4-5 mL of the culture (Tetraselmis sp., HL) into a sample cuvette.
• Zero the spectrophotometer with 4-5 mL enriched sea water medium.
• Calibrate the spectrophotometer to 664 nm .  Record the reading.  Before making the next measurement, be sure to zero the spectrophotometer again with enriched sea water.
• Repeat the measurement using 647 nm for the calibration.  Record the reading.
• Calculate the absorbence of chlorophyll a using the following equation:

[Chlorophyll a] (in mg/L) = (11.93 x absorbence 664 nm) – (1.93 x absorbence 647 nm)

Note:  The peak absorbence for chlorophyll a is at a frequency of 647 nm and 664 nm.

If the absorbence if less than .1, you may need to concentrate the mixture.  To do this, centrifuge the mixture (about 50 mL) for ten minutes.   Remove the excess water with a pipette being careful not to disturb the algae that has settled to the bottom of the centrifuging tube.  You will have to correct for concentration by multiplying the above equation by your ending volume of algae mixture divided by the beginning algae mixture.

5. How to conduct a cell count using a hemocytometer:

• Take a 5 mL sample of algae and add one drop of Lugol’s solution in order to fix the algae.
• Fill a hemocytometer (microscope slide with a grid) with the culture using a Pasteur pipette.
• The hemocytometer is divided into nine large squares, each 1 mm on a side.
• Count the number of cells of algae in four of the nine squares.
• Average the number of cells in these four squares.

6.  Collect samples on a regular basis.  Shake or stir sample bags to ensure distribution of algae in sample.  Do a cell count and spectrometer on all algae samples.