Biological field and laboratory methods for measuring the quality of ...

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BIOLOGICAL METHODS *Fritsch, F. E. 1956. The structure and reproduction of the algae. Volumes I and II. Cambridge University Press. Geitler, 1. 1932. Cyanophyceae. In: Rabehnorst's Kryptogamen-Flora, 14: 1-1096. Akademische Verlagsgesellschaft m.b.H., Leipzig. (Available from Johnson Reprint Corp., New York.) Glezer, Z. 1. 1966. Cryptogamic plants of the U.S.S.R., volume VII: Silioflagellatophyceae. Moscow. (English Transl. Jerusalem, 1970) (Available from A. Asher & Co., Amsterdam.) Gran, H. H., and E. C. Angst. 1930. Plankton diatoms of Puget Sound. Umv. Washington, Seattle. Hendey, N. 1. 1964. An introductory account of the smaller algae of British coastal waters. Part V: Baccilariophyceae (Diatoms). FIsheries Invest. (London), Series IV. Huber-PestalozzI, G., and F. Hustedt. 1942. Die Kieselalgen. In: A. Thienemann (ed.), Das Phytoplankton des Susswassers, Die Binnengewassllr, Band XVI, Teil II, Halfte II. E. Schweizerbart'sche Verlagsbuch-handlung, Stuttgart. (Stechert, New York, repnnted 1962.) *Hustedt, F. 1930. Die Kieselalgen. In: 1. Rabenhorst (ed.), Kryptogamen-Flora von Deutschland, Osterreich, und der Schweiz. Band Vii. Akademische Verlagsgesellschaft m.b.h., Leipzig. (Johnson Reprint Co., New York.) *Hustedt, F. 1930. Bacillariophyta. In: A Pascher (ed.), Die Suswasser-Flora Mitteliuropas, Heft 10. Gustav Fischer, Jena. (UniversIty Microfilms, Ann Arbor, Xerox.) Hustedt, F. 1955. Marine littoral diatoms of Beaufort, North Carolina. Duke Univ. Mar. Sta. Bull. No.6. Duke Univ. Press, Durham, N. C., 67 pp. Irenee-Marie, F. 1938. Flore Desmidiale de la region de Montreal. Lapraine, Canada. *Patnck, R., and C. W. Reimer. 1966. The diatoms of the United States. Vol. 1. Academy of Natural Sciences, Philadelphia. Tiffany, 1. H., and M. E. Britton. 1952. The algae of Illinois. Reprinted in 1971 by Hafner Publishing Co., New York. Tilden, J. 1910. Minnesota algae, Vol. 1. The Myxophyceae of North America and adjacent regions including Central America, Greenland, Bermuda, the West IndIes and Hawaii. Univ. Minnesota. (FIISt and unique volume) (Reprinted, 1969, in Bibliotheca Phycologica, 4, J. Cramer, Lehre, Germany.) 4.1.2 Quantitative analysis ofphytoplankton To calibrate the microscope, the ocular must be equipped with a Whipple grid-type micrometer. The exact magnification with any set of oculars varies, and therefore, each combination of oculars and objectives must be calibrated by matching the ocular micrometer against a stage micrometer. Details of the procedure are given in Standard Methods, 13 th Edition. When counting and identifying phytoplankton, analysts will find that samples from most natural waters seldom need dilution or concentration and that they can be enumerated 8 directly. In those samples where algal concentrations are extreme, or where silt or detritus may interfere, carefully dilute a 10-ml portion of the sample 5 to 10 times with distilled water. In samples with very low populations, it may be necessary to concentrate organisms to minimize statistical counting errors. The analyst should recognize, however, that manipulations involved in dilution and concentration may introduce error. Among the various taxa are forms that live as solitary cells, as components of natural groups or aggregates (colonies), or as both. Although every cell, whether solitary or in a group, can be individually tallied, this procedure is difficult, time consuming, and seldom worth the effort. The unit or clump count is easier and faster and is the system used commonly within this Agency. In this procedure, all unicellular or colonial (multi-cellular) organisms are tallied as single units and have equal numerical weight on the bench sheet. The apparatus and techniques used in counting phytoplankton are des;:;ribed here. Sedgwick-Rafter (S-R) Counting Chamber The S-R cell is 50 mm long by 20 mm wide by 1 mm deep; thus, the total area of the bottom of the cell is 1000 mm 2 and the total volume is 1000 mm 3 or one m!. Check the volume of each counting chamber with a vernier caliper and micrometer. Because the depth of the chamber normally precludes the use of the 45X or 100X objectives, the 20X objective is generally used. However, special long-working-distance, higherpower objectives can be obtained. For the procedure, see Standard Methods, 13th Edition. Place a 24 by 60 mm, No. I coverglass diagonally across the cell, and with a largebore pipet or eyedropper, quickly transfer a I-ml aliquot of well-mixed sample into the open corner of the chamber. The sample should be directed diagonally across the bottom of the cell. Usually, the cover slip will rotate into place as the cell is filled. Allow the S-R cell to stand for at least IS minutes to permit settling. Because some organisms, notably blue-green algae, may
float, examine the underside of the cover slip and add these organisms to the total count. Lower the objective lens carefully into position with the coarse focus adjustment to ensure that the cover slip will not be broken. Fine focus should always be up from the cover slip. When making the strip count, examine two to four "strips" the length of the cell, depending upon the density of organisms. Enumerate all forms that are totally or partially covered by the image of the Whipple grid. When making the field count, examine a minimum of 10 random Whipple fields in at least two identically prepared S-R cells. Be sure to adopt a consistent system of counting organisms that lie only partially within the grid or that touch one of the edges. To calculate the concentration of organisms with the S-R cell, for the strip count: ex 1000 mm 3 No. per m! = L X D X W X S where: C = number of organisms counted (tally) L = length of each strip (S-R cell length), mm 0= depth of a strip (S-R cell depth), mm W= width of a strip (Whipple grid image width), mm S = number of strips counted To calculate the concentration of organisms with the field count: ex 1000 mm 3 No. per m! = A X D X F where: C = actual count of organisms (tally) A = area of a field (Whipple grid image area), mm 2 0= depth of a field (S-R cell depth), mm F = number of fields counted Multiply or divide the number of cells per milliliter by a correction factor for dilution (including that resulting from the preservative) or for concentration. 9 PLANKTON COUNTING Palmer-Maloney (P-M) Nannoplankton Cell The P-M cell was especially designed for enumerating nannoplankton with a high-dry objective (45 X). It has a circular chamber 17.9 mm in diameter and 0.4 mm deep, with a volume of 0.1 ml. Although useful for examining samples containing a high percentage of nannoplankton, more counts may be required to obtain a valid estimate of the larger, but less numerous, organisms present. Do not use this cell for routine counting unless the samples have high counts. Pipet an aliquot of well-mixed sample into one of the 2 X 5 mm channels on either side of the circular chamber with the cover slip in place. After 10 minutes, examine the sample under the high-dry objective and count at least 20 Whipple fields. To calculate the concentration of organisms: ex 1000 mm 3 No. per m! = A X D X r where: C = number of organisms counted (tally) A = area of a field (Whipple grid image), mm 2 o = depth of a field (P-M cell depth), mm F = number of fields counted Bacterial Counting Cells and Hemocytometers The counting cells in this group are preciselymachined glass slides with a finely ruled grid on a counting plate and specially-fitted ground cover slip. The counting plate proper is separated from the cover slip mounts by parallel trenches on opposite sides. The grid is ruled such that squares as small as 1/20 mm (50 IJ.) to a side are formed within a larger l-mm square. With the cover slip in place, the depth in a Petroff Hausser cell is 1/50 mm (20IJ.) and in the hemocytometer I/l 0 mm (lOOM). An optical micrometer is not used. With a pipet or medicine dropper. introduce a sample to the cell and at high magnification identify and count all the forms that fall within the gridded area of the cell. To calculate the number of organisms per milliliter, multiply all the organisms found in the gridded area of the cell by the appropriate factor. For example, the multiplication factor
Page 1 and 2: ... EPA-610/4-13 -001 July 1973 BIO
Page 3 and 4: • FOREWORD Man and his environmen
Page 5 and 6: Name Anderson, Max Arthur, John W.
Page 7 and 8: The role of aquatic biology in the
Page 9 and 10: FOREWORD 1 PREFACE CONTENTS BIOLOGI
Page 11 and 12: BIOMETRICS 1.0 INTRODUCTION 1.1 Ter
Page 13 and 14: BIOLOGICAL METHODS sample or a plan
Page 18: TABLE 1. RAW DATA ON PLANKTON COUNT
Page 33: BIOLOGICAL METHODS y POSITIVE INTER
Page 40: PLANKTON 1.0 INTRODUCTION . 2.0 SAM
Page 43 and 44: sampling is usually sufficient. In
Page 45 and 46: Several sampling methods can be use
Page 47: 100X. When combined with the ocular
Page 51 and 52: of counts to numbers per ml is quit
Page 53 and 54: pipet with distilled water into a c
Page 56: BIOLOGICAL METHODS Calculate the ch
Page 59 and 60: PLANKTON REFERENCES Holmes, R. W. 1
Page 61 and 62: PERIPHYIOI
Page 66 and 67: BIOLOGICAL METHODS Diatom Species P
Page 68 and 69: BIOLOGICAL METHODS Patrick, R. 1957
Page 70: MACROPHYTON 1.0 INTRODUCTION . 2.0
Page 73 and 74: 3.0 REFERENCES MACROPHYTON Blackbur
Page 75 and 76: I '" MACROINVERTEBRATES 1.0 INTRODU
Page 77 and 78: 1.0 INTRODUCTION The aquatic macroi
Page 79 and 80: the limitations of available sampli
Page 81 and 82: Because of the extreme spatial and
Page 83: predetermined depth. Grabs with spr
Page 86 and 87: BIOLOGICAL METHODS Because of the s
Page 88 and 89: BIOLOGICAL METHODS fauna and hydrol
Page 90: BIOLOGICAL METHODS technique, or co
Page 94: BIOLOGICAL METHODS Equitability "e,
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BIOLOGICAL METHODS TABLE 7. CLASSIF
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BIOLOGICAL METHODS TABLE 7. (Contin
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TABLE 7. (Continued) MACROINVERTEBR
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MACROlNVERTEBRATE REFERENCES 33. Ll
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MACROINVERTEBRATE REFERENCES Hauber
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MACROINVERTEBRATE REFERENCES Robert
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FISH
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Deepwater seInIng usually requires
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BIOLOGICAL METHODS (Lonchocarpus ni
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BIOLOGICAL METHODS Figure 4. Gill n
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BIOLOGICAL METHODS the major univer
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BIOLOGICAL METHODS 7.0 BIBLIOGRAPHY
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Freshwater: Northeast FISH REFERENC
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FISH REFERENCES U.S. Department of
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BIOASSAY 1.0 GENERAL CONSIDERATIONS
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BIOLOGICAL METHODS When making wast
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BIOLOGICAL METHODS TABLE 1. STOCK C
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BIOLOGICAL METHODS the concentratio
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BIOLOGICAL METHODS Dimick, R. E., a
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Laboratory in Newtown, Ohio. Groups
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taining some yellow pigment, coarse
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Appendix A Test (Evansville, Indian
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2.3 Food Use a good frozen trout fo
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BIOLOGICAL METHODS 3.5 Methods When
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APPENDIX
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Item Source Cat. No. Unit Approx. C
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2.3 Fish Sources of information on
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Definitions In its original concept
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To Minims _ Liquid Ounces _ Gills _
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