Photo-Electric Colorimetry as a Method of Quantitative Phytoplankton Analysis

Photo-Electric Colorimetry as a Method of Quantitative Phytoplankton AnalysisAuthor(s): Allan TuckerSource: Transactions of the American Microscopical Society, Vol. 75, No. 4 (Oct., 1956), pp.422-427Published by: Wiley on behalf of American Microscopical SocietyStable URL: http://www.jstor.org/stable/3223614 .

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H. W. LEVI H. W. LEVI

length, 2.2 mm.; carapace, 1.0 mm. long, 0.7 mm. wide; first femur, 1.4 mm.; patella and tibia, 1.6 mm.; metatarsus, 1.0 mm.; tarsus, 0.6 mm.; second patella and tibia, 1.1 mm.; third, 0.7 mm.; fourth, 1.0 mm.

The genitalia and smaller size distinguish this species from A. analyticum.

Type locality: Male holotype, female allotype, one male and two female paratypes from Barro Colorado Island, Panama Canal Zone, June 16 to July 15, 1934 (A. M. Chickering) in the Museum of Comparative Zoology.

Records: Tamaulipas: 27 mi. north of Villa Juarez, April 17, 1938 (L. I. and A. M. Davis), 2 9. Chiapas: La Zacualpa, Aug. 1909 (A. Petrunkevitch), 2 9. Canal Zone: Barro Colorado Island; 18-25 Aug., 1939, 1 9; 28-31 July, 1934, 1 9; 1-4 July, 1950, 1 9 ; Old Plantation, 30 July, 1950, 1 9; Old Plantation, 13 July, 1954, 1 c; Barbour Point, 14 Aug., 1954, 1 9. Peru: Piura: north of Mallares, Rio Chira, Jan. 4, 1942 (D. and H. Frizzell), 9 9, 1 c.

LITERATURE CITED

ARCHER, A. F. 1946. The Theridiidae or comb-footed spiders of Alabama. Pap. Alabama Mus. Nat. Hist., 22: 1-67.

CAMBRIDGE, F. O. P.- 1902. Arachnida-Araneidea Vol. 2, in Biologia Centrali- Americana.

COMSTOCK, J. H. 1912. The spider book. Doubleday, Page and Co., Garden City. GERTSCH, W. J. 1949. American spiders. Van Nostrand Co., New York. MCCooK, H. C. 1893. American spiders and their spinning work, Vol. 3,

Philadelphia. SIMON, E. 1891. Voyage de M. E. Simon au Venezuela. Ann. Soc. Ent. France,

60: 11. 1894. Histoire naturelle des araignees, Paris, Vol. 1. 1903. Ibid., Vol. 2.

PHOTO-ELECTRIC COLORIMETRY AS A METHOD OF QUANTITATIVE PHYTOPLANKTON ANALYSIS1

ALLAN TUCKER

Department of Natural Science, Michigan State University, East Lansing, Mich.2

The pigment extraction technique of plankton analysis is being used by many workers at present because of its simplicity and rapidity in estimating phytoplankton productivity at a given time. In a previous paper (Tucker, 1949) the writer compared statistically the densities of extracted pigment with the counted units of phytoplankton in comparable samples collected from the Bay of Quinte, a narrow body of water opening into Lake Ontario. In that study, those samples from which pigment was to be extracted were filtered through Whatman's number 44 filter paper.

'Contribution from Department of Zoology, University of Michigan and Univer- sity of Michigan Biological Station. A portion of a dissertation prepared under the direction of Prof. F. E. Eggleton and submitted in partial fulfillment of the require- ments for the degree of Doctor of Philosophy.

2This study was aided by a grant from the All-College Research Fund of Michigan State University.

length, 2.2 mm.; carapace, 1.0 mm. long, 0.7 mm. wide; first femur, 1.4 mm.; patella and tibia, 1.6 mm.; metatarsus, 1.0 mm.; tarsus, 0.6 mm.; second patella and tibia, 1.1 mm.; third, 0.7 mm.; fourth, 1.0 mm.

The genitalia and smaller size distinguish this species from A. analyticum.

Type locality: Male holotype, female allotype, one male and two female paratypes from Barro Colorado Island, Panama Canal Zone, June 16 to July 15, 1934 (A. M. Chickering) in the Museum of Comparative Zoology.

Records: Tamaulipas: 27 mi. north of Villa Juarez, April 17, 1938 (L. I. and A. M. Davis), 2 9. Chiapas: La Zacualpa, Aug. 1909 (A. Petrunkevitch), 2 9. Canal Zone: Barro Colorado Island; 18-25 Aug., 1939, 1 9; 28-31 July, 1934, 1 9; 1-4 July, 1950, 1 9 ; Old Plantation, 30 July, 1950, 1 9; Old Plantation, 13 July, 1954, 1 c; Barbour Point, 14 Aug., 1954, 1 9. Peru: Piura: north of Mallares, Rio Chira, Jan. 4, 1942 (D. and H. Frizzell), 9 9, 1 c.

LITERATURE CITED

ARCHER, A. F. 1946. The Theridiidae or comb-footed spiders of Alabama. Pap. Alabama Mus. Nat. Hist., 22: 1-67.

CAMBRIDGE, F. O. P.- 1902. Arachnida-Araneidea Vol. 2, in Biologia Centrali- Americana.

COMSTOCK, J. H. 1912. The spider book. Doubleday, Page and Co., Garden City. GERTSCH, W. J. 1949. American spiders. Van Nostrand Co., New York. MCCooK, H. C. 1893. American spiders and their spinning work, Vol. 3,

Philadelphia. SIMON, E. 1891. Voyage de M. E. Simon au Venezuela. Ann. Soc. Ent. France,

60: 11. 1894. Histoire naturelle des araignees, Paris, Vol. 1. 1903. Ibid., Vol. 2.

PHOTO-ELECTRIC COLORIMETRY AS A METHOD OF QUANTITATIVE PHYTOPLANKTON ANALYSIS1

ALLAN TUCKER

Department of Natural Science, Michigan State University, East Lansing, Mich.2

The pigment extraction technique of plankton analysis is being used by many workers at present because of its simplicity and rapidity in estimating phytoplankton productivity at a given time. In a previous paper (Tucker, 1949) the writer compared statistically the densities of extracted pigment with the counted units of phytoplankton in comparable samples collected from the Bay of Quinte, a narrow body of water opening into Lake Ontario. In that study, those samples from which pigment was to be extracted were filtered through Whatman's number 44 filter paper.

'Contribution from Department of Zoology, University of Michigan and Univer- sity of Michigan Biological Station. A portion of a dissertation prepared under the direction of Prof. F. E. Eggleton and submitted in partial fulfillment of the require- ments for the degree of Doctor of Philosophy.

2This study was aided by a grant from the All-College Research Fund of Michigan State University.

422 422



PHOTO-ELECTRIC ANALYSIS OF PHYTOPLANKTON

The filter paper containing plankton residue was placed in a small beaker and covered with 15 cc. of acetone. The tops of the beakers were tightly covered with wax paper to prevent evaporation. After three hours, the acetone in the beakers, now containing the extracted pigment in solution was filtered through a rough filter paper to remove any particles of the first filter paper. This acetone was compared in a Leitz No. 2509 visual colorimeter to an arbitrary color standard suggested by Harvey (1934). This color standard consisted of a mixture of 25 mg. of potassium chromate and 430 mg. of nickel sulfate dissolved in a liter of water, the color intensity of this solution constituting one "Harvey U nit" of pigment.

During the course of an investigation by the author to study the relation of phytoplankton periodicity to the nature of the physico-chemical environment in certain Michigan lakes (Tucker, 1952), it became apparent that more data should be collected to further test the reliability of the pigment extraction method as a means for evaluating phytoplankton abundance. Therefore duplicate series of plankton samples were collected at various times and depths during the summers of 1950 and 1951 from Douglas, Lancaster and Lansing Lakes; one series for counting, the other for the purpose of extracting pigment. The samples were collected with a 10-liter Birge and Juday plankton trap with No. 25 silk bolting cloth. They were concentrated to approximately 15 or 20 ml., depending upon the concentration of plankton in the lake at the time, and transferred to 1-ounce packer bottles. About eight drops of formalin were added as a preservative to those samples in which the plankters were to be counted. The duplicate samples, from which pigment was to be extracted were brought back to the laboratory in the fresh condition.

In this investigation, pigment was extracted with acetone as before, but the density of its color was determined by the use of an industrial model of the Klett-Summerson photo-electric colorimeter with a red light filter No. 66 having an approximate sprectral range of 640-700 mu. The colorimeter is provided with rectangular shaped solution cells of various sizes and test tubes of different volume capacities. None of the acetone pigment solutions analysed in this investigation had a greater volume than 15 cc. so that 20 cc. test tubes specially designed for the machine were used. The scale reading is a measure of the optical density of the colored solution as determined by the photo-electric cell. The optical density of the solution according to Beer's Law is theoretically proportional to the concentration of the colored substance. Therefore the scale readings are directly proportional to the concentration of the substances being determined. A useful feature of this colorimeter is the logarithmically spaced scale divisions. This eliminates the necessity of preparing a calibration curve from the scale readings. The machine is constructed in such a way that the pointer can be adjusted to zero. In order to establish a position for the zero, a Klett-Summerson test tube containing 15 cc. of pure acetone was placed in the colorimeter. The position to which the pointer had moved was designated as zero. The test tube was removed and another test tube containing 15 cc. of the acetone pigment solution to be measured was placed in the colorimeter. When the pointer had ceased to move, a reading was taken, which was the measurement of the pigment color density in Klett Units.

The degree of correlation between density of pigment and units of count in comparable samples was determined statistically by means of scatter diagrams and linear regression lines (Fig. 1). The comparisons

423



TABLE I

Statistical Analysis Showing Relationship Between Number of Units of Phytoplankton Counted and Density of Pigment Extracted*

Number of

Items

40

12

12

Formula of

Regression Line

log. Y = 4.39726 + 1.46665 log. X

log. Y = 4.11619 + 1.35441 log. X

log. Y = 3.85793 = 1.32421 log. X

Standard Error of Estimate

+ .211

= .118

= .080

Coefficient of

Correlation

.81

.95

.88

Probability of correlation as great

through chance alone

less than .00001

less than .00001

less than .00001

*X = Density of extracted pigment in Klett units per liter Y = Phytoplankton count in units per liter

Lakes

Douglas ....

Lancaster..

Lansing....

Figure

A

B

C

z C-

ci

Cl M tt



PHOTO-ELECTRIC ANALYSIS OF PHYTOPLANKTON

were made between the logarithms of the absolute values of the samples in order to reduce the large spread and consequent large absolute variation which would result if the absolute values themselves were used. The formulae employed were taken from standard textbooks of statistics. The results of the statistical analysis are summarized in Table I.

The coefficients of correlation for samples from Douglas, Lancaster and Lansing lakes were .84, .95, and .88 respectively. In the Bay of Quinte analysis (Tucker, I.c.) the coefficient of correlation was .83 when considering all of the samples collected during the summer of 1945. When considering only those samples from the Bay of Quinte whose composition was 90 percent or over of diatoms, the coefficient of correlation was .84. The correlation in Douglas Lake is about the same as that for the Bay of Quinte when considering just the Quinte samples containing the high percentages of diatoms, although in Douglas Lake the diatoms make up

800,000- 00000

DOUGLAS LAKE LANCASTER LAKE LAKE LANSING

400,000

200,000

100,000 D= 80,000

60,000 _ / / / - I 40,000- 00X / /

20.000 - 4o,ooo

u zi-

-J - 20,000 -

o 6,000 -

> 4,000-

2,000

A B C 1,000

.1 . ..4 .6.81.0 2.0 4.0 7.0 .2 .4 .6.81.0 2.0 4.0 7.0 .2 .6 .1.0 2.0 4.0 7.0 3.0 .0 10.0 3.0 5.0 10.0 & 0 .0 10.0

EXTRACTED PIGMENT IN KLETT UNITS PER LITER

FIG. 1. Relation between density of extracted. pigment (measured in Klett Units per liter) and units of count per liter in comparable 10 liter samples of plankton collected from Douglas, Lancaster and Lansing lakes. (Displayed on double log. scale).

only between 54.3 and 82.7 percent with an average of 67 percent. This confuses the picture especially if one attempts to generalize that the approximation of the coefficient of correlation to unity depends on the number of extraneous factors involved. There would seem to be less extraneous factors in those Quinte samples containing 90 percent or over of diatoms than in the complete series of Douglas Lake samples. The coefficient of correlation, however, is the same.

In the author's previous paper (Tucker, I.c.) the results of a laboratory controlled experiment are discussed. In this experiment, pigment was extracted from samples of different concentrations of a pure culture of green algae (Ankistrodesmus). A statistical comparison was made between the color densities of the extracted pigment samples and the volumetric concentrations or corresponding geometric dilutions of the culture. The coefficient of correlation here was .97. In the Lancaster Lake samples, considering the complete series, the coefficient of correlation was .95. This appears to be quite high but upon considering the percentage composition of the samples, it is seen that they are composed

425



ALLAN TUCKER

almost entirely of two groups of algae, namely Chrysophyceae and diatoms. In spite of this, it is difficult to explain why a coefficient of correlation so much higher is found here than in the Douglas Lake samples.

When considering the complete series from the Bay of Quinte, i.e. not excluding those samples containing less than 90 percent composition of diatoms, the coefficients of correlation determined in that study were lower than those determined in the present study. This difference could be due to elimination of the personal error involved in estimating color densities through a visual colorimeter. In the study of the Bay of Quinte samples, pigment densities were measured in Harvey Units; that unit being the color density of an arbitrary color standard. In this study, pigment densities were measured in Klett Units, which is a measure of the distance that the pointer of the photoelectric colorimeter deviates from zero due to the color density of the pigment being measured.

During the course of this investigation it was decided to collect data which would indicate the percentage of loss of plankton through a net as compared to the amount retained by a centrifuge. Consequently pigment was also extracted from the concentrates of duplicate series of plankton samples, one series concentrated by filtration through a plankton net, the other by centrifuging. Assuming that the centrifuge retained 100 percent of the pigment, the net was found to retain between 12 and 28.6 percent with an average of 17.5 percent. This value is similar to that determined gravimetrically by Birge and Juday (1922) who found that the net was able to remove from the water only one-quarter to one-fifth of the organic matter that could be removed by the centrifuge.

A detailed critique of the pigment extraction method as a means of quantitatively analysing phytoplankton abundance has already been published (Tucker, I.c.). The results of the present investigation merely confirm the author's previous conclusion, that because of the many variables involved, this method even with the use of a photo-electric colorimeter can be used only as a general indicator. In order to determine food supply available to plankton feeders, knowledge of the particular classes and genera of algae present in a body of water must be obtained. This same type of data is necessary in carrying out studies pertaining to contamination by algae of water supplies. The only way that this kind of information can be obtained is by the use of a microscope.

SUMMARY

1. Duplicate series of plankton samples were collected from three Mich- igan lakes, one series for counting plankton, the comparable series from which to extract pigment.

2. The results of these two types of analyses were compared statistically and coefficients of correlation were calculated.

3. The use of a photo-electric colorimeter in determining the amount of pigment present in plankton samples is described.

LITERATURE CITED

BIRGE, E. A., and JUDAY, C. 1922. The inland lakes of Wisconsin. The plankton. I. Its quantity and chemical composition. Wis. Geol. and Nat. Hist. Surv. Bull. 64, Sci. Ser. No. 13: 1-222.

HARVEY, H. W. 1934. Measurement of phytoplankton population. Jour. Mar. Biol. Assoc., 19: 761-773.

426



PHOTO-ELECTRIC ANALYSIS OF PHYTOPLANKTON PHOTO-ELECTRIC ANALYSIS OF PHYTOPLANKTON

TUCKER, A. 1949. Pigment extraction as a method of quantitative analysis of

phytoplankton. Trans. Amer. Micros. Soc., 68: 21-33. 1952. The relation of phytoplankton periodicity to the nature of the physico-

chemical environment in certain Michigan lakes. (Doctorate dissertation, University of Michigan). University Microfilms, Ann Arbor, Michigan.

NOTES ON STIGONEMATACEAE FROM SOUTHEASTERN UNITED STATES1

C. S. NIELSEN

Florida State University, Tallahassee

Eight genera are generally recognized as constituting the Stigone- mataceae of the blue-green algae. The family is distinguished from others by the method of branch origin of the filaments, cell shape, and characteristics of the sheath. Published reports of collections of this particular group are few from southeastern United States. Speci- mens collected and studied by the author and his students are cited subsequently; collections by others and those unavailable for examina- tion at this time are referred to in the citation of the literature and in the catalogue of species. It is planned to treat these geographically in a more comprehensive study later.

One of the eight genera, Loefgrenia Gom., is not represented. The monotypic genus was described from specimens collected in the province of Sao Paulo, Brazil and Drouet (1938) collected it again in that country. A report of its appearance in Tennessee was made by Silva and Sharp (1944); these specimens, however, have been referred to Entophysalis Lemaniae (Ag.) Dr. & Daily by Drouet. Our observations concur with the referral.

Unless otherwise indicated all specimens are deposited in the personal herbarium of Dr. Francis Drouet (D), the cryptogamic herbarium of the Chicago Natural History Museum (C), and the herbarium of the Florida State University (F).

The author is greatly indebted to Dr. Drouet for his species deter- minations and for his encouragement since the initial collecting in 1948.

KEY TO GENERA A. Sheath distinct, definite at m argins ...................................... B

B. Filaments not agglutinated in a compact cushion .................... C C. Trichomes consistently uniseriate ............................ D

D. Heterocysts terminal or lateral; branches of 2 forms: (1) cylindrical; (2) definitely attenuate to a hair. Plants usually growing on old shells ............. . Mastigocoleus

DD. Heterocysts intercalary; branches not attenuate. Plants aquatic, delicate, pliant, green ..........2. Hapalosiphon

CC. Trichomes of most filaments multiseriate ..................... E E. Heterocysts more often lateral, occasionally intercalary.

Branches arising from all sides of main filaments and approximating them in width, only occasionally produc- ing hormogonia in apices of branches ...... 3. Stigonema

Contribution number 67, Botanical Laboratory, Florida State University.

TUCKER, A. 1949. Pigment extraction as a method of quantitative analysis of

phytoplankton. Trans. Amer. Micros. Soc., 68: 21-33. 1952. The relation of phytoplankton periodicity to the nature of the physico-

chemical environment in certain Michigan lakes. (Doctorate dissertation, University of Michigan). University Microfilms, Ann Arbor, Michigan.

NOTES ON STIGONEMATACEAE FROM SOUTHEASTERN UNITED STATES1

C. S. NIELSEN

Florida State University, Tallahassee

Eight genera are generally recognized as constituting the Stigone- mataceae of the blue-green algae. The family is distinguished from others by the method of branch origin of the filaments, cell shape, and characteristics of the sheath. Published reports of collections of this particular group are few from southeastern United States. Speci- mens collected and studied by the author and his students are cited subsequently; collections by others and those unavailable for examina- tion at this time are referred to in the citation of the literature and in the catalogue of species. It is planned to treat these geographically in a more comprehensive study later.

One of the eight genera, Loefgrenia Gom., is not represented. The monotypic genus was described from specimens collected in the province of Sao Paulo, Brazil and Drouet (1938) collected it again in that country. A report of its appearance in Tennessee was made by Silva and Sharp (1944); these specimens, however, have been referred to Entophysalis Lemaniae (Ag.) Dr. & Daily by Drouet. Our observations concur with the referral.

Unless otherwise indicated all specimens are deposited in the personal herbarium of Dr. Francis Drouet (D), the cryptogamic herbarium of the Chicago Natural History Museum (C), and the herbarium of the Florida State University (F).

The author is greatly indebted to Dr. Drouet for his species deter- minations and for his encouragement since the initial collecting in 1948.

KEY TO GENERA A. Sheath distinct, definite at m argins ...................................... B

B. Filaments not agglutinated in a compact cushion .................... C C. Trichomes consistently uniseriate ............................ D

D. Heterocysts terminal or lateral; branches of 2 forms: (1) cylindrical; (2) definitely attenuate to a hair. Plants usually growing on old shells ............. . Mastigocoleus

DD. Heterocysts intercalary; branches not attenuate. Plants aquatic, delicate, pliant, green ..........2. Hapalosiphon

CC. Trichomes of most filaments multiseriate ..................... E E. Heterocysts more often lateral, occasionally intercalary.

Branches arising from all sides of main filaments and approximating them in width, only occasionally produc- ing hormogonia in apices of branches ...... 3. Stigonema

Contribution number 67, Botanical Laboratory, Florida State University.

427 427



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Photo-Electric Colorimetry as a Method of Quantitative Phytoplankton Analysis