Bacterial
Abundance
in the Sea
of the ・Hiuchi-Nada
Area
Hideo MiYOSHI
Labor at oりof Aquatic Environment , Departjnent of Agriculture
Abstract : As a part of the JIBP-PM programme, bacteriologicalexaminations were carried out to provide information on bacterial abundance in the Hiuchi-Nada area. Bacterial countings were done by the following three methods : direct microscopic method, extinction dilution method ; and agar pour plate method。
As a whole the apparent number of bacteria decreased according to the method employd in the following order ; direct microscopic count ; extinction dilution count ; agar pour plate count. Significant correlations were observed among the corresponding counts. Irrespective of counting methods employed during the warm water period, the bacterial population was large especially in the topmost few meters of water ; but during the cold water period, the vertical localization
of bacteria was less pronounced. The bacterial population was large in polluted waters。 The volume of these cells fell within the range of 0.1-0.4μ3 with an average volume of 0.26 μ3. Based on these data, the bacterial biotnass in the waters was estimated.
Introduction
As a part of the JIBP-PM
programme,・
the Hiuchi-Nada
area, the central part of the Seto
Inland Sea,
was investigated from an ecological viewpoint. The
waters had
been con-taminated by increasing effluentsfrom the surrounding land, and bacterial biomass seemed
to be imporant
in view of the nutrient budget of the waters. Then
attempts were made
to
estimate the number
and volume
of bacteria in the waters.
As a matter of course, apparent
number
of bacteria was influenced greatly by the counting method
employed".
Thus
two
cultural and one direct microscopic methods
were
employed
for estimating the number
of
bacteria。
The
outline of this work
has been reported^', and the present paper describes the details
of this work.
Materials and Methods
Sampling Water samples were collected at 5 771 depth intervals from surface to bottom by
using Van Dorn samplers.
Bacterial counting After collecting each sample, bacterial counting was undertaken within
l hour by the following three methods :
£)irectmicroscopic・method− An appropriate dilution of 2 Z sample was filtered through a
membrane filter of 0.2μporosity, and the cells retained on the filter were counted by the
procedures described by Lumpkins and Arveson".
Eエtinction dilutionmethod−An appropriate series of decimal dilutions of 2 Z sample was
inoculated into 5 replicate tubes containing 2216 E broth. After 2−3 weeks of incubation
at 30°C, a most probable number count was estimated for the combination of positive and
negative tubes from the table of MPN index.
dishes, and the dishes were poured with molten 2216 E agar medium
and mixed thoroughly.
After 2−3 weeks of incubation at 30°C, an agar pour plate count was
estimated from the
colonies developed.
Estimation
of bacterial biomass
The measurement
of bacterialsize was done under a
mi-croscope with the aid of conventional micrometers, arid the volume of cellswas calculated in
the same manner as described in“Methods
in Aquatic Microbiology”*>. The
specificgravity
of the bacterialcells was presumed to be about 1, so the wet weight of the bacterial cells
can be readily estimated from the volume
of cell. Accordingly,
the dry weight of bacteria
can be caluculated from the wet weight by assuming
that an average wet cellcontains 20 %
dry matter 5,6) ●’
Results and Discussion
Fig. 1. The Hiuchi-Nada area in the Seto Inland Sea, showing locations of sampling.
Date
Bacterial countings of each sample collected from the・ sea of the Hiuchi-Nada area (Fig.
1) were carried out by the direct microscopic method, the ・extinction dilution method, and the agar pour plate method respectively. The results are shown in Table 1。
Irrespective of counting method employed, during the warm water period, bacterial DO-pulation was large especially in the topmost few meters 0f water; but during the cold water period, the vertical localization of bac-teria was less pronounced. Bacterial popula-tion was large in the coastal regions especially in area adjacent to Shikoku Island。
As shown in Figs. 2 and 3, significant correlations were observed between the direct microscopic counts and the two cultural
Table 1Number and hiomass・ofthe hacteriodetected by direct microscopic me£hod、extinction dilutio、1 method、or agar 夕ol'Γplatemethod. Samples mere collectedfrom the Seaof the HiucHi-Nada area Station June 25 1972 4 6 Depth (m) 0 5 0 1 1 5 2 0 2 5 0 0 5 3
Bacterial numbers estimated by : pour plate method 43333320000000 1111111 ××××××× 7406030 −一一・摯・・一ひ641117 伊03 11×× 00 &︱ Extinction dilution method (MPN/ml) 2.4×105 9.0×103 4.3×103 2.3×103 2.3×103 1.5×103 4.3×103 2.4×104 4.3×103 Direct nicroscopii method 【cells/m】) 0 6 0 6 0 4 0 4 0 4 0 3 0 3 1 1 1 1 1 1 1 × × × × × × × 6 2 0 4 4 7 / 0 r S 2 C i C ^ < -s l I r t v D 0605 11×× 25 Lt Pour plate フcount (g/m3) 3.0×10-3 3. SXIO-" 2.1×10-" 8.3×10-5 5.2×10“5 6.8×10-5 3・.6×10-5 3.1×10-3 4.7×10て4 Extinction dilution count (g/ 「) 1.3×10-2 4.7×10-4 2.2×lO-" 1.2×lO-" 1.2×lo-" 9.8×10-5 2.2×10-4 1.3×10-3 2.2×lo-* calculated Direct . microscopic count (g/m3) 1.9X10-1 1.1×10-1 1.6×10-3 1.3×10-3 1.3×10-3 3.0×lo-" 3.4×10-" 2 3 一 I v o c ^ ×10-2 ×10-2
Jan. 26/27 1973 4 6 9 佃 8.8×lo-" 9.9×10“4 9.4×10-" 1.3×10-3 7.8×10-4 1.3×10゛‘3 7.8×10-" 2.0×10-3 2.3×10 ̄3 1.4×10-3 3.2×10-3 3.2×10'3 3.2×10-3 3.0×10“3 7.8×10-4 9.4×10-4 6.8×lO-" 7. 8 X lO"" 1.0×10 ̄3 4.6×10-4 5.7×10-4 8.3×10-4 4.8×lO-" 2.0×10-3 1.3×10-3 8.8×10-4 1.0×10“3 3443﹄一 ・・ 10101010 ×××× 4236 L^ QOL Table 1.(Continued)
Date
StationDepth
(m)
Bacterialnumbers estimated by、:
Bacterial dry weights calculated'!from: − ,Pour plate method (c. f. u. /ml)
Extinction
dilution
method
`(MPN/ml)
Directmicroscopic method(cells/ml) Pour plate `ヽcount・ (g/m3)Extinction
dilution
count
(g/
「)
Direct-microscopic count、 (g/ 「)'・ June 25 1972 9 12 17 19 21 23 0 5 10 15 0 51 10 15 0 5 10 15 0 5 10 15 0 5 10 0 5 10 15 3.9× 2.8× 6.9× 2.2× 1.6× 2.5× 1.3× 2.1× 2.7× 6.3× 2.3× 1.0× 4.4× 4.3× 1.1× 2.0× 5.0× 1.2× 8.0× 3.6× 9.0× 2.3× 1.4× 0 4 0 3 0 3 0 3 0 4 0 3 0 3 0 3 , 0 4 0 3 0 3 0 3 0 4 0 3 0 3 0 2 0 4 0 4 0 2 0 4 0 3 0 3 0 3 4.3× 2.3× 1.5× 4. &× 4.6× 4.3× 1.5× 9.3× 9.3× 4.3× 3.9× 9.0× 9.3× 4.6× 1.5× 9.0× 4.6× 9.5× 4.3× 2.4× 2.4× 2.3× 9.0× 0 4 0 3 0 4 0 3 0 4 0 3 0 3 0 2 0 4 0 3 0 3 0 2 0 4 0 3 0 3 0 2 0 4 0 3 0 3 0 4 0 3 0 3 0 2 3.3× 1.7× 2.2× 1.3× 2.1× 6.3× 4.5× 9.0× 6.3× 6.3× 1.4× 1.5× 3.0× 5.1× 6.0× 4.5× 8.7× 4.5× 6.0× 1.6× 1.2× 6.9× 6.1× 0 6 0 5 0 4 0 4 0 5 0 4 0 4 0 3 0 6 0 5 0 4 0 4 0 6 0 4 0 3 0 3 0 6 0 5 0 4 0 6 0 5 0 4 0 4 2.0× 1.5× 3.6× 1.1× 8.3× 1.3× 6. 8X l.1× 1.4× 3.3× 1.2× 5.2× 2.3× 2.3× 5.7× 1.0× 2.6× 6.2× 4.2× 1.9× 4.7× 1.2× 7.3× O - 3 0 - 4 0 ゛ 4 0 - 4 0 - 4 0 - 4 0 - 5 0 - 4 0 - 3 0 - 4 0  ̄ 4 0 - 5 0 ` 3 0 - 4 0 - 5 0 - 5 0 - 3 0 - 4 0 - 5 0 - 3 0 - 4 0  ̄ 4 0  ̄ 5 2.2× 1.2× 7.8× 2.4× 2.4× 2.4× 7.8× 4.8× 4.8× 2.2× 2.0× 4.7× 4.8× 2.4× 7.8× 4.7× 2.4× 4.9× 2.2× 1.3× 1.3× 1.2× 4.7× O  ̄ 3 0 “ 4 0 - 4 0 - 4 0 - 3 0 ' 4 0 - S O - 5 0  ̄ 3 0 - 4 0 - 4 0  ̄ 5 0  ̄ 3 0 - 4 0 - 5 0  ̄ 5 0 - 3 0 - 4 0 - 4 0  ̄ 3 0 - 4 0 - 4 0 - 5 1.7× 8.8× 1.1× ■6.8× 1.1× 3.3× 2.3× 4.7× 3.3× 3.3× 7.3× 7.8× 1.6× 2.7× 3.1× 2.3× 4.5× 2.3× 3.1× 8.3× 6.2× 3.6× 3.2χ 011 O ̄3 073' O ̄4 0-2 0-3 043 074 O ̄1 O-2 O-4 o-4 o-1 O-3 o-4 O ̄4 o-1 O-2 O-3 O-2 o-3 O ̄3 o-3 O u n 0 − 1 5 2 0 12 17 19 2 1 5 0 0 5 0 0 5 0 5 0 5 0 L T S 2 3 1 1 1 1 1 0 5 1 0 1 5 2 0 O L r t C 3 1 15 O u -j e = j L T i 1 1 322Z222 0000000 1111111 ××××××× 400001z0 `eeS一一一1987836 1.6×104 1.5×104 6.2×103 4 Q り 4 a Q v 0 0 0 0 1 1 1 1 × × × × O O S C 3 C ノ ` 争 一 一 一 l O S C ︱ v D 1.4X10' 8.0×102 5.0×102 8.0×102 2.4×103 3.6×103 2.4×103 3.4×103 3.4×103 8.5×102 1.4×103 6.0×102 6.0×102 0 3 0 3 0 3 0 3 1 1 1 1 × × × × 0 7 2 3 0 d c ︱ s o r Z 3.4×103 3.4×103 1.5×103 1.5×103 1.6×103 1.0×1G3 1.6×103 3.4×104 2.6X10'' 2.6×104 9. 9X10' 6.6×103 2.2×103 1.5×103 9.8×102 9.8×103 9.8×103 6.6×103 6.6×103 6.6×103 1.6×103 3.4X103 1.6×103 2.6×103 9.8×103 9.8×103 9.8×103 1.7×104 1.7×1 1.9×1 1.8×1 2.4×1 1.5×1 2.4×1 1.5×1 4 4 4 4 4 4 4 0 0 0 0 0 0 0 3.8×104 4.5×104 2.6×104 6.1×104 6.2×104 6.2×404 5.7×104 1.5×104 1.8×104 1.3×104 1、5×104 4344300000 11111 ××××× 08163 ・一Iい・ n/″n5119 4 4 4 4 0 0 0 0 − − 1 1 × × × × ' O r o \ O ' ︱ I I I ● ・ 2 1 1 3 一 一 ・ 一 ( v ) x f -^ n 1×10-4 1 7xlo ̄51 2×10 ̄5 6×10-5 4. 2X10"‘5 1.6×10'5 3.4×10-s 8.3×!O-4 7.3×10-" 3.2×10-" 5.2×10-" 5.2×lO-" 3.6×10-4 3.2×10`4 7.3×10-5 4.2×10 ̄5 2.6×10 ̄5 4.2×10-s 1.3×lo-" 1.9×10-" 1.3×10-4 1.8×10-4 1.8×10 ̄4 4.4×10-5 7.3×10 ̄s 3.1×10-5 3.1×10 ̄5 4.2×lO-" 4.0×10-" 3.2×10-4 3.8×10-4 1.8×lo-" 1.8×lo-" 7.8×10-5 7.8×10-= 8.3×10-5 5.2×10-5 8.3×10 ̄5 1.7×10-3 1.4×10 ̄3 ・1.4×10 ̄3 1.1〉ぐ10-3 1.1×10 ̄3 5. 2X10-' 5.2×10 ̄4 3.4×lO-i 1.1×10-4 7.8×10-5 5.1×10-4 5.・1×lO-" 5.1×10-41 3.4X10-" 3.4×10-' 3.4×10-4 8.3×10-5 1.8×10-" 8.3×10 ̄5 1.4X10-" 5.1×lo-" 5.1×10-4 5.1×10 ̄4 8.8×10-4♂ 4 0 ♂ ♂ 1 1 1 1 一 E / N d W ' l u コ o 0 U O I l コ 一 i p U O U 3 U I U ( 3 1 0 1 ,。j11‥‘ よ為/ぬ6 &/
loqY = a8952 loqX −0.4373 r = 0.694
1♂ 103 1{r 105 1♂
Direct microscopic count, eelIs/ml
1 0 7
Fig. 2. Relationship between the direct microscopic counts and the extinction dilu- tion counts. A s June 1972, △:Jan. 1973 一 E / n p j u n o D a j e i d ﹄ コ o l 1 ♂ loqY =1.0046 10q x − 1.1969 r = 0.743 轟 轟 Directmicroscpiccount,cell/ml Fig. 3. Relationship between the direct microscopic counts and the agar pour plate counts.
A: June 1972 △:Jan. 1973. よ● ’ 7
method counts (the extinction dilution counts and the agar pour plate counts), but the direct microscopic counts were larger than the cultural method counts. This latter tendency seemed to be especially true for the water samples of high bacterial density.
The counts obtained by the direct microscopic method should correspond to the total bac-terial cells, but are obliged to include the numbers of dead cells and non-biological particles indistinguishable from bacteria. The counts obtained by the cultural methods should corres-pond to those of heterotrophic members of bacteria. Part of aquatic bactera are believed to be inactive*', and all members of heterotrophic bacteria cannot always proliferate under a given cultrual condition. Therefore, the differences between the direct microscopic counts and the cultural method counts were 一 E ` コ ` Q 、 ) u n o 3 a i e i d ﹄ コ O a 0 1 loqY = 1.2038 log X-1.0025 「z U80 ( ; 1♂ 1♂ 「 1♂ 10s 107 Extinctiondilutioncount. MPN /ml Fig. 4. Relationship between the extinction dilutioncouuts and the agar pour plate counts.
ム:June 1972, △:Jan. 1973.
unavoidable。
As shown in Fig. 4, there was a close correlation between the agar pour plate counts and the extinction dilution counts.
As a whole, the agar pour plate counts were ’a little smaller than the extinction dilution counts. It is noteworthy that
the correlation coefficient between the agar pour plate counts
and the extinc-tion dilution counts was significantly higher than the coefficients between ‘the direct microscopic counts and the
cultural method counts。
The underestimation in agar pour plate counts was mainly due to the thermal
103
sensitivity of bacteria" The difference between the paired cultural method counts was not so large as compared with a previous report'' which was carried out tentatively with various types of seawater samples. The discrepancy may depend.on the difference in bacterial flora。 Thirty-three hundred and ten cells of bacteria were chosen randomly from various depths of water in the whole waters of the Hiuchi-Nada area. and the volumes of these cells were determined. As illustrated in Fig. 5, the volume of most bacterial cells fell within the range of 0.1-0.4 μ3 with an average volume
of 0.26 μ3ヽ The average volume was somewhat larger than that of a previous report5).Thelarger value may be asso-ciated with the fact that the waters con-tained a relatively large amount of nu-trients available for bacteria。
Based on the average volume, the dry weights of bacteria in each sample are also shown in Table 1.
’Of course, the distribution and the abundance of bacterial biomass should vary directly with the bacterial counts. Irrespective of counting methods emplo-yed, the waters had a relatively abun-dant biomass of bacteria. High values of bacterial biomass seemed to be associa-ted with the“eutrohpic”conditions of the waters S 、Aouanuaji 9A!>B│ コ E コ U 0.1 0.2 0.3 0j4 05 0.6 Volume of cell, 1』3
Fig. 5. Cumulative frequency distribution
of bacterial volumes. Samples were collee・
ted from the sea of the Hiuchi-Nada area
at June '11, Aug. '72, and Jan. '73.
Acknowledgements
lam
very gratefulto Mr. Masato
Kondo,
Chief of Oceanography
Section, Nansei Regional
Fisheries Research Laboratory,; Mr.
Haruyuki
Koyama,
Assistant Prof. 0f Hiroshima
Uni-versity; and the skippers and crews of the Shirafujimaru and Toyoshiomaru
for their help
in samplings.
Thanks
are also given to Mr.
Koichi Nakamoto
for his help in the direct
microscopic counting.
References
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2) Hogetsu, K., Hatanaka, M., Hanaoka, T., and Kawamura, T. (ed.), Productivity of Biocenoses in Coastal Regions of Japan, p. 267-283, Univ. Tokyo Press, Tokyo (1977).
3) Lumpkins, E. D. and Arveson, J. S., Improved technique for staining bacteria on membrane filters./1かZ.Microbiol..16, 433-434 (1968).
4) Rodina, A. G., Methods in Aquatic Microbio】ogy【Translated, edited, revised by Colwe】I, R. R. and Zambruski, M. S. ) p. 157-162, Univ. Park Press, Baltimore (1972).
ed. by Oppenheimer, C. H・,p. 3-24, Charles C. Thomas Publisher, i】linois(1963).
6) Sorokin, T. I. and kadota, H. (ed. ), Techniques for the Assessment of Microbial Production and Decomposition in Fresh Waters, p. 50,Blackwell ScientificPublications, Oxford (1972). 7) ZoBell, C. E., and Conn, J. E., Studies on the thermal sensitivityof marine bacteria. J. Bacteriol・,40, 223-238 (1940).