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Enumeration and biomass of phytoplankton

Sample collection

Phytoplankton in the open water of a lake or stream often is sampled by means of water bottles such as the Van Dorn or Kemmerer sampler. These samplers are lowered open to a specific depth and then are closed by means of a weighted messenger that is dropped along the cable to trip the closing mechanism.

Whenever possible, phytoplankton species should be examined while alive, particularly delicate species of flagellated algae. Algae may be kept for several hours without appreciable deterioration when kept cold during transportation to the laboratory.

Normally, samples are preserved for long term storage. The best preservative is Lugol's solution, added to samples to yield a 1% final concentration. The adsorption of iodine from Lugol's solution by the cell also promotes settling when the sedimentation-inverted microscopy technique is used.


1. Before going out in the field, add 2 squirts of Lugols solution into an appropriate number of sample jars. Make sure you also have an empty labeled jar to collect a live sample without Lugols.

2. Use a water sampler to collect a water sample from a depth of 1 m.

3. Fill your sample jars to the brim.

Phytoplankton quantitative enumeration – Sedimentation technique

If the system is very productive and there is lots of phytoplankton in the sample, they may be viewed directly by adding 1 ml to a Sedgwick-Rafter cell as described below for zooplankton. More commonly, however, it is necessary to concentrate the sample for easier counting. Sedimentation chambers, developed by Utermöhl in the 1930s, are the most common means for concentrating phytoplankton samples. However, these are too expensive unless you are a professional phytoplanktologist. An alternative is to use a 12-well tissue culture plate as a sedimentation chamber. These are relatively inexpensive and easily “borrowed” from the lab down the hall.

As stated above, viewing live samples is always preferable to working with preserved samples, however it may not be feasible to do so. In many cases, the Lugol’s solution which is used to preserve the algal cells and make them heavier so they will sink to the bottom of a counting chamber stains the cells and gives them an unnatural color that makes them more difficult to identify. Sometimes, using material that has been collected with a zooplankton net yields a sample that is easier to work with.


1. You will need one 12-well tissue culture plate for each depth sampled. Thoroughly mix your preserved phytoplankton sample by mixing. Use a Hensen-Stemple pipette to transfer exactly 5 ml of the sample to each of four sample wells in a 12-well tissue culture plate. If you have a live sample, transfer 5 ml of the live sample to one well.

2. Prior to viewing the preserved sample, take a look at the live sample so you know what species to expect. After settling, carefully view the preserved samples with an inverted microscope. Scan the entire bottom of each of the four wells and identify the phytoplankton present to the genus level.

3. Calculate the # cells/ml.

Evaluation of phytoplankton biomass

A number of different methods have been employed to estimate the biomass of phytoplankton populations including measures of fresh and dry weight, cell volumes, and organic carbon. In vivo chlorophyll analysis is the fluorescent detection of chlorophyll a in algal and cyanobacterial cells in water. In this technique, the excitation light from the fluorometer passes through untreated sample water and excites chlorophyll a within the cells. Environmental conditions, presence of interfering compounds, cellular physiology, morphology, and light history all can influence the relationship between in vivo fluorescence and the concentration of chlorophyll a. These factors cause in vivo fluorescence to be a semi-quantitative tool. Despite its semi-quantitative nature, in vivo fluorescence data can supply valuable information on the spatial and temporal distribution of chlorophyll concentrations quickly and easily. To obtain quantitative data, the in vivo fluorescence data must be correlated with extracted chlorophyll a data that can be obtained through the extraction and measurement of the pigment from grab samples on a laboratory fluorometer, spectrophotometer or HPLC.

The Aquafluor is a hand held fluorometer that is ideal for measuring in vivo chlorophyll in the field. When you calibrate the Aqaufluor as described below, you are assigning a value (Cal Standard Value) to the fluorescent intensity of a Solid secondary standard. The Cal Standard Value you assign will only be a relative value compared to the actual chlorophyll concentration. The Solid Standard simulates the in vivo fluorescence of a 10 µg/L marine diatom culture.

The Aqaufluor calibration reads a Blank solution and automatically subtracts it from the sample readings (zero point). The best “true” blank is the natural water that has been filtered through a GF/F or membrane filter in order to remove the algal cells but to retain the dissolved components. However, distilled water can be used for the Blank since the in vivo readings are semi-quantitative.


1. Press the "ON/OFF" button. The instrument will turn on and count down for 5 seconds.

2. Press the "A/B" button to choose the CHL channel.

3. Press the "STD VAL" button. Use the up and down arrows to adjust the standard value to the desired value (10 µg/l). Holding either arrow button pressed down will activate a faster scrolling of the value.

4. Press "ENT" to accept the value and to return to the Home screen.

5. Press the "CAL" button and Press "ENT" to start the calibration.

6. Insert your blank and press "ENT". The Aquafluor will average the blank reading for 10 seconds.

Note – Do not Blank without a liquid sample because it can result in a falsely high blank value. Fill the Cuvette at least 2/3rds full to insure the sample meniscus is above the light path.

7. Insert the Solid Secondary standard and press "ENT". The reading will be averaged for 10 seconds. 8. Press "ENT" when the calibration is complete to accept the calibration. If "ENT" is not pressed within 10 seconds, you will be asked if you want to abort the calibration. Press the up or down arrow to abort or accept the calibration respectively. In addition, the calibration can be aborted at any time by pressing "ESC"