This article was downloaded by: [North Carolina State University]On: 11 May 2013, At: 14:19Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

British Phycological JournalPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/tejp19

Enumeration of natural MicrocystispopulationsC.S. Reynolds a & G.H.M. Jaworski aa Freshwater Biological Association, The Ferry House,Ambleside, Cumbria, LA22 0LPPublished online: 24 Feb 2007.

To cite this article: C.S. Reynolds & G.H.M. Jaworski (1978): Enumeration of naturalMicrocystis populations, British Phycological Journal, 13:3, 269-277

To link to this article: http://dx.doi.org/10.1080/00071617800650331

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions

This article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,systematic supply, or distribution in any form to anyone is expressly forbidden.

The publisher does not give any warranty express or implied or make anyrepresentation that the contents will be complete or accurate or up to date. Theaccuracy of any instructions, formulae, and drug doses should be independentlyverified with primary sources. The publisher shall not be liable for any loss, actions,claims, proceedings, demand, or costs or damages whatsoever or howsoevercaused arising directly or indirectly in connection with or arising out of the use ofthis material.

Br. phycoL J. 13:269-277 I September t978

E N U M E R A T I O N O F N A T U R A L M I C R O C Y S T I S P O P U L A T I O N S

By C. S. REYNOLDS and G. H. M. JAWORSKI

Freshwater Biological Association, The Ferry House, Ambleside, Cumbria, LA22 0LP

Three methods for estimating the ceil concentrations of natural, colonial Microcystis populations are described, and the results are compared statistically. Ultrasonic disruption of colonies is most conveniently applied to dense populations and gives results comparable with those obtained by alkaline hydrolysis. Excessive exposvre to ultrasonic waves may destroy cells. A regression relating cell concentration to mean colony volume can, subject to conditions described, give crude estimates of Mieroeystis at low population densities or in mixed populations.

Programmes monitoring phytoplankton in lakes and reservoirs usually involve the estimation of changes in the concentrations of algae in space and time. Direct counting methods (e.g. Utermohl, 1931; Gilbert, t942; Lund, 1951, 1959; Lund &Talling, 1957; Lund, Kipling & Le Cren, 1958; Youngman, 1971; Will6n, 1976) are essential if changes in specific algae present in mixed populations are to be followed. Whereas single-celled organisms, and many simple colonial forms are enumerated with comparative ease, cell concentrations of the blue-green algae which form large, dense, mucilage-bound colonies (Coelosphaerium, Gomphosphaeria, Microcvstis) can scarcely be estimated by direct counts. In many ecological studies populations of these organisms are necessarily cited as colonies per unit volume of lakewater. Inherent variations in the cell:colony ratio, even within individual populations, reduce the com- parability of colony counts and limit the scope of any data related to the specific biomass of these algae.

Several existing methods potentially overcome these difficulties of quantifi- cation. Populations which are virtually monospecific may be most conveniently quantified in terms of their particulate volume [measured electronically on a Coulter Counter: see Evans & M cGill (1970)], of their pigment content (usually chlorophyll a), carbon content or dry weight. The possibility of using the con- centration of phycoerythrin extracted from water samples as an index of the blue-green algal biomass of mixed plankton populations has been discussed by Watanabe (I 977). Pre-treatment of samples with alkali hydrolyses the mucilage binding the colonial algae sufficiently for individual cells to be distinguished microscopically (e.g. Reynolds, t973). More recently, disintegration of blue- green algal colonies has been achieved by exposing them to ultra-sonic waves enabling cell counts to be made (Cronberg, Gelin & Larsson, 1975; Coveney et al., 1977; Dr T. J. Lack, personal communication). From comparisons of the mean diameters of Microcystis colonies in suspensions roughly fractionated according to size with estimates of the cell concentrations in corresponding

269

0007-1617J78/0901-0269 $02.00/0 © 1978 British Phycological Society

Dow

nloa

ded

by [

Nor

th C

arol

ina

Stat

e U

nive

rsity

] at

14:

19 1

1 M

ay 2

013

270 C. S, REYNOLDS AND G. H. M. JAWORSKI

samples t r ea t ed wi th p o t a s s i u m h y d r o x i d e so lu t i on ( the ac tua l effect ive c o n - c en t r a t i on was ~ 2 . 5 ~ by weight ) , R e y n o l d s (1973) o b t a i n e d a r eg res s ion re l a t ing the n u m b e r o f cells in a g iven c o l o n y to its ave r age l inear d imens ions .

I n th is pape r , we c o m p a r e s o m e es t ima tes o f n a t u r a l Microcystis p o p u l a t i o n s o b t a i n e d , in each case, by a t least t w o o f these m e t h o d s . W e a lso c o m m e n t u p o n the i r use in rou t ine m o n i t o r i n g .

M A T E R I A L S A N D M E T H O D S

The natural Mieroeystis populations used in the present account were collected from Rostherne Mere, Cheshire (N.G.R. SJ745843) and from the experimental enclosures ["Lund Tubes", A and B: see Lack & Lurid (1974)] installed in Blelham Tam, Cumbria (NY 366005); mention is also made of populations derived from Crose Mere, Shropshire (SJ 430305) and Slapton Ley, Devonshire (SX 823430). All population data quoted refer to the algal con- centrations in integrated samples collected with a vertical polyethylene tube (Lund& Talling, 1957) of 5 m (or 4-5 m: R ostherne Mere only) in length. Reference is also made to observations on Mieroeystis culture L155, originally isolated from Esthwaite Water, Cumbria and maintained in colonial condition for 9 years in a modified ASM-I medium (see Appendix). Chlorophyll a concentrations were calculated from the absorbance of hot 90 % methanol extracts, according to the equation of "Falling & Driver (1963).

Except where stated, the organisms have been ascribed to Microeystis aeruginosa Kiitz. emend. Elenkin, but a considerable range in colony size, shalze, numerical cell density and in the thickness of the peripheral mucilage was evident, both from lake to lake, and from season to season; these variations will be described in a future paper.

METHODS OF ESTIMATING CELL CONCENTRATION

(a) Alkaline hydrolysis of mucilage 100 mI aliquots of a well-shaken sample or prepared algal suspension were placed in 100 ml

conical flasks, to which a single pellet ( ~ 0-3 g) of sodium or potassium hydroxide was added. Smaller volumes of more dense suspensions were diluted with clean tapwater or filtered lake water to 100 nal. To ensure complete disruption of the more mucilaginous colonies, we have found it necessary to heat the alkaline suspension in a thermostatic oven for 30 rain at c. 90 °. Depending upon the original thickness and consistency of the peripheral mucilage, the hydro- lysis could be carried out overnight without heating. Cold treatment caused minimal shrinkage to the cells, but separation of cells was often incomplete; nevertheless, the treated colonies were sufficiently diffuse to permit individual cells to be distinguished and direct counts to be made.

(b) Ultrasonic disintegration of colonies Rapid (generally < 1 rain) disruption of the colonial structure of Mieroeystis was effected

by ultrasonic vibrations (,-~ 12 ¢~m, 20 kHz), transmitted in an MSE 150 W Disintegrator, equipped with a titanium probe (MSE: Crawley, England). Disintegration was less rapid in our initial experiments which were carried out in an Elliott Accoustica ultrasonic bath (Elliott Bros: London). The resultant suspensions were homogeneous and directly ready for counting Microscopically, no mucilage or cell debris was observable after 1 rain exposure; judging by the immediate colour change in the algal suspensions at the start of ultrasonieation, collapse of gas vacuoles normally occurs almost instantaneously. To test for possible damage to cells by cavitation, we carried out a series of ultrasonic exposures on stationary phase clones of Mierocystis L155 in which the colonies had become diffuse; the cells were present singly or in small loose groups, permitting direct counts to be made on the untreated material. Suspen- sions were also enumerated after timed ultrasonications. We demonstrated that older, isolated cells, at least, may not withstand more than one minute's ultrasonic exposure before their concentration may be significantly reduced (see Fig. 1). In practice, howevel, we have found that i rain is usually sufficient to complete the disruption of the colonies, so that cell destruc- tion need not provide a serious problem.

(c) Regression solution According to Reynolds' (1973) average relationship, based on observations on algae col-

lected from Crose Mere, Rostherne Mere and Slapton Ley, the number of cells (y) in a healthy, quasi-spherical colony of Mieroeystis aeruginosa can be approximated from its diameter (x).

Dow

nloa

ded

by [

Nor

th C

arol

ina

Stat

e U

nive

rsity

] at

14:

19 1

1 M

ay 2

013

Natural Microcystis populations 271

I00

§ 50

._c

o 25

.L \ n T

I I I I ,, I I ~ 30 60 90 120 t80 360 600

T i m e ( s )

FIG. i. Semilog plot of the percentages of cells in stationary-phase Microcystis cultures surviving timed exposures to ultrasonic treatment, e , O, D, ~ , represent separate experiments. Vertical bars indicate the extreme values of the 95 ~ confidence limits of the individual determinations.

3 0 0 0 0

I0 000

3000

IOO0 d z

300

I00

30

3

6

~o/

oe oS lid

?

/ /8 l l ..... I0 30 IO0 300

Colony diometer (Fm)

Fio. 2. The regression of the mean number of cells per Microcystis colony against mean colony diameter (see text). The material was prepared from algal collections from Crose Mere (O) Rostherne Mere ( e ) , and Slapton Ley ([]).

Dow

nloa

ded

by [

Nor

th C

arol

ina

Stat

e U

nive

rsity

] at

14:

19 1

1 M

ay 2

013

272 c . S . REYNOLDS A N D G. H. M, JAWORSK!

The regression equation of the line (shown in Fig. 1 of Reynolds, 1973) has been solved as log loy ~ 2.99 loglo x- log~o 626, and is replotted here in log/k~g format, as Fig. 2. The method, originally designed to quantify developing Microcystis populations when still present at low densities, and often subdominant to other algae, has several drawbacks to more wide- spread application. Firstly, colonies are not always spherical or even nearly so; second (and most important) the spacing of cells within the mucilage envelope is highly variable; and third, the relationship does not hold for populations in which there is a wide variation in colony diameter (and hence, volume on which the cell complement essentially depends). However, we have been able to use the regression to evaluate a large number of Microcystis populations, subject to the following modifications: (1) the peripheral mucilage is ignored for the purposes of colony mensuration, "'colony diameter" being taken as the diameter of the part of the colony occupied by cells; (2) non-spherical colonies are treated as cylinders or ovoids to obtain estimates of their volume; (3) that the "mean diameter" used in the final computation is the diameter of a sphere equal to the mean colony volume. It is necessary to select, at random, not less than 30 colonies to obtain a reasonable estinaate of mean colony volume. The product of the mean number of cells per colony and the mean colony concentration gives the estimated population density; alternatively the diameter of the sphere equal to the cumulative volume can be used to calculate the population. All the data considered here have been calculated by the former method. Equally the mean number of cells could be read off directly from the curve in Fig. 2.

COUNTING AND STATISTICAL TREATMENT

Following methods (a) and (b), we have estimated the numbers of free cells, either in a simplified counting chamber (Lurid, 1959) based on a minimum of 100 fields, or by counting the cells on the bottom of a 1 ml Utermohl sedimentation chamber on the inverted microscope (Lund et al., 1958). Optical resolution of alkali-treated cells is improved by the use of phase- contrast light microscopy. The former is less tedious; the latter may be more accurate, especially if (as in method (a)) colonial disruption is incomplete and the cells retain a clumped distribution. To achieve complete sedimentation it may often be necessary to collapse residual gas vacuoles (this problem does not arise in ultrasonicated cells). We have found that this can be easily achieved by introducing the neutralized suspension into a syringe, which was then closed with a finger and tapped smartly on the bench top; this sudden application of hydrostatic pressure imitates the effect of Klebahn's (1895) classical "Hammer, Cork and Bottle" tech- nique of collapsing gas vacuoles (see also Walsby, 1972).

The margins of error involved in enumerating algal populations are well-known (e.g. Javornicky, 1958; Lund et al., 1958; Will6n, 1976). Assuming random distribution of the organisms we have calculated the 95 ~ confidence limits of our estimates as:

100 ±2~%. Errors arising during sampling or as a direct result of the preparations for counting, and

their impact upon the mean estimates are compared below. The variances and the sample means have been respectively subject to F- and t-test procedures (e.g. Elliott, 1977).

R E S U L T S

E s t i m a t e s o f na tu ra l Microcystis p o p u l a t i o n s , s imu l t aneous ly o b t a i n e d by the a lka l ine hydro lys i s a n d r eg res s ion so lu t ion m e t h o d s are c o m p a r e d in T a b l e I. In each case, t h e conf idence l imi ts o f t he m e a n s ove r l ap , a n d in all bu t t h ree cases, t he m e a n va lue o b t a i n e d by o n e m e t h o d lies w i th in the conf idence l imi ts o f t h e m e a n o f the o ther . N e i t h e r m e t h o d cons i s t en t ly g a v e l a rger m e a n est i- ma te s t h a n the o ther , a n d wi th in t he l imits i m p o s e d , the re is no s ignif icant d i f fe rence be tween the overa l l m e a n s fo r e i t he r m e t h o d .

T a b l e I a lso inc ludes t he r e l evan t d a t a used to test t he nul l hypo thes i s t h a t t he t w o m e t h o d s d id n o t y ie ld s ignif icant ly d i f ferent results . T h e r a t io o f t h e va r i ances ( F = 3.727) is sma l l e r t h a n any cr i t ica l v a l u e (3.79 fo r 7 degrees o f f r e e d o m each , u p p e r 5 ~ po in t s ) c i ted in t he B i o m e t r i k a tables (Pea r son & Har t l ey , 1966, Tab l e 18); t ha t is, the va r i ances a re n o t s ignif icantly d i f ferent at t he 5 % level.

Dow

nloa

ded

by [

Nor

th C

arol

ina

Stat

e U

nive

rsity

] at

14:

19 1

1 M

ay 2

013

Natural Microcystis populations 273

TABLE I. Microcystis population estimates: comparison of the alkaline hydrolysis and regression solution methods

Estimated population (1) Alkaline (2) Regression

Sample source hydrolysis solution

(a) Blelham B, 0-5, 6.IX.76 53844±34622 631464-16304 (b) Blelham B, 0-5, 20.VI.77 51044- 2650 37484- 2650 (c) Blelham B, 0-5, 4.VII.77 22273± 4233 137884- 8720 (d) B[elham B, 0-5, 11.VII.77 52036±17101 1089564-51362 (e) B!elham B, 0-5, 29.VII.77 124925- 7009 11533 5_ 5157 (f) BIelham A, 0-5, 29.VIII.77 224684- 3852 231984- 6314 (9) Blelham A, 0-5, 5.IX.77 228664- 2150 21685 4-15641

*(h) Rostherne Mere, 0 m, 22.IX.77 432964-10332 45512± 6372

Mean (37) 29297"38 4-10243-63 36445"75 ~ 14042-50 Variance (S z) 331,518,312 1,235,625,830

Degrees of freedom 14 (F = ) ($2)2/($2)1 3"727 Student's t 0"948

* Original sample contained occasional colonies ( < 4 ~ by number) of M. aeruginosa f. flos-aquae. Cells will have been included in the alkaline hydrolysis enumeration but not in the regression solution.

TABLE II. Microcystis population estimates: comparison of the ultrasonic disintegration and regression solution methods

Estimated population (1) Ultrasonic (2) Regression

Sample source disintegration solution

(a) Blelham B, 0-5, 4.X.76 (b) Blelham B, 0-5, 11.X.76 (c) Blelham B, 0-5, 18.X.76 (d) Blelham B, 0-5, 25.X.76 (e) Blelham B, 0-5, 2.XI.76 (f) Blelham B, 0-5, 9.XI.76 (9) Blelham B, 0-5, 23.XI.76

*(h) Rostherne Mere, 0-4.5, 30.VII.77 t(j) Rostherne Mete, 0 m 22.IX.77

1528304-41954 2435754-62891 172451 q- 27509 218434 4- 60582 120391 4- 28989 177595 _t_ 63794 113489 4-17748 201310 4- 76088 106192 4- 21431 64203 4-46524 1057104-21334 110921 +26316 48117+21653 558444-19256

194481 4-32414 217535±33668 367024-11043 455124- 6372

Mean (37) 116707"00±24897'22 148325.444-43943.44 Variance (S 2) 2,725,203,213 6,255,102,000

Degrees of freedom 16 (F = ) ($2)2/(SZ)1 2.295 Student's t 1-001

* Original material preserved in Lugol's iodine prior to enumeration. t From same suspension as sample (h), Table I.

Dow

nloa

ded

by [

Nor

th C

arol

ina

Stat

e U

nive

rsity

] at

14:

19 1

1 M

ay 2

013

274 C. S. REYNOLDS A N D G. H. M. JAWORSKI

Similarly, solution of Student's t gives a value 0-948, which is smaller than values for 14 degrees of freedom tabulated for probabilities 2Q < 20~£ (e.g. Pearson & Hartley, 1966, Table 12). Thus, the null hypothesis is not disproved, and the means derived by the two methods are not significantly different.

Estimates of the cell concentrations of Microcystis populations using ultra- sonic disintegration are similarly compared with simultaneous estimates, based on the regression solution, in Table II. Again, the confidence limits of cor- responding pairs overlap, although in only half the cases do they encompass the mean value obtained by the other method. In eight cases out of nine, the regression solution gave a greater mean estimate than did the ultrasonic dis- ruption method. Although the difference between the totals of the means is relatively greater than in the earlier comparison (Table I), it is still not signifi- cant. The ratio of the variances (F = 2-295) for 8 degrees of freedom indicates (Pearson & Hartley, 1966, Table 18) that the variances are not significantly different at the 10~ level. Similarly, solution of t (= 1.001) shows that the difference between the mean values (if1, -rz2) is not significant at the 20% level.

60

"~ 4O

20

40 80 120 160 Cells [-I x 106

F]G. 3. The relationship between the cell concentration of dominant Microcystis populations in Blelham Enclosure B, 1976, and the chlorophyll content of corresponding water samples.

Since the alkaline hydrolysis and ultrasonic disintegration method yield results which are statistically similar to the regression solution, it follows that they are themselves similar to each other. We have subjected only one sample (the Rostherne Mere surface dip, 22:IX:77; h in Table I; j in Table II) to simultaneous analysis by all three methods, which indeed gave comparable results; the true population probably lay in the range 39-48 x 10 3 cells ml-L The ultrasonic disruption technique gave the lowest results; it is possible that regular use might consistently underestimate populations, but the errors involved should not exceed those attributable to counting.

Dow

nloa

ded

by [

Nor

th C

arol

ina

Stat

e U

nive

rsity

] at

14:

19 1

1 M

ay 2

013

Natural Microcystis populations 275

During October and November 1976 the phytoplankton of Blelham Enclosure B consisted almost entirely of Microcystis. Weekly estimates of cell concentra- tion, using the ultrasonic disintegration technique, have been compared with the chlorophyll a concentrations in the original samples. The correlation (see Fig. 3) is highly significant (r = 0.841, P < 0.01) and suggests consistent performance of the method. The regression coefficient is an expression of the mean chloro- phyll content of the Microcystis population, 0.37 pg chl a (10 6) cells -1. Incidentally, this estimate may be compared with the chlorophyll content of the Rostherne Mere dip sample (equivalent to 15.85+0-I1 pgl-~) , or between 0.37_+0.11 (estimated by alkaline hydrolysis) and 0.43+0.27 Ftg (106) cells-1 (ultrasonic disintegration).

DISCUSSION

All three methods tested gave estimates of natural, colonial, Microcystis populations which are mutually similar, or at least, whose differences do not exceed the confidence limits of the counting methods employed. When two or more methods are compared, overlapping confidence limits are more likely to embrace the true population density than to either under- or over-estimate it to a similar extent. The alkaline hydrolysis method seems to be the least likely to give an erroneous estimate of the live cell concentration of colonial blue-green algae, because it does not destroy intact cell walls. Its major defect may lie in the incomplete dispersion of cells in the treated suspension; this can be over- come by counting a larger number of sample fields. In view of the convenience and rapidity with which homogeneous cell suspensions may be prepared, ultrasonic disintegration would provide a more attractive alternative in most laboratories. According to our results the method can give acceptable estimates of cell concentration. However, it is essential that exposure to ultrasonic waves should be for the minimum time judged to bring about the required degree of colonial disruption and, generally, should not exceed 1 rain. Another potential advantage of this method is that it can be used on some preserved materials.

The regression solution method is perhaps the least useful of the three alterna- tives, largely because of the variation in colony shape, size and cell density, both between individuals and between populations, and because the calculations involved are cumbersome. It may be most practical when several species or varieties of blue-green algae are present and the colonial habit is required for diagnostic purposes, or when developing Mierocystis populations are still sub- dominant to other algal species, and might not be easily distinguishable after colonial disruption. All three methods are su~ect to the efficiency of sampling of natural blue-green algal populations.

A C K N O W L E D G E M E N T S

We are most grateful to Dr J. W. G. Lund, C.B.E., F.R.S., for permission to sample the Blelham Enclosures, and to the Nature Conservancy Council who allowed us to visit Rostherne Mere National Nature Reserve. We would particularly like to thank Dr T. J. Lack of the Water Research Centre who supplied us with unpublished details of his technique for ultrasonic disintegration of Microcystis, and Miss C. Kipling and Dr Lurid who read and commented upon the manuscript. The assistance provided by Mr M. J. Nield, both in the field and in the laboratory is also gratefully acknowledged.

Dow

nloa

ded

by [

Nor

th C

arol

ina

Stat

e U

nive

rsity

] at

14:

19 1

1 M

ay 2

013

276 C. S. REYNOLDS A N D G. H. M. JAWORSKI

APPENDIX

The modified culture medium, based on the ASM-1 medium of Gorham et al., 1964 and developed by G. H. M. Jaworski, in which Microcystis L155 has been maintained in colonial condition since its isolation in 1969.

Stock A. NaNO3 1-7 g MgSO4.7H20 0"49 g MgCI2 . 6H20 0.41 g CaCl2 .2H20 0.29 g Distilled water to 2 litres.

Stock B. KH2PO4 0.87 g Na2HPO,, 0-705 g Distilled water to 1 litre.

Stock C. H3BOa 2.48 g MnCl~. 4H20 1.39 g (NH4)6MoTO24.4H20 1-00 g Distilled water to 1 litre.

Stock D. EDTA. Na2 2.00 g FeCI3.6H~O 1.66 g Distilled water to 1 litre.

The modified ASM medium is then made up from the stock solutions as follows:

Stock A 200 ml Stock B 20 ml Stock C 1 ml Stock D 4 ml Distilled water 775 ml.

REFERENCES COVENEY, M. F., CRONBERG, G., ENELL, M., LARSSON, K. & OLOFSON, L., 1977. Phytoplankton,

zooplankton and bacteria-standing crop and production relationships in a eutrophic lake. Oikos, 29: 5-21.

CRONBERG, G., GELIN, C. & LARSSON, K., 1975. Lake Trumen restoration project II. Bacteria, phytoplankton and phytoplankton productivity. Verh. int. I/erein. theor, angew. Limno/., 19: 1088-1096.

ELLtOTT, J. M., 1977. Some methods for the statistical analysis of samples of benthic inverte- brates. Scient. Pubis Freshwat. biok Ass., 25 (2nd Edition), 160.

EVANS, J. H. & McGILL, S. M., 1970. An investigation of the Coulter Counter in "biomass" determinations of natural freshwater phytoplankton populations. Hydrobiologia, 35: 401-419.

GILBERT, J. Y., 1942. The errors of the Sedgewick-Rafter Counting Chamber in the enumera- tion of phytoplankton. Trans. Am. microsc. Soc., 61: 217-226.

GORHAM, P. R., McLACHLAN, J., HAMMER, U. T. & KIM, W. K., 1964. Isolation and culture of toxic strains of Anabaena ties-aquae (Lyngb.) Breb. Verh. int. Verein. theor, angew. LimnoL, 15: 796-804.

JAVORNI~Y, P., 1958. Revise n6kter~ch meted pro zjig{ovfini kvantity fytoplanktonu. Sb. vys. Sk. chem.-technoL, Praze, 2: 283-367.

KLEBAHN, H., 1895. Gasvakuoten, ein Bestandteil der Zellen der Wasserbliitebildenden Phycochromaceen. Flora, Jena, 80: 241-282.

LACg, T. J. & Lur,tD, J. W. G., 1974. Observations and experiments on the phytoplankton of Blelham Tam, English Lake District. I. The experimental tubes. Freshwat. bioL, 4: 399-415.

LUND, J. W. G., 1951. A sedimentation technique for counting algae and other organisms. Hydrobiologia, 3: 390-394.

LUND, J. W. G., 1959. A simple counting chamber for nannoplankton. Limnol. Oceanogr., 4: 57-65.

LtmD, J. W. G., IOPLINQ, C. & LE CREN, E. D., 1958. The inverted microscope method of estimating algal numbers and the statistical basis of estimations by counting. Hydro- biolo.qia, 11: 143-170.

LUND, J. W. G. & TALLING, J. F., 1957. Botanical limnological methods with special reference to the algae. Bet. Rev., 23: 489-583,

Dow

nloa

ded

by [

Nor

th C

arol

ina

Stat

e U

nive

rsity

] at

14:

19 1

1 M

ay 2

013

Natural Microcystis populations 277

PEARSON, E. S. & HARTLEY, H. O., 1966. Biometrika tables for statisticians, Vol. I 3rd Edition. Cambridge University Press, Cambridge.

REYNOLDS, C. S., 1973. Growth and buoyancy ef Microcystis aeruginosa Kiitz. emend. Elenkin in a shallow eutrophic lake. Proc. R. Soc. B, 184: 29-50.

TALLING, J. F. • DRIVER, D., 1963. Some problems in the estimation of chlorophyll a in phytoplankton. Proceedings, Conference on pr#nary production measurement, marine and _freshwater, Hawaii, 1961. U.S. Atomic Energy Comm., TID 7633:142-146.

UTERM6HL, H., 1931. Neue Wege in der quantitativen Erfassung der Planktons. Verh. int. Verein. theor, angew. L#nnoL, S: 567-597.

WALSBY, A. E., 1972. Structure and function of gas vacuoles. Bact. Rev., 36: 1-32. WATANABE, M. E., 1977. Phycoerythrin in the deeper water layer of a stratified eutrophic lake:

an application of bile pigment in determining the standing crop of blue-green algae. Int. Revue #es. Hydrobiol. Hydrogr., 62: 549-556.

WILL~N, E., 1976. A simplified method of phytoplankton counting. Dr. phycol. J., 11 : 265 278. YOUNGMAN, R. E., 1971. Algal monitoring of water supply reservoirs and rivers. Tech. Memo.

Water Res. Ass., 63: 26.

(Accepted 18 April 1978)

Dow

nloa

ded

by [

Nor

th C

arol

ina

Stat

e U

nive

rsity

] at

14:

19 1

1 M

ay 2

013