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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.

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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

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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

Station

Depth

 (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

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  ♂     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

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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

1) Jannasch, H. W. and Jones, G. E.. Bacterial populations in the sea water as determined by  dぼerent methods of enumeration. LtTjtnol.Ocean・gr., 4, 128-139 (1959).

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).

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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).

(Manuscript

received, September

14, 1977)

Table 1Number and hiomass・ofthe hacterio detected by direct microscopic  me£hod、extinction dilutio、1 method、or agar 夕ol'Γplatemethod
Fig. 2. Relationship between the direct  microscopic counts and the extinction dilu‑
Fig. 5. Cumulative frequency distribution  of bacterial volumes. Samples were collee・

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