MARINE BACTERIA OF CARDIGAN BAY. I. BACTERIA OF AN OFF-SHORE AREA

BY MABYN HUDLESTON

B o b r ~ y Department, U?Liversitp College of Wnlus, .-lberysttuyttt.

SU-Y: A study of marine bacteria from a fixed off-shore area in Cardigan Bay showed there was little significance in the variation between numbers from different lev& within 8 fathoms deep. There was a suggestion of seasonal variation in surface water in colony counts at 15-18'. In general, the bacterial content was highest over the summer months, but showed little direct correlation with temperature, pH or light.

Strains were mainly obligate halophytes and small Gram-negative rods predominated (96%). Nearly 50% of strains isolated liquefied gelatin; otherwise, biochemical reactions were weak. The proportion of pigmented strains was low.

Alien bacteria were present in irregularly varying numbers in all samples and showed no seasonal variation. Coli-aerogenes strains were recorded in small numbers, a t all depths and in all seasons, but Bmt. coli type I only rarely occurred.

M A I ~ ~ N E bacteriology is a highly specialized field and at present is most intensively studied in California, Massachusetts and New South Wales : the literature has been summarized adequately by ZoBell (1946). There appears to have been little work on the subject in this country, apart from that of Lloyd (1!)30, 1931) and Mare (1942), except in connexion with the fish industry (e.g. Shewan, 1944, 1945; Shewan h Hodgkiss, 1951).

This preliminary paper deals with some observations of marine (and other) bacteria in off-shore water in Cardigan Bay. The area from which samples were taken is 84 fth deep and situated 1& nautical miles off the rocky coast and 2 nautical milea south of Aberystwyth. (Normal national grid reference 2215579). This was chosen as being free from any direct influence of fresh water from rivers or streams and clear of pollution from direct sewage ontfall. The stretch of coast is free from industrial contamination, except for a few local trawlers and pleasure boats. The whole area of Cardigan Bay is shallow, the 25 fth contour lying outside the Bay. The latter is open to high winds and rough seas, which may cause vertical mixing in shallow areas; care was therefore taken to fix sampling times a few days after more or less calm weather. Any upwelling of water through local effects would tend to spread nutrients and also bacteria.

EXPERIMENTAL METHODS

Sampling. Samples were obtained by means of a deep water sampling apparatus, almost identical with that used by Wilson (1919). The 3 oz samples were conveyed to the laboratory and examined within 1 hr. Samples were taken at approximately monthly intervals during June 1949 to March 1950 inclusive; 7 series were examined, each consisting of 4 separate samples from different depths; surface, 2, 44 and 8 fth.

Bacteria in of-shore water 23

Water temperature. Experimental readings were made by means of a deep sea thermometer, but it proved satisfactory to draw up an extra bottle of water for each depth and take the temperature immediately with an ordinary laboratory thermometer.

Hydrogen ion concentration. An extra sample of water was obtained from each level for the purpose of ascertaining pH, using a colorimeter. According to Ibanez (1930), 4 drops of a saturated solution of mecuric chloride added to each 100 ml of the sample prevents any change due to bacterial action. However, there was no change within the hour before sample examination, even without a stabilizer.

Dissolved oxygen in sea water. MacArthur's method (1916) was adopted and a modified apparatus used, devised by Hudleston (1949). The method depends on treating the water with a mixture of alkaline potassium iodide and manganese chloride, while air is carefully excluded. The resulting precipitate of Mn(OH), rapidly reacts with the free 0, dissolved in the water, forming %(OH),. On acidification, the hydriodic acid reduces the manganic ion again, giving an equivalent amount of iodine which may be titrated with thiosulphate. The solubility of 0, is expressed in terms of the volume in ml at NTP.

Bacteriological analysis. The sample was thoroughly shaken by hand for 5 min before plating, in order to break up aggregates of bacteria, and inocula of 1 and 10-1 ml plated. In all cases, triplicate plates were made and the arithmetic mean recorded. Agar was poured at the lowest possible temperature to avoid killing heat sensitive bacteria. Given slow cooling and air temperature about ll", this was possible at 37"; 40" was more often used. Colony counts of course merely indicate the number of aerobic bacteria that will grow on the particular medium chosen. Such counts give values less than the total content and their main use is for comparative purposes under standardized conditions. Direct microscopical counts were consistently higher, but figures are not recorded here.

In an attempt to provide a reasonably complete picture of all aerobic and facultative anaerobic life, three media were used :-

Sea water agar. This was as used by ZoBell(l941) but with the amount of phosphate doubled. This appeared to induce stronger growth and intensity of pigmentation. This medium. which gave reproducible results, was as follows: 'Aged' (matured) sea water 1 1.; Bactopeptone 5-0 g; Ferric phosphate 0.2 g; Bactoagar 15.0 g; pH adjusted to 7%. ' Aged ' or ' rotted ' sea water was prepared from water left standing in glass Winchester bottles, in the dark, at room temperature, for 8 weeks. This reduced and stabilized the organic content and gave reproducible results.

Marine strains of bacteria were found to show tolerance over a wide temperature range, sometimes between 6 and 25" and the maximum rate of growth was often at about 20". However, 15-18' incubation for 14 days gave most consistent results and more marked pigment formation, and was employed throughout. Also, other plates were made and incubated for 21 days at 0-3".

Standard nutrient agar. The Ministry of Health (1939) medium was used, with a pH of 7.6, to give counts for probable soil and water forms. Incubation waa at 37" for 3 days. 22" for 5 days and 0-3" for 21 days.

24 Mabyn Hudleston

MacCmkey's broth. The Ministry of Health (1939) medium was used to indicate the presence or absence of coli-aerogenes bacteria. Quintuplicate amounts of 10, 1 and lo-' ml of sea water were inoculated directly into MacConkey's broth. One set of tubes was incubated at 37" for 2 days and another set at 44" for 2 days, the latter being maintained in a carefully regulated water bath.

Examination of individuul strains. A number of representative colonies from sea water agar, incubated 14 days at 15-18", were streaked on sea water agar slopes, standard nutrient agar slopes and potato wedges. These were incubated for 24 hr (or until growth was sufficient) and the morphology and Gram-reaction ascertained. The following biochemical tests were applied: formation of acid and gaa from glucose, lactose and sucrose in 5 days at 3U" and 14 days at 22"; litmus milk reaction in 5 days a t 30" and 14 days at 22"; liquefaction of gelatin (approx. 20"), the production of indole from tryptophane broth and reduction of nitrates by means of the Greiss test (Feigl, 1947).

RESULTS AND DISCUSSION

Marine bucteria ard some physiological factorb

The criterion by which marine bacteria were recognized for the purposes of this investigation, was that such organisms grew on sea water agar from initial isolation within 14 days at 15-18', but did not grow or showed very scanty growth on standard nutrient fresh water agar, within the same time at 22". The comparatively few organisms that gave good growth on the latter were probably soil or fresh water forms partly adapted to marine life or marine forms with euryhaline characteristics.

The data recorded in Table 1 show that the temperature of the water in the sampling area varied with the season a t the 4 depths, but showed very little variation in any one sample. The temperature at 8 fth was not always lower than that at the surface. Although water temperature showed a normal rhythmic seasonal cycle, there was little correspondence in bacterial content, except in surface water. There was usually no marked difference in sample counts taken from different depths, although there was possibly a tendency for greater numbers to be present at the surface in milder temperatures. November sampling showed a, marked reversal of this, although it was not continued in the two following series where much colder conditions prevailed.

There is some evidence of seasonal fluctuation in surface water; counts from 15-18" incubations showed highest totals over the 3 summer months and fell over September, November, January and March. Counts from 0-3" incubation followed much the same trend. However, with so few samples and in one year only, it is perhaps premature to form any conclusions.

Fred, Wilson & Davenport (1924) failed tb find any definite seasonal changes in Lake Mendota over three years, while Henrici (1938) showed that certain increases followed those of plankton increase during certain months. Lloyd (1930) found the number of bacteria in the Clyde sea area was constant throughout the year. except for the surface layer where the bacterial content fluctuated widely; but there was no evidence of seasonal variation.

Table 1

Depth Date pH (fW

June 8.3 July 8.6

8.0 Mar. 8.1 June 8.3 July 8.4 Aug. 8-4

NOV. 8.5 Jan. 8.0 Mar. 8.1 June 8-3 July 8.4 Aug. 8.4

NOV. 9.0 Jan. 8.1 Mar. 8-1 June 8.4 July 8.4 Aug. 8.5

NOV. 9.0 Jan. 8-1 MEW. 8.1

Bacteria in o$-shore water

Cornprison of colony counts from sea water with depth, ternpera.ture and season

2 5

Water temp. ("C)

16.0 17.0 21.0 16.7 10.7 4.0 9.6

16.2 16.1 20.3 15.6 11.0 4.0 9.2

16.0 16.0 20.0 15.6 11.0 4-0 9.1

16.0 16.2 18-7 15.5 11-7 4.9 9.0

Plate count/ml MacConkey's broth A

I , /1OOmlSsmple Sea water agar Standard agar & ,-~->-iGTiZZ 14 davs 21 davs 5 davs 3 davs 21 davs asroeenes twe I 15-liO

463 530 627 77 25 23 56

408 300

18 105 190 33 84

37.5 650 408

71 365 54 62

363 210 117 94

1,240 85 79

0-3; 715 804 820 99

111 203 279 316 384

10 13 12 38 58

27 1 276 381

9 3

1 1 54

204 120 30 17

2 50 45

225 195 200 219 28

320 107 123 86

200 30

100 255 101 150 41

700 371 306 560 220 163 52

670 4

305 2,700

312 179

37; 25 40 64 1

820 20 31 12 0 0 0

850 13 26

7 100 30 0

2.50 7

17 1

272 0

240 1,700

10 16

0-3; 2 d a G 37" 2 & y ~ 4 4 ~ 40 4 41 2 46 4 0 2

52 50 34 2 45 1 62 50 11 0 9 0 0 2 2 14

123 0 116 0 52 0 2 0 0 10

243 6 72 4 5 0

25 0 38 0

2 4 0 0 0 6 1 17

17 0 12 0

0 0 0 0 5 0 0 0 0 0 0 4 0 0

0 0 0 0 0 0 0 0 0 0 0 8 0 0

The temperatures and bacterial counts indicate that the water is probably not stratified within the depth of 8 fth; whether this is due to upwelling or tidal streams is uncertain. Rough seas are frequent, which may keep the water in circulation within the fixed distance of 13 nautical miles off-shore. Samples from greater depths would probably have given more interesting results.

In future work on marine bacteria, i t would be advisable to take weekly samples (admittedly not possible in a stormy region such as Cardigan Bay) and at the same periods, consider all factors which may bear on fluctuation of numbers, in particular the question of predatory protozoa (Waksman & Hotchkiss, 1937; Mare, 1942). There is evidence, a t least for polluted water, that protozoa and microcrustaceans live on and markedly reduce the numbers of bacteria (Purdy & Butterfield, 1918). Again, microbial antagonism may limit numbers ; also the fact that dissolved organic matter, available as food for bacteria, is in great dilution. no more than 2-5 mg/litre (Harvey, 1945).

Pigment formation by marine bacteria (and also by alien forms) was much less evident than other workers have recorded. Only 3% of the colonies at 15-18" exhibited definite pigmentation that was reproducible on subculture. This is far below the

26 Mabyn Hudleston

69.4y0 from sea water sources quoted by ZoBell (1946). In all cases, yellow and orange pigments predominated, with a few fluorescent. brown or black, pink or red, in that order of frequency.

Agar digesters were met with infrequently. Hydrogen ion concentration. The samples showed only slight variation in pH

value, the averages being: surface, 8.3: 2 fth, 8.2; 4 fth, 8.3; 8 fth. 8.5. In 3 cases only did the pH rise to 9 or over and i t never fell below 8.0. For ocean water, Harvey (1945) stated that the values were rarely as low as 8.0 and. in the upper limits, rarely exceeded 8.3.

Waksman (1934) stated that the concentration of 0, hrts an important effect on bacterial development and activities and that consumption of 0, takes place a t the expense of carbohydrates in the water and in the decomposition and oxidation of the organic nitrogenous constituents.

Estimations of free 0, were not undertaken at the same time as sampling, but a trial was carried out during September from 5 sets of 5 samples taken (a) near sunset, when the average amount of free 0, in the water was 5-4 ml/litre at 15.5" and (b) at dawn, when the amount of free 0, was 5.3 ml/litre a t 14.5". There waa thus a loss of 0.1 ml/litre during the hours of darkness (salinity 33.2"/,).

Harvey (1945) stated that ocean water with a salinity of 34-32"/,, contained 5.86 ml O,/litre a t 15". the solubility increasing with decrease in temperature and salinity.

Some characteristics of marine bacteria. All the colonies (98) from a sea water agar plate incubated for 14 days a t 15-18" were subcultured and the 93 strains which grew on subculture were subjected to more detailed examination. The 5 strains which died out were deep colonies and probably anaerobes. A small number of these anaerobic organisms developed on nearly every occmion after incubation of sea water inoculations into sea water agar enriched with glucose and sodium thio- glycollate, with methylene blue as an indicator (ZoBell, 1946). A glass disc over the medium maintained anaerobic conditions.

Among the 93 aerobic strains Gram-negative rods were dominant and comprised 967,,. Short rods (64%) were twice as common as long rods (32%). Cocci were few (4%). Pigmented colonies formed 23%. Proteolytic bacteria amounted to 43% of the total strains. Just over half of the proteolytic cultures completed liquefaction of gelatin in 5-10 days, 19% took 11 d a y s 4 weeks and 30% took 6 weeks or longer. ZoBell (1946) stated that '75% of marine bacteria are proteolytic. The aerobic strains were weakly saccharolytic. a few formed acid, 6% from glucose and sucrose, none from lactose, but there was never gas; occasionally there was fair to good growth in sugar media, without visible reaction beyond turbidity. Litmus milk gave varied results. Only 18% showed any reaction at all: 9% gave acid, 3% acid with clot, 6% formed alkali, 4% reduced the indicator and 1% caused peptoni- zation. There was no production of indole in tryptophane broth and only 6% of the strains reduced nitrate; another 8% gave indecisive results. Only 2 organisms showed slight growth in peptone broth and on nutrient agar. None of the strains produced any reaction in MacConkey's broth. Ropy growth was exhibited by 10 strains and was marked in 4.

Free oxygen.

27 Bacteria in of-shore water

The majority of the 93 cultures stored at 0-3" on sea water agar survived for 4 yeam and growth was resumed in favourable temperatures (15-18').

There is some interest in comparing the findings of Taylor (1042) for fresh water bacteria in lakes, with those of the author for sea water. Both agree that the majority are Gram-negative rods, 05% in fresh water, 96% in sea water; also spore formers are few and, in general, biochemical reactions weak. On the other hand, gelatin liquefiers form a much higher proportion of the marine organisms (43%) than those in lakes ( 2 4 O 4 , ) . Marine pigment producers were fewer than in fresh water strains.

Alien bacteria

For the purpose of this paper, alien bacteria are defined aa those which grew vigorously on nutrient agar at 22" or 37"; the majority failed to grow on sea water agar. These were probably derived from soil and fresh water : they were not studied in detail. There was a tendency for counts of these organisms to be higher a t greater depths. Counts from 37" incubations were less than half those a t 22".

Coli-aerogenes bacteria were detected in 100 ml amounts of all series of samples, the most probable number ranging from 1-501100 ml. It is significant that Ruct. coli type I was found in 3 samples only.

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

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28 Mabyn Hudleston

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(Received 25 Ja.nuary, 1954)