MARINE BACTERIA OF CARDIGAN BAY. 11. BACTERIA IN ROCK POOLS

BY MABYN HUDLESTON

Botany Department, University College of Wales, Aberystwyth

SUMMARY: Two pools of different characteristics were examined for bacterial content, marine and alien. Comparative monthly counts showed little consistency over the sampling periods and no definite seasonal variation. Temperature, pH and light had little direct influence. Chromogenic strains were few. Most marine bacteria were small Gram-negative rods; a high proportion liquefied gelatin, but other biochemical reactions were weak.

The amount of dissolved oxygen increased with exposure of the pools but decreased in a third pool devoid of visible flora and fauna. All three pooh showed a marked increase in marine bacterial content during intertidal exposure. The coli-aerogenes organisms content was low and, 88 in the case of soil and fresh water, the strains showed no definite seasonal variation.

Two TYPICAL rock pools situated on the foreshore of Aberystwyth were examined at monthly intervals with the object of determining the constitution of the marine bacterial flora, the alien bacterial flora, including coli-aerogenes organisms, together with the varying relationships of temperature, hydrogen ion concentration and dissolved oxygen content. In addition a third pool, devoid of visible flora and fauna, was used to determine the amount of increase in bacterial content (if any) during exposure and also for dissolved oxygen estimations.

These rock pools of the intertidal zone lie in the softer mudstone depressions between projecting ridges of grit. They are caused by differential erosion of the ' Aberystwyth Grits ' formation, which forms part of the upper Llandovery rocks of the Silurian system.

EXPERIMENTAL METHODS

Pools examined. Two pools, A and B, both free from sand and mud, were examined. Their characteristics are given in Table 1, together with those of a third pool C , used only for estimations of the dissolved oxygen content.

Sampling. The apparatus consisted of a 300 ml sterile, screw-top, glass bottle which was filled from the pool by hand in the recommended way. Samples were not collected if rain had fallen while the pool was exposed. Samples were collected 7 hr after the retreat of the tide, at monthly intervals over a period of 3 years from pool A and 16 months from pool B. Samples over one 12-month period are available for comparison in A and B. All samples were examined within half an hour. The media used and methods of testing were similar to those described by Hudleston (1955) except that inocula of 10-1 dilutions were used in addition to 1 ml.

Dissolved 0, content. Five tests were made during September 1952 under similar conditions of light, temperature and weather, (a) ' pre-exposure,' immediately

3 0 Mabyn Hudleston

following the retreat of the tide from the pool and (b) ' post-exposure.' just before the pool was submerged. Exposure time amounted to approximately 7 hr. Triplicate experiments were carried out in each case and the average recorded; the variation between these triplicates was in the order of 0.2-1.8 ml O,/litre of sample.

Table 1 . Characteristics of rock pooh A , B and C (all free from sand nnd mud)

Pool Depthof Physical water (in.)

A 18

B 9

C 9

characters

Steepsided triangular cavity. 5 x 2 ft, uurface

{area

Parallel ridges of rock forming shallow elongated pools often inter- communicating. 1 x 6 ft surface

as B above. 1 x 5 ft surface

I [area

{are,

Dominant macroscopic

(a) Flora Abundant Enteromwpha compema Ulva lactuca Corallina officinalis attached to side facing maximum light, filling half the pool Ascophyllum nodosum completely covering the pool

(b) Fauna Scanty Balanwr balanoides (Barnacle) Patella v u l g a l a (Limpet) Littorina littorea (Periwinkle) aa A above together with Actinea equina (sea anemone)

Above the Ascophyllum zone and devoid of visible flora and fauna

RESULTS A N D DISCUS~ION

Physiological factors

Temperature. During the three years of observation, for A and also for B, the temperature curves followed a similar trend (Tables 2 and 3). In these small. exposed pools, water temperature was closely related to air temperature and under certain conditions the range may be over 6" during 7 hr exposure. This is in sharp contrast to the state of affairs in off-shore water where the diurnal range is slight. Water temperature in pool B was frequently higher than in A, possibly due to the blanketing effect of the Ascophyllum in acting as protection from wind. The numbers of bacteria/ml plotted against temperature gave curves (not given) of doubtful value, since the intervals of sampling were too long, but obviously there was little or no relation between bacterial content and temperature. Increase in numbers may at times have corresponded with increase in temperature, but this was not consistent : even optimum temperatures did not always result in high counts. It is possible that this condition is even more conducive to the multiplication of protozoa, for example, which probably have some adverse effect on bacterial content, as in the soil. I n January, February and March 1947 low counts coincided with low tempera- ture in the case of marine strains only: the pools had been thinly iced over shortly before sampling and the pH was also low. This may have been coincidence, however, since in other periods of low temperature this relationship did not appear.

Year end

month

1946-7 June

Aug.

Oct. Nov. Dec. Jm. Feb. Mar. Apr. May

June J d Y Aug. Sept. Oct. Nov. Dec. Jan. Feb. Mar. Apr .

J d Y

Sept.

1948-9

May

1950-1 June July Aug.

Oct. Nov. Dec. Jan. Feb. Mar. Apr. May

Sept .

Light.

Bacteria in rock pools

Table 2. Tempemtures, p H , and c e m w of bacteria in pool A : 3 ymr.9 (Average of triplicate plates)

P=

9.0 9.0 9.0 8.0 8.2 8.6 8-6 8.0 8.0 8.0 8.9 9.0

9.0 9.0 8.9 9.0 8.9 8.9 9.0 8-6 8.9 8.9 9.0 8.9

8.9 8.6 8.9 8-4 8-4 8.2 8.4 8.2 8.4 8.9 8.8 8.4

Water temp. ("C)

18.0 15.0 18.0 15-0 14-0 10.0 10.0 4.0 3.0 4.0 8.5

13.5

24.6 22.0 22.0 17.0 14.5 10.5 6.5 6.0 6.0

10.0 17.0 20.0

19.0 17.5 19.0 18.2 12-0 3.0 6.5 3.0 6.0 7 -0

10.4 16.0

Marine bacteria/ml Colonies/ml (sea water agar) (standard agar) ___h__, n . . 14 days 21 days 5 days 3 days 21 days 1.5-17' 0-3" 22" 37" 0-3'

190 2,312 1,512 1,316 2,052 2,800 1,420

33 93

140 1,196

520

462 166 177 279 74

328 1,016

132 508 278 379

1,672

40 716 562 598 159 184 747 468 508 436

1,840 ,524

145 581 4 270 >5,000 103 179 3,064 162 40 48 40 60 >5,000 120

145 > 5,000 > 5,000 310 > 5.000 > 5,000

35 48

167 4-48

10

69 50 83

105 0

200 548 273 92

221 261 792

532 318 336 64

636 67

884 316

92 232 66

330 1,564

302 4,200 4,948

21 520 36 89 12 10 80

430 270 27

104 7

441 1,128

6 956 70 19

180 67

270 49

1.080 24 >5;000

1 500

10 0

40

0 19 5

14 2

10 9

60 49

1 136 10

4 10 2

56 20 19 28 2

49 3

15 >

15 20 16 20

119 110 73 30 16 85 47

6

6 12 9

18 0 3

25 106 54 22 4

15

632 26 30 21

120 5

110 69 54 58 16 3 5

3'

.MaoConkey's broth (MPN/100 ml)

coli- Bact. wli aerogenes

0 20 50 40 20 20 20 26

0 0

50 0

0 20 26 0 0 0 0

YO 4 0 0

10

10 0

30 0 5 0

35 8 4 0

10 0

type 1

0 140 100 17 0

25 25

4 0 ( 1 0 0

0 0 0 0 0 0 0 8 0 0 0

25

25 0 0 0 8 0

20 0 0 0 0 0

Although bacteria normally develop more rapidly in the dark during artificial cultivation, the counts were by no means consistently greater in pool B, where Ascophyllum formed an effective screen from light, than in the exposed pool A. Light does not appear to be an effective factor even when combined with optimum temperature.

Hydrogen ion concentration. The pH of pool A ranged from 8.0 to 9.0. Pool B ranged from 8.8 to 8.9 (with 2 exceptional readings).

The relationship between algae and the surrounding water has not a~ yet been stated with any certainty; investigators have differed widely in their findings as to the relative effectiveness of different algae in photosynthesis and the consequent

3 2 Mabyn Hudleston

alkalinity of the water. The records from a series of pH determinations in Aberystwyth pools with differing algal content gave a range of averages from 8.6 in ' bare ' pools, with 8.8 in Ascophyllum pools, to 10.0 in pools with Enteromorpha or Ulva, at approximately the same time and under similar conditions.

Dissolved 0, content. Table 4 shows the results of the estimations of dissolved oxygen. The temperatures of the samples ranged between 16-18".

Table 3. Temperature, pH, and census of bacteria in pool B : 16 months (Average of triplicate plates)

Month 1950-1

May

J d Y

Sept .

June

Aug.

Oot. Nov . Deo. Jan. Feb. Mar. Apr.

June

Aug.

May

J d Y

temp (sea water ag&) ("C). &

14 days 21 days 15-17' 0-3'

PH Water Marine bacteriaiml Colonies/ml MacConkey's broth (standard agar) (MPN/lOO ml)

5 - 5 e i G Z 22" 37" 0-3' arogenes type1

8-8 8.9 8.9 8.8 8.9 8.9 8.8 8.8 8.2 8.9 8.9 8.8 8-6 8.9 8.6 8.8

13.3 16.0 21.0 19.5 17-5 12.5 4.0 5.5 4.0

11.0 10.0 16.5 20.0 17.0 19.5 20.0

511 366 996 384

1,440 1,130 1,196 1,004

388 604 276 453

1,892 30

387 2,368

5,000 78 20

118 558 149 592 220 384 246 154 247

3,812 784 584

1,464

1,136 3 5 2

49 2 111 2 499 34

59 7 60 6 44 4 50 3 39 8 55 29 11 4

256 77 36 2

132 154 664 267

200 0 0

13 3 0

17 3 3

35 38 21 58

504 7 5

10 35

0 35 35

7 20 0 0 0

25 0 0 0 0

70

6 10 0 0 0 0 0 0 0 0 0 0

25 0 0 0

Table 4. Dissolved oxygen at NTP (ml/l) in sea water before and after

Sample 1 2 3 4 5

exposure in rock pools for 7 hr in daylightt

A A r - - - - A A a* b* a b a b a b a b

Pool A 8.5 9.7 7.2 9.0 7.0 8.1 6-8 9.6 7-6 9.0 Pool B 7.3 9.7 7.6 8.1 6.6 7-8 5.5 7.6 6-4 7.6 Pool c 6-5 4.6 8.5 4.5 6.5 4.6 5.6 4.0 6.5 4.5

* a, ' pre-exposure ' water; b, water after 7 hr exposure. t Means from triplicate tests.

The Table indicates that after 7 hr exposure in daylight the water in A and B showed an increase in oxygen content in every case. The increase may have been due largely to the photosynthetic processes of the algae present and this seems confirmed by the results from pool C, which had no visible living organisms; here, dissolved 0, was always less after 7 hr exposure. It seems reasonable to assume that this reduction was due mainly to consumption by microscopic life during respiration. Also, in hot weather, the water in type C pools attains a high temperature and some 0, loss must thereby occur.

Bacteria in rock pools 3 3

Marine bacteria

Increase of marine bacteria during exposure. In order to test the assumption that an increase in the bacterial content actually does take place during exposure of a pool, the following experiment was undertaken. Ten series of triplicate, sea water agar plates were inoculated from ' pre-exposure ' water and a corresponding series after 7 hr exposure, using water from pools A, B and C. In all cases there was a definite increase of the marine bacteria in the water after exposure. Table 5 shows that the increase was greatest in pool C, which was devoid of visible life, and least in A, with an abundant growth of algae.

Table 5. Increase of marine bacteria in pools during intertidal exposure of 7 hr (Average Gf triplicate tests in a series of 10)

Pool Range of increase Average increase (%I ( %)

A B C

28 - 69 77-101 72-127

52 84 98

Numbers and types of marine bacteria. Consideration of the bacterial counts in Tables 2 and 3 (and also of graphs, which are not given) strongly emphasizes the danger of making didactic statements concerning the bacterial content based on only a single year's work. The latter can give a reasonable picture for that year only. The three years of samples from pool A (incubation 15-17") showed little consistency, apart from an increase in numbers in July (2 years) and in November or December. There appeared to be no relation between number of bacteria, temperature and pH. It is possible that some relationship might emerge if the number of samples taken a t each date was sufficiently large to cancel out the error of random sampling. Grand totals for the three years amounted to 15,421, 8,155 and 10,348 respectively.

A comparison of the counts for pools A and B, a t the same incubation temperature shows corresponding increases in spring and midsummer. In general, counts/sample were distinctly higher for B, where Ascophyllum covered the pool, although not so consistently as to suggest that light was a deciding factor. The grand total was 16,702. The. pH was more even throughout the year in B, usually 8.8-8.9, and the water temperature normally higher.

The counts from incubations at 0-3" for A and B did not correspond; the numbers were normally below those at 15-17'.

Strains which produced pigment were not numerous; 19, 4 and 5% in the three years samples from pool A and 6% from B. The relatively high percentage recorded for the first year may well have been influenced by the presence of two isolated unusually high counts of fluorescent strains. ZoBell & Feltham (1934) used a similar medium and stated that 69.4% of the total bacteria from sea water or marine mud were pigment producing. Yellow or orange pigmented forms predominated in pools, followed by greenish fluorescent strains and a few red, brown and black colonies.

34 Mabyn Hudleston

About 4% of the colonies produced some liquefaction of the agar medium at 15-11" but not at 0-3". Most of these agar digesters resembled Pseudomonas.

Some morphological and physiological data were obtained concerning 62 marine strains from pool A and 68 strains from B. An attempt was made to find some method of arbitrary classification but the number of groups was always so high that the result was little better than individual description. It seems likely that pleomorphism among marine bacteria is more prevalent than has been thought.

Certain data are recorded in Table 6, including the corresponding findings from off-shore water for purposes of comparison. As in off-shore water, the majority of strains (93.5%) were small Gram-negative rods. This resembles the results obtained by ZoBell (1946), who stated that 95% of the bacteria from sea sources were Gram- negative rods. Similar results were obtained by Taylor (1942) for the bacterial flora of lakes and streams. Cocci were relatively infrequent and spore formers were rare.

Table 6. Sowbe characteristics of marine strains isolated from sea water agar

Source No. of Pigmented Composition of flora, (yo) strains colonies A ,

Gram- Long Short rods, Cocci negative rods, 1:3 below 1:3 rods or over to oval

Pool A 62 I 1 98 10 89 1 Pool B ti1 ' 8 89 8 89 3 Off -shore 93 2 3 96 3 2 64 4

Gelatin liquefaction was slow, none of the cultures showing any trace in 10 days but in 6 weeks 93% of the cultures from pool A and 53% from B showed very marked liquefaction. In contrast, about 22% of the strains from off-shore water exhibited rapid action (within 14) days) although only 43% liquefied in 6 weeks. An average of 73% from pools compares well with ZoBell's 75% for marine bacteria as a group. This figure drops to 43% in Aberystwyth off-shore water and 24% (Taylor, 1942) in fresh water sources.

Saccharolytic action was weak; not more than loo/, of the cultures formed acid in glucose, lactose and sucrose within 8 days a t 22" or 5 days at 30". Similarly, fermentation of litmus milk a t 22 and 30" was not a marked characteristic of the cultures; a few produced some peptonization or an alkaline reaction.

Nitrate reduction tests gave indecisive or negative results, and MacConkey's broth was not fermented. All the strains grew readily on sea water agar a t 15-17', but only 12 showed

slight growth on standard nutrient agar. in nutrient peptone broth and on potato wedges at 22".

dlien bacteria

Tables 2 and 3 record the counts of ' soil and fresh water ' strains obtained by methods described by Hudleston (1955). These bacteria are of random occurrence and aa expected were more numerous in pools than in off-shore water. In nearly half the samples, they exceeded the number of marine bacteria.

Bacteria in rock pools 35

During the first year for pool A, the counts appeared to follow certain trends of the weather. In general, the highest numbers occurred following gales and storms, but this also applied, with less apparent reason, to the marine organisms. A drop in numbers coincided with a particularly cold, severe spell of weather. together with low pH and destruction of Ulua by storms. The connexion, however, may be more apparent than real, for these coincidences did not hold in the other two years; admittedly, the weather was much less violent. Pigment producing bacteria were again relatively uncommon, constituting only !)yo of the cultures examined.

(loli-aercgenes Docteriu in s e a water. These organisms occurred less consistently than might have been expected considering the possible excretal contamination from seagulls. The MPN of coli-aerogenes organisms and Bact. coli type I/100ml of sample are indicated for pools A and B in Tables 2 and 3 . The data are re-assembled in Tables 7 and 8, together with relevant facts concerning off-shore water.

Table 7. The incidencr of coli-uerogenes organisms in samples of seu mter taken 14 miles off-shore in Cardigan Bay and from rock pool^ in the inta&hl

zone at Aberystwyth

Seevoii No. of Coli-aerogenes organisms/100 ml Buct. coli type I/100 ml n samdes - , -\

( 1 1-10 Off-alwre aamplea 1949-50 June-August 12 6 I

Sept.-Nov. 8 0 ,5 Dec.-Feb. 4 3 1 Mar.-May 1 3 1 Totals 'ti 12 12 Percentage 100 42.9 42.!)

Hock pool sankples 1946-7, 1948-9. 1!+5&1 June-August 16 6 "' Sept.-Nov. 12

Mar.-May 12 8 - >

) f

) 3 Dec.-Feb. 12

Totals 52 24 9 Percentage 100 46.2 17.3

-

11-50

1 3 0 0 4

14.3

I

3 2

17 32.7

>

;,.so ( 1 1-5 (i-10

0 12 b 0 0 5 2 1 0 4 0 0 0 4 0 0 0 25 2 1 0 89.3 7 . 1 3.6

1 1 1 0 2 0 9 0 1 1 8 1 1 0 10 0 0 2 38 1 4 3.8 7 3 . 1 1.9 7.7

Table 8. Presence of coli-aerogenes bacteria in off-shore water and in rock pools

source No. of Per cent. samples with: samples r -JL----7

Coli-aerogenes Bact. coli type I bacteria present present in

in 100 ml 100 ml Off -shore 28 57.1 10.7 Rock pools 52 53.8 26.9

> 10

0 0 0 0 0 0

3 2

2 9

17.3

> -

The proportion of samples containing coli-aerogenes bacteria was much the same in both off-shore water and in rock pools, while the incidence of B a t . coli type I waa markedly heavier in rock pools (26.9% of samples against 10.770), where the

36 Mabyn Hudleston

MF" were mostly between 25 and 140/100 ml of sample. There seemed little or no evidence of seasonal variation. In three cases, the counts of Bact. wli type I were higher than for other coli-aerogenes, an unusual feature; differences of this magnitude are not often found in untreated fresh water (Thomas & Jones, 1953).

Grateful acknowledgement is made to Mr. S. B. Thomas, N.A.A.S., Trawscoed, for reading and criticizing this paper and for various facilities afforded during the investigation.

REFERENCES

HUDLESTON, M. (1955). Marine bacteria in Cardigan Bay. I. Bacteria of an off-shore area. J . appl. Bact. 18, 22.

TAYLOR, C. B. (1942). Bacteriology of fresh water. 111. The types of bacteria present in lakes and streams and their relationship to the bacterial flora of soil. J. Hyg. , C m b . , 42, 284.

THOMAS, S. B. & JONES, G . E. (1953). Pers. c m . ZOBELL, C. E. & FELTHAM, C. E. (1934). Preliminary studies on the distribution and

characteristics of marine bacteria. Bull. Scripps. Inst. Oceamgr. Tech. Ser. no. 3, 279. ZOBELL, C. E. (1946). Marine Microbiology. Waltham, Maas: Chronica Botanica Co.

(Received 25 January, 1954)