W. Thomas Shoaf | Geology Reports

USGS1977

The quantitative determination of chlorophylls A and B from freshwater algae without interference from degradation products

Chlorophyll was efficiently extracted from freshwater algae with dimethyl sulfoxide. The extract was chromatographed on commercially available thin-layer cellulose sheets, 2 percent methanol and 98 percent petroleum ether being used as the solvent. Chlorophylls a and b , and phaeophytins a and b were cleanly separated from each other and other degradation products. No artifacts were formed. Recoveries of chlorophylls a and b were 98 and 96 percent as measured by spectrophotometry or fluorometry.

Journal of Research of the U.S. Geological Survey↗

USGS1976

The measurement of adenosine triphosphate in pure algal cultures and natural aquatic samples

Three methods for the extraction of adenosine triphosphate (ATP) neutral dimethyl sulfoxide (DMSO), boiling tris buffer, and butanol-octanol extraction were equally effective on the alga Chlorella vulgwia . Dilution of extracted ATP samples was linear. Filtration of different volumes of samples resulted in proportional values for ATP in the extracts. Measurement of activity by either peak height or integration of the area under the peak were equally sensitive and reproducible. The assay of ATP sample was inhibited by mercuric chloride > cadmium chloride > calcium chloride > potassium or sodium phosphate, and by high concentrations of the extractant DMSO. Analysis of ATP in aquatic environments led to the problem of transferring a sample from the field to the laboratory without obtaining a change in ATP concentration. Membrane filtration of the sample followed by chilling at 4°C, slow freezing at 20° C, or freezing on dry ice were ineffective in maintaining a constant level of ATP. Chilling caused a marked increase in ATP, whereas slow freezing caused a significant loss of ATP. Freezing on dry ice was variable but generally resulted in large losses of ATP. Quick freezing by immersion of filter and algae in liquid nitrogen and storage on dry ice maintained a constant ATP level. Field extraction of the ATP followed by quick freezing in an acetone-dry-ice bath maintained the ATP in a convenient and stable form.

Journal of Research of the U.S. Geological Survey↗

USGS1976

An evaluation of methods for measuring algal growth

Algal growth was expressed as milligrams dry weight per litre with use of the electronic particle counter for determining cell number and cell volume. A gravimetric constant in milligrams dry weight per cubic micrometre was determined. This constant was applicable throughout the growth curve since there was a linear relationship between total cell weight and total cell volume. Cell volume must be considered in measuring growth because there is a change in average cell volume in addition to an increase in cell numbers throughout the growth curve. Chlorella vulgaris and Selenastrum capricornutum exhibited similar growth curves when growth was expressed as cells per litre or as milligrams dry weight per litre. Chlamydomonas reinhardtii appeared to have a lower algal growth potential when measured in cells per litre but had the same growth potential as C. vulgaris and S. capricornutum when expressed as milligrams dry weight per litre. The growth rate for Scenedesmus quadricauda was less than the other three, regardless of how the results were expressed. The image analysis system was used to count different types of algae in a mixed culture. C. reinhardtii , C. vulgaris , and Anacystis nidulans were not grossly distorted by the sample preparation method and could be distinguished from one another by variations in morphology. The counts could be separated by computing the ratio of area to length for each particle and by allowing the computer to sort and count each of the three types. Typical ratios ranged from 5 to 7 for G. reinhardtii , 2 to 4 C. vulgaris , and 0.2 to 1.3 for A. nidulans . The number of types to be counted in a mixture was limited only by the ability to apply shape factors without having an overlap between each type. Both the image analysis and electronic particle counter produced equal count and size measurements. Fluorescence was a sensitive measure of growth on a laboratory prepared medium since there was a logarithmic relationship between fluorescence and cells per millilitre for both C. vulgaris and S. capricornutum . Fluorescence measurements were not satisfactory for determining growth in samples of varying chemical composition (which occurs in natural samples) because the chlorophyll-to-cell ratio varied with media composition. This was especially pronounced with C. vulgaris .

Journal of Research of the U.S. Geological Survey↗