Steps

1.Filter cells onto a glass fiber filter. Add 5 drops of saturated MgCO3 during filtration.

2. Measure out 10 mL 90% acetone.

3. Put filter into glass motar with some of the acetone. Grind filters using a reinforced glass mortar and teflon pestle. A hand drill can be used cautiously. Wear work gloves and glasses.

4.Grind until the filter is totally macerated.

5. Centrifuge in a 15 mL tube at 2000 rpm.

6. Read the extract on a spec at 645, 665 and 750

Calculations

Using the following estimates will give you the amount of chlorophyll in a 1 cm cuvette.

Use the OD7750 to correct for turbidity of the centrifuged extract. It should be 0.010 absorbance units or less.

The following two equations are the same.

(OD665-OD750) * 11.6 = ug Chla/mL in the cuvette

(OD665-OD750)/0.089 = ug Chla/mL in the cuvette

These values are the same as used by Myers (****) and in the trichromatic method in Phycological Methods (****). They are based on the absorption coefficient of chlorophyll in acetone, which is E for a 1 cm cell for 1% solution = 890 absorbance units (Kirk ****). This means that a 10^-6 solution (1 ug/mL) would have an absorbance of 0.089.

If the sample has Chlorophyll b, you should use the trichromatic method

first subtract the turbidity blank (OD750) from the other readings at 665, 645 and 630

Chla = 11.6*OD665 - 1.31*OD645 - 0.14*OD630

Chlb = 20.7*OD645 - 4.34*OD665 - 4.42*OD630

Chlc = 5.0*OD630 - 4.64*OD665 - 16.3*OD645

You can see how the initial calculation of Chla is similar to the simple equations given above. The other terms in the Chla equation are to correct out for the spectral overlap from Chlb and Chlc. The same goes for Chlb and Chlc.

Correcting for the concentration of chlorophyll in your culture is a simple dilution problem. Some descriptions of the chlorophyll method contain the dilution factors into the equations. This makes for complications if you use different dilution factors.

Example

References

Hansmann, E. (1973) Pigment Analysis. In: Handbook of Phycological Methods:Culture Methods and Growth Measurements. [Ed] Janet Stein. Cambridge Press.

Jensen, A. (1978) Chlorophylls and carotenoids. In: Handbook of Phycological Methods: Physiological and Biochemical Methods. [Ed] Johan Hellebust and J.S.Craigie. Cambridge University Press.

Kirk, John T.O. 1994. Light and Photosynthesis in Aquatic Ecosystems. 2nd Ed. Cambridge University Press.

January 16, 2000