The “oxidase reaction” (using p-amino-dimethyl-aniline oxalate as the reagent) has been used to distinguish oxidase-negative from oxidase-positive bacteria from the sea, when grown on membrane filters. By this means, it has been shown (a) that under conditions of stable stratification of  the sea as in the tropics, a relationship exists between the percentage incidence of oxidase negative bacteria in the flora and the depth of the water; (b) that the maximum value for this percentage incidence (100) is reached at or immediately below the upper limit of the oxygen minimum layer; (c) that this percentage value (expressed as Oxⁿvalues) may be used to demonstrate the movements of water masses during upwelling. Such upwelling as indicated by theoretical findings and by temperature determinations along two transects off the west coast of Ceylon during the north east monsoon, has been confirmed by the distribution of Oxⁿvalues at these transects

De Silva, N.N.

Aquatic Commons

Bull. Fish. Res. Stn., Ceylon. Vol. 16, No.2, pp. 1-10, JJecember, 196J 
Marine Bacteria as Indicators of Upwelling in the Sea 
By 
N. N. DE SILVA 
(Fisheries Research Station, Colombo 3, Ceylon) 
AJ3STRAOT 
THE ' oxidase reaction ' (using p-amino-dimethyl-aniline oxalate as the reagent) has been used to distinguish 
oxidase-_negative from oxidase-positive bacteria from the sea, when grown on membrane filters. By this means-
iii has been shown (a) that under conditions of stable stratification of the sea as in the tropics, a relationship 
exists between the percentage incidence of oxidase negative bacteria in the :flora and the depth of the water; 
(b) that the maximum value for this percentage incidence (100) is reached at or immediately below the upper 
limit of the oxygen minimum layer; (c) that this percentage value (expressed as Oxn values) may be used to-
demonstrate the movements of water masses during upwelling. 
Such upwelling as indicated by theoretical findings and by temperature determinations along two transects 
off the west coast of Ceylon during the north east monsoon, has been confirmed by the distribution of Oxn values ai£ 
these transects. 
Introduction 
Many of the classical techniques used at present for the study of oceanic upwelling have been 
found, especially under tropical conditions, to be of limited value. These techniques are based 
on chemical estimations of chlorides, phosphates, nitrates, silicates and oxygen, as well as 
physical measurements of temperature, of the water of the deeper layers of the sea and that of 
th~ surface. However many of these techniques suffer from the same kind of disadvantage as 
that pointed out by Cooper (1960) in the use of salinity determinations during studies of deep-
sea oceanography-that the interpreted differences are not much greater than the errors of 
measurement and that an exact measure of the statistical significance of this error cannot. 
readily be made. The type of difficulty in demonstrating upwelling with these techniques is. 
evident, for instance, in the work of Rochford (1962). He found that though phosphate deter-
mi11ations indicated two regions of upwelled water off North West Australia, south of Timor and 
in the East Arafura Sea, in both instances determinations of salinities and temperatures we:re 
very variable and gave no indication of upwelling. 
The phenomenon of upwelling is of special significance under tropical conditions, parti-
cularly due to its correlation with regions of increased productivity in the sea. In addition there 
is reason to believe that coastal upwelling of cold deeper water influences fish behaviour signi-
fi.cantly; for fish are known to react to temperature differences of even 0.03°C. (Bull, 1952). 
Theoretical calculations based on the work of Sverdrup and his colleagues (Sverdrup,. 
1930; 1938; Sverdrup & Fleming, 1941) and of Yoshida (1955) indicated that conditions of wind 
force and direction during the North East monsoon were favourable for upwelling on the west 
coast of Ceylon. An attempt was therefore made to confirm these theoretical findings, especially 
by using certain characteristic properties of marine bacteria as indicators o;f such water movement_ 
Methods 
At about 20 stations between 50 and 100 miles off-shore, bacterial sampling of the water was: 
done at various depths from the surface down to 200 meters. A similar number of stations was: 
chosen cetween 25 to 30 miles off the shore. These stations were chosen for correlation between 
depth and bacterial flora after bathythermographic readings had indicated a stabilised layer of 
discontinuity at depths between 200 to 250 meters. 
For the study of upwelling, sampling stations were chosen along two transects at right 
angles to the shore, one off Balapitiya (6o38'N), and the other off Chilaw (7°38'N) on the, 
west coast of Ceylon. The sampling stations were so located on these transects that about half 
of them were within and about half beyond the edge of the continental shelf. Each transect 
2-R 11560 (10/63) 
M.A.IUNE B.A.OTERI.A. AS INDICATORS OF UPWELLING 
itsclf was sited as far away as possible from major river mouths to minimise :fluviatile inter-
ference wi±h hydrological conditions. At the Balapitiya transe(lt the :North East Monsoon 
·current system had stabilised itself after ·rounding Dondra ·Point· at· the southern tip·· of the 
·:Slana. At the Chilaw tran.f)ect this current system had swung North West .and had weakened 
~onsiderably. 
·water was collected at interv-al~:; down to 1epths of 200 meters at sampling stations beyond 
'the continental shelf and to appropriate lesser depths at the inshore stations, as indicated in 
Figs. 2, 3, 4 and 5. Nansen bottles were used and, immediately they were hauled in, temperatures 
were re.eorded and the wnter~ sampled for bacterial flora. Bacterial counts wore done using the 
millipore membrane filter technique (Kri::>s, Rukina .& Kovozhilova, 1952) o:t::t 25 mls. of water 
{or onappr.oprittte dilutions of surface and other samples down to depths of 50 m., sterile sea-
'l>Vater being the diluent). The filtration vYas carried out within 5 to 10 minutes of sampling. 
'1'4e membranes were incubated in aluminium tin::;, one in each, on filter pads impregnated with 
ZoBell's liquid medium, 2213 (.ZoBel!, 1946), with o.ged sea water as base. 
1]Jhe _colonies were cou;nted after incubution at 20°C. for 3 days, and at 28-30"C. for 
auother 3 days. Colonies were picked up at. random from duplleate samples for generic deter-
minati-Qns. of the bacteri<L The '' oxidase '' reaction of- the colonies on the membrane was 
determined by placing the membrane, with colonies intact, on a dl'Op ( abou.t 2 mls.) of a freshly 
prepared 5% soln. of p-amino-dimethyl-analiue oxalate (Difco) in distilled water for about a 
minute. \Vhen the positive colonies had taken on a dark pink eolour (which later turned to 
black, .especially i.n the centre) the membrane -vyas plaeed on a clean glas::; dish anJ. both types 
of coloT:Jies counted at a· magnification of 10 t.:mes with a Zeiss stereoscopic microscope. The 
.::-elat.ive abundance of oxidase negative and oxida:::e positive colonies W<.cts calculated and express-
ed aR the percentage of oxidase negative colonies in the total count. An average o.t two or mor8 
dU.plieate .counbs was used for computing the final value of this petcentage. 
Results 
i!'rom .. samples o£ sea water at locations where stable stratification occurred as iDdicatecl by 
bathythenr10graphic readings, it was possible to demonstrate a relationship, wh::.ch is statistically 
sig6.;1'ica.ut, between the. proportion of oxidase negative l.htcteria in the total vi:tble count and 
depth. C:own to and below t,he layer of discontinuity (Fig. J.). This rehtionshi:o can be used for 
estir.."!ating the depth of the d.iscontilwity layel'-t,he depth at which the percentage o£ oxida;;e 
negative bl:hcteria in the flora (Ox.n value) reaches 100 corresponding <tpproximately to the upper 
li.rn.ii. ·:A this layer. 
~T'emperature J.etermlllatious along the transects showed upwelling (:Figs. 2 & 4). A ·com-
parison oi Figs. 2 & 3 and 3 & 4, of the temperatwe profiles at. the transects with the distdbution 
of the Ox"" values on those same transects, showed that these latter values are an even clearer 
iuciication of ·bhis upwelling. 
From both temperature aud Ox.n values at the Balapitiy::;t transect (Figs. 2 & 3); a picture 
emei"ges. of a stream of water in cross-section, upwelling obliquely across the . face of the steep 
contirumtal slope. Lowest temperatures and highest Oxn values are found in the core of the 
stream., ·Temperatui:e- n1.easuren:ienfs did not clearly indicate the emergence of thi,; stream at 
-~he smface, but the Oxn values do, Yery markedly. The transect off Chilaw presents a somewhat 
d~fferent picture, o£ a stream of water rit>ing almost vertically from about 100 m. below the 
:Slll'face" 
Diwussion 
Siuce the pioneering work of Ekman in 1902 on upwelling, several other workers. have .examined 
·the basis of this phenomenon (Thorade, 1909; l.\icEwen, 1912: Defant, 1936;· Sverdrup, 1930; 
1938; Sverdrup & 'Fleming, 1941; Yoshida, 1955). An application of recent views, those of Yoshida 
(1955) ar1sing out of his stuJ.y·of upwelling off tlie Californian coast, to condition in Ceylon, ·led 
"i.1s to the .conclusion tbat there should be upvn:lliug of W8,ter in nanow bands within relatively 
small distancee f,t;om the shores. 
N. N. DESILVA 3 
In view of the limitations already referred to in the classical techniques for the demons-
. tration of movements of water masses, the possibility of using bacteria as indicators for 'this 
purpose was explored in this study. 
The use of bacteria as indicators of water movements is relatively recent (Kriss, 196Q). 
Jt has been shown that even slight variations in the stratification of the water can be detected 
by determining differences in the abundance of these microorganisms. Kriss and his colleagues, 
however, have relied, except for their broad division of the bacterial flora into ' autotrophs ' and 
· heterotrophs ', upon the total numbers of bacteria rather than on the presence of particmlar 
,groups to characterise the different water masses. They did consider the possibility of using 
morphological types, but reported that " due to the scarcity of microbial forms; chiefly. rods 
and cocci, the morphological characteristics of bacteria could not .be relied on as mdicators in 
in hydrological studies " (Kriss, 1960). This difficulty need not be . expected as regards the 
biochemical and physiologica1 properties of bacteria. These not only show a wide· range of variation 
but they are also sufficiently specific to permit of easy differentiation. · It is precisely such a 
property of the bacterial flora, namely the nature of their cytochromes, that has been success-
fully used in the present investigation to characterise water masses. 
Earlier studies in another connection had shown that the cytochromes, which mediatE) in 
·electron transfer during cellular ·metabolism in a majority of bacteria, are sensitively adapted 
to the oxidation-reduction conditions of the environment. Even within a single group of bacteria 
(the fluorescent pseudomonads) such environmentally determined variations could be demons-
tratd (de Silva & Swain, unpublished). 
Kriss, Lebedeva and Mitzkevitch (1960) ha,ve indicated from the numbers of heterotrophs, 
that the deep waters of the tropical Indian Ocean are probably rich in organic matter. With a 
relatively stable thermocline which has been shown to occur under tropical conditions (Watts, 
1958), it is to be expected that large amounts of decaying organic matter will accumulate at or 
below that layer, producing a zone characterised by a deficiency of oxygen and a low oxidation-
reduction potential. Vinogradov and Voronina (1961) have in fact demonstrated the existence in 
the Arabian Sea of a sharply defined 0 2-minirrium layer with its upper limits about 125 to 200 
meterR below the surface. This limit probably coincides with the layer of sudden density change 
reported by earlier workers (Carruthers et af, 1959; Neiman, 1960); and it is therefore to be 
expected that the bacteria characteristic of these waters will have cytochromes adapted to operat-
ing under conditions of low oxygen tension. The results of the present il!llvestigation confirm this. 
The validity of the observed relationship between Oxn values and depth will, of course, 
have to be confirmed by further investigations in widely different oceanographic conditions: but 
it is interesting to note that extrapolation of curves A & B in Fig. 1 bo the Oxn value of 100, 
in the absence of upwelling, corresponds with fair accuracy to the expected depth of the 
upper limit of the oxygen minimum layer. It is also noteworthy that the maximum Oxn values 
appear inshore in much shallower waters than they do away from the continental shelf. This 
therefore might be an indication of the upward slope of the oxygen minimum layer near the 
continental shelf observed by Carruthers et al (1959) off the Indian coast. If a straight line 
relationship obtains between Oxn values and depth-under conditions of stable stratification, then 
determination of Oxn values at a few depths will provide, by graphical extrapolation, a fHir 
·estimate of the upper limits of the layer of discontinuity and oxygen minimum. 
The work reported here is the first experimental demonstration of upwelling induced by 
monsoon winds off the coast of Ceylon. La Fond has also reported similar upwelling along the 
·east coast of India and has corelated it with the replenishment of plant nutrients in the sea (La 
Fond, 1954; Bhavanarayana & La Fond, 1957). Recently Wyrtki (1962) has demonstrated bhat 
upwelling occurs along the coast of Java and Sumatra on the right flank of the South East 
Equa.torial Current, during the South East Monsoon. These investigations along with the data 
now presented, appear to indicate that monsoon wind induced upwelling is one of the main factors 
responsible for the seasonal enrichment of the sea in the trop.ical and sub-tropical reg-ions of the 
Indian Ocean. 
4 MARINE BACTERIA AS INDICATORS OF UPWELLING 
Acknowledgements 
The author wishes to thank the officers and crew of the Japanese research vessel ' Kuroshio 
MalU ' for providing laboratory facilities on board their vessel and for their co-operation during 
the conduct of this research; to Messrs N. L. R. Munasinghe and A. H. W. Mendis of the 
Fisheries Research Station, Ceylon, for technical assistance and to Dr. A. C. J. Weerekoon for 
many f'timulating discussions on this research and for advice in the preparation of this paper. 
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Oceanography, Vol. 1, pp. li1.7-121. 
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Scripps Inst. Oceanographic, Bull., VoL 4, pp. 2&1-378. 
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pp. 17-34, 16---76. 
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N. N. DE SILVA 5 
VINoGRADOV, M. AND VoRONINA, N. M. (1962). Influence of t·he oxygen deficit on the distribution of plankton 
in the Arabian Sea. Deep Sea Res. Vol. 9, pp. 523-530. 
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WYRTKI, K. (1962). The upwelling in the region between Java and Australia during the South East monsoon. 
Aust. J. Mar. Freshw. Res. Vol. 13, pp. 217-225. 
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Proc. UNESCO Symp. on Physical Oceanography, Tokyo, 1955. 
ZoBELL, C. E. (1945). " Marine Microbiology" Chronica, Botan:ica Co., Waltham. Mass. U.S.A. 
6 
100 
(/) 50 
w 
:;:) 
..J 
~ 
c: 
X 
0 
Fig. 1. 
lOOm 
MARINE BACTERIA .AS INDICATORS OF UPWELLING 
200m 
DEPTH 
300m 
Correlation of Oxn values (=percentage of oxidase negative 
bacteria in the total viable count) with depth of the sea. 
A= Inshore stations, 25-30 miles from the coast; B= Off 
shore stations, 50-100 miles from the coast. 
N, N. DE-SILVA 
100 
"' a: 
... 
... 
"' :=;; 
~ 
% 200 li: 
"' 0 
Fig. 2. 
28·1 27-7 27·8 27·8 21·9 
OIS7AHCE F"ROM SHORE !LONGITUOEI 
:distribution of temperature measurements at the 
transect off Balapitiva. 
7 
8 
100 
IJ) 
a: 
"' .... 
"' 
"" 
z 
"' .... Q. 
UJ 
c 
200 ' 
r 
00 
Fig. 3. 
MARIKE BACTERIA AS INDICA'l'ORS OF UPWELLING 
/ 
' .. 
,... ... -~ ... ~ GO·&o% 
• 2~L30.,._._,,''\. • _: ....... ...!-"'""•--... \ 
.,.!--:---.J~ ';,-r 
' [ • ..i\·30'J,. 
'""-; .. -- .... -...... 
. . 
. . . 
.... ------/~ 7~-150~ ; ........ \ 
'\..... _., ... "',) 
DISTANCE FROM SHORE: ( LONGJTUOEI. 
Distribution of percentage oxida.se negative 
bacteria (Oxn values) at the transect off 
Balapitiya. 
N. N. DE SILVA 
·o 
100 
U> 
cr 
... 
... 
.... 
~ 
0: 
X 
.... 
Q. 
"' a 
200 
Fig. 4. 
27·9 25-1 
.....__ 
47 
~- ;/ 
~ 
~ 
. . . 
~ 
___/5·0 
.. J4·9 
14·6 14·1 
42' 79"Z7' 
DI;;TAf'I~E FROIIII SHORE ( LONGITUDE): 
Distributio:r;J, of temperature measurements at 
transect off Cbilaw. 
10 
"' a: 
"' ... 
"' :E 
.:!:" 
::: 
.... 
... 
.. 
0 
MARINE B.A.OTERI.A. .AS INDIO.A.TORS OF UPWELLING 
Or---~~------~--~~~~----~--~--~~------
_,;;' .; 
100 
200 
.,.', ,, 
, ' _,.'I 
/ I • , •I 
;' • ,' 8!5-95% t' I 
I I I I 
I 1 I 1 I 1 I I 
I t• • I f 
1 J I 
I f /• 
: I I / 
J , , 
\ / I : .... ___ .,~,, 
J· ' 
: 65-85~ i/ 
. / 
~ . ,' 
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... ........ ___ ....... ,' 
,,-a.s.,. 
79"ZT' 
Fig. 5. Distribution of percentage oxidase negative·bactena 
(Oxn values) at the transect off Chilaw. 


Marine bacteria as indicators of upwelling in the Sea

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Marine bacteria as indicators of upwelling in the Sea

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