73-5276

SECHLER, Gary Evans, 1939-ON TIIE MICROBIOLOGY OF SLIME LAYERS FORMED ONlMMERSED MATERIALS IN A MARINE ENVIRONMENT.

University of Hawaii, Ph.D., 1972Microbiology

University Microfilms, A YJ:ROX Company, Ann Arbor, Michigan

THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED.

ON THE MICROBIOLOGY OF SLIME LAYERS

FORMED ON IMMERSED MATERIALS

IN A MARINE ENVIRONMENT

A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE

UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

IN MICROBIOLOGY

SEPTEMBER, 1972

By:

Gary Evans Sechler

Dissertation Committee:

Kaare R. Gundersen, Chairman

Leslie R. Berger

Barbara Z. Siegel

Sidney J. Townsley

Sanford M. Siegel

PLEASE NOTE:

Some pages may have

indistinct print.

Filmed as received.

University Microfilms, A Xerox Education Company

iiiABSTRACT

A primary film or slime layer forms on immersed surfaces in

marine waters; these primary films were assayed for the presence of

various microorganisms.

In a preliminary study, surface swabbing and filtration techniques

were used to identify and quantitate heterotrophic bacterial densities.

From these data the succession patterns of representative isolates

from seven (tentatively identified) genera common to marine coastal

waters were followed. No individual isolate or group dependence upon

the chemical composition of the test panel surfaces (which included

steel, aluminum, zinc, plexiglass and wood) was found. However, the

appearance of various isolates and their relative density were found to

vary with the composition of the test material luring the first few

days following immersion.

Two methods were developed to assay the primary film layers

formed on opaque materials immersed in the sea. Each method was

designed for expedient field testing and utilized light microscopy of

intact, removed, slime layers.

In the first method, ultrathin Teflon membranes with micropores

were fitted over surfaces of glass, aluminum, phosphor-bronze, zinc,

wood and steel. Corrosion products from the metals were shown to

diffuse through the membrane pores. Bacteria adsorbed to excised

membranes on all surfaces except phosphor-bronze by one day; active

proliferation occurred by four days following immersion. Diatoms

attached sparingly during the first day but appeared on most all

surfaces by four days. Qualitative differences in the bacterial and

iv

diatom communities suggested that the surface chemistry may influence

microbial attachment.

The Parlodion filming technique is the major analytic method

used in these studies. It provides a view of the microorganisms

exactly as they appear on the test surface. Using this method,

quantitative comparisons of bacteria, diatoms and extraneous particulate

matter were made for extended periods on aluminum, stainless steel,

Monel, glass, plexiglass and phosphor-bronze. Bacteria and diatoms

were nonrandomly distributed while extraneous particles showed a

(generally) random distribution.

Adsorption and attachment sequences consistently began with

bacteria, followed by diatoms on all test panel surfaces.

Adsorption of bacteria appeared to be dependent upon several

factors: the relative polarity and electronegativity of the test

material, the ability to synthesize and excrete slimy materials, and

the motility or chemotactic response of the attaching bacteria. The

evolution of increasingly complex surface ecosystems were compared for

all test materials.

vTABLE OF CONTENTS

ABSTRACT iii

LIST OF TABLES vii

LIST OF FIGURES viii

I INTRODUCTION 1

(i) In Situ Formation of Primary Films (Slime Layers) 2

on Immersed Glass Slides

(ii) In Situ Biotic Succession following the Formation 2

of Primary Films on Immersed Glass Slides

(iii) In Situ Attachment of Macroorganisms to Immersed 4

Test Materials

(iv) The Development of Primary Films on Immersed Glass 4

Slides: Laboratory Studies

(v) The Importance of Primary Films in the Marine 5

Ecosystem

6

7

7

Role of the Material

Attraction of

Attachment of

Formation of a Primary Film:

Formation of a Primary Film:

Microorganisms

Formation of a Primary Film:

Microorganisms

(ix) Interactions of the Primary Film with the Immersed 9

Material

(vi)

(vii)

(viii)

II METHODS AND MATERIALS

(i) Field Test Site

(ii) Test Panels

(iii) Test Panel Preparation

12

12

14

19

vi(iv) Post Immersion Processing of Test Panels 20

(v) Rationale 21

(vi) Surface Swabbing Technique: Comparative Bacterial 21

Numbers from Chemically Diverse Surfaces

(vii) Surface Swabbing Technique: Bacterial Succession 23

Patterns on Chemically Diverse Surfaces

(viii) The Teflon Membrane Technique: Preliminary

Laboratory Tests

(ix) The Teflon Membrane Technique: Field Tests

(x) The Parlodion Filming Technique

(xi) The Parlodion Filming Technique: Field Tests

III RESULTS

(i) The Interaction of Primary Films with Test

Panels

(ii) Surface Swabbing Technique

(a) Determination of the Average Bacterial

Density on Immersed Test Panels Following

Colony Formation on Modified Growth Media

(b) Bacterial Succession Patterns on Test

Panels During a 40-Day Immersion Period

(iii) Teflon Membrane Technique Field Study

(iv) Parlodion Filming Technique

IV DISCUSSION

APPENDIX I

LITERATURE CITED

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34

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63

92

98

100

viiLIST OF TABLES

TABLE NO. PAGE

I GALVANIC SERIES OF METALS USED IN FIELD EXPERIMENTS 18

II GROWTH OF BACTERIA ON TEST PANELS AFTER PLATING ON 46VARIOUS MEDIA

III BACTERIAL GENERA ISOLATED BY SWABBING METHODS 48

IV DIVERSITY RATIOS CALCULATED FROM SURFACE BACTERIAL 55SUCCESSION PATTERNS

V BACTERIAL AND DIATOM DENSITIES OF TWO SAMPLING 62INTERVALS ON TEFLON MEMBRANES OVERLYING VARIOUS TESTSURFACES

VI DETERMINATION OF THE RANDOMNESS OF MICROSCOPIC 73BACTERIAL COUNTS (CHI-SQUARE TESTS)

VII VARIATION IN BACTERIAL DENSITY ON IMMERSED TEST 75PANELS ASSAYED BY THE PARLODION FILMING TECHNIQUE

VIII VARIATION IN DIATOM DENSITY ON IMMERSED TEST PANELS 78ASSAYED BY THE PARLODION FILMING TECHNIQUE

IX VARIATION IN EXTRANEOUS PARTICLE DENSITY ON IMMERSED 81TEST PANELS ASSAYED BY THE PARLODION FILMINGTECHNIQUE

viiiLIST OF FIGURES

FIGURE PAGE

1 GEOGRAPHIC LOCATION OF THE FIELD SAMPLING SITE 13

2 APPEARANCE OF THE PLEXIGLASS TEST PANEL AFTER VARIOUS 41PERIODS OF IMMERSION

3 APPEARANCE OF THE ALUMINUM TEST PANEL AFTER VARIOUS 42PERIODS OF IMMERSION AT THE TEST DEPTH

4 APPEARANCE OF THE STEEL TEST PANEL AFTER VARIOUS PERIODS 43OF IMMERSION AT THE TEST DEPTH

5 APPEARANCE OF THE ZINC TEST PANEL AFTER VARIOUS PERIODS 44OF IMMERSION AT THE TEST DEPTH

6 APPEARANCE OF THE WOOD TEST PANEL AFTER VARIOUS PERIODS 45OF IMMERSION AT THE TEST DEPTH

7 DEVELOPMENTAL PATTERN OF HETEROTROPHIC SURFACE BACTERIAL 47POPULATIONS ON A NUMBER OF CHEMICALLY-DIVERSE MATERIALSDURING A 40-DAY IMMERSION PERIOD

8

9

10

11

12

13

OCCURRENCE OF VARIOUS BACTERIAL ISOLATES AT EACHSAMPLING INTERVAL ON PLEXIGLASS TEST PANELS

OCCURRENCE OF VARIOUS BACTERIAL ISOLATES AT EACHSAMPLING INTERVAL ON ALUMINUM TEST PANELS

OCCURRENCE OF VARIOUS BACTERIAL ISOLATES AT EACHSAMPLING INTERVAL ON STEEL TEST PANELS

OCCURRENCE OF VARIOUS BACTERIAL ISOLATES AT EACHSAMPLING INTERVAL ON ZINC TEST PANELS

OCCURRENCE OF VARIOUS BACTERIAL ISOLATES AT EACHSAMPLING INTERVAL ON WOOD TEST PANELS

NUMBER OF BACTERIAL ISOLATES PRESENT AT EACH TESTINTERVAL ON THE SURFACE OF EACH TEST MATERIAL DURINGA 40-DAY IMMERSION PERIOD

49

50

51

52

53

54

14 NUMBER OF DAYS PRESENT BY THE VARIOUS ISOLATES RECOVERED 56BY SWABBING METHODS ON 1-5 TEST SURFACES

15 TYPICAL MICROSCOPIC FIELDS OF MICROORGANISMS REMOVEDFROM VARIOUS TEST SURFACES BY SWABBING AND PREPAREDBY THE MILLIPORE FILTRATION-STAINING TECHNIQUE

57

FIGURES (CONTINUED)ix

PAGE

16

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27

28

29

STAINED TEFLON MEMBRANE PREPARATIONS FROM CERTAINMATERIALS AFTER 1 AND 4 DAYS OF IMMERSION

STAINED TEFLON MEMBRANE PREPARATIONS FROM CERTAINMATERIALS AFTER 1 AND 4 DAYS OF IMMERSION

MICROGRAPHS OF THE PRIMARY FILM OR SLIME LAYER FORMEDON IMMERSED GLASS SLIDES EXAMINED BY THE PARLODIONFILMING TECHNIQUE

PLEXIGLASS TEST PANEL SURFACE MICROBIOTIC CHANGESOCCURRING AS A FUNCTION OF TIME OF IMMERSION FROMPARLODION MOUNTS

STAINLESS STEEL 304 TEST PANEL SURFACE MICROBIOTICCHANGES OCCURRING AS A FUNCTION OF TIME OF IMMERSIONFROM PARLODION MOUNTS

ALUMINUM 5052 TEST PANEL SURFACE MICROBIOTIC CHANGESOCCURRING AS A FUNCTION OF TIME OF IMMERSION FROMPARLODION MOUNTS

MONEL TEST PANEL SURFACE MICROBIOTIC CHANGES OCCURRINGAS A FUNCTION OF TIME OF IMMERSION FROM PARLODIONMOUNTS

PHOSPHOR-BRONZE TEST PANEL SURFACE MICROBIOTIC CHANGESOCCURRING AS A FUNCTION OF TIME OF IMMERSION FROMPARLODION MOUNTS

SOME ATYPICAL OR UNUSUAL OBSERVATIONS ON PARLODIONMICROSCOPIC MOUNTS t INCLUDING MEMBERS OF MANY PHYLANOT QUANTITATED IN THESE STUDIES

TOTAL SURFACE BACTERIAL POPULATIONS ON CHEMICALLY­DIVERSE MATERIALS DURING 120 DAYS OF IMMERSION

BACTERIAL DEVELOPMENTAL PATTERN ON VARIOUS MATERIALS

TOTAL SURFACE DIATOM POPULATIONS ON TEST PANELS DURING120 DAYS OF IMMERSION

DIATOM DEVELOPMENT ON IMMERSED TEST PANELS

EXTRANEOUS PARTICLES DEPOSITED ON TEST PANELS DURING120 DAYS OF IMMERSION

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1I

INTRODUCTION

A primary film (often called a slime layer) rapidly develops on

any common material immersed in a marine environment. This slime layer

is potentially important for the subsequent development of a marine

zoological community on the immersed material [ZoBell (1937; 1938;

1939); Sheer (1945); Aleem (1957); and Edmondson (1944)]. Several

idealized biotic succession patterns have been proposed to describe the

attachment of macroorganisms to the immersed material: in most of these

studies colonization by microorganisms and formation of a primary film

were postulated to be essential for the subsequent development of the

complete marine ecosystem.

The general purpose of this thesis is to characterize the

formation of primary films on a variety of materials immersed in a

marine environment. A primary film is a surface microcosm composed of

microorganisms (bacteria. diatoms. etc.). extracellular slime. and

inorganic as well as organic particulate matter (Christie and Floodgate.

1966). Underlying this research is the assumption that the

colonization of immersed materials by microorganisms is crucially

important to the entire marine ecosystem.

Previous field studies examining the microbiology of primary

films utilized transparent materials (primarily glass slides) immersed

in marine environments. In this thesis. two new techniques are

introduced that permit characterization of the primary films formed on

immersed. chemically diverse opaque materials (metals. wood. etc).

Using these techniques. I am able to describe the formation of primary

2

films on immersed samples of aluminum, zinc, phosphor-bronze, steel,

wood and glass (for comparison with earlier studies).

In order to have a framework in which to discuss my results, I

shall examine the relevant concepts that have emerged from the earlier

studies on transparent materials. In addition, some earlier studies on

the biological and chemical events that precede or accompany primary

film formation are reviewed. I shall minimize the controversy about

whether these primary films are important for the attachment of

macroorganisms. Instead I shall concentrate on a description of these

slime layers, emphasizing their possible (though not proven) functions.

(i) In Situ Formation of Primary Films (Slime Layers) on Immersed

Glass Slides

The development of microorganisms on glass slides immersed in

natural aquatic environments was first reported by Naumann (1925).

However, this method was not used extensively until the studies by

Henrici (C. Henrici, 1933; 1936; Henrici and Johnson, 1935) on fresh

water microorganisms and by ZoBell (ZoBell and Allen, 1933; 1935) on

marine microorganisms. These workers demonstrated the proliferation on

immersed glass slides of numerous bacteria and other morphologically

distinct microorganisms which were not common in enriched fresh or sea

water.

(ii) In Situ Succession Following the Formation of Primary Films on

Immersed Glass Slides

ZoBell (1937; 1938; 1939) proposed a unique biotic sequence

during the formation of a primary film on an immersed glass surface.

3Initially the glass surface adsorbs and concentrates nutrients from

sea water [this was confirmed by subsequent experiments (ZoBe11, 1943);

see section (vi) for further discussion]. These adsorbed nutrients in

turn attracted bacteria from the surrounding water [see section (v)].

Numerous successful colonies then developed on the immersed glass

surface. In this context it is of interest that ZoBe11 (1943) found

that 47 marine bacterial species (out of 96 surveyed) showed a tendency

to attach to glass surfaces; only 29 species were strictly sessile.

The next event in the biotic succession observed by ZoBe11 was

the attachment and proliferation of diatoms. This resulted in a large

increase in the biomass. At this point the primary film (surface

microcosm) began to offer increasing amounts of food, protection, and

assistance in physical attachment---resu1ting in a complex biotic

succession pattern involving larger, more complex organisms.

Sheer (1945) and A1eem (1957) elaborated upon these events,

proposing idealized schematic biotic succession stages. Invariably

bacterial colonization preceded the development of diatom communities.

Although differing in minor details Tegarding the order of attachment,

Sheer and A1eem indicated that sessile algae and protozoans were

followed by hydroids, bryozoans, and other higher invertebrates

(including barnacles). It is important to note that representatives

from each group never totally disappeared after the initial appearance

of the group.

In apparent contrast to ZoBe11's results, Skerman (1956)

concluded that dense settlements of bacteria observed on glass slides

immersed in New Zealand marine waters were fortuitous, adventitious

4

forms from contaminating sewage. No active growth was observed on the

immersed glass slides. The bacterial populations declined rapidly

within a few days following immersion, although colonization by

bactivorous sessile protozoans was observed.

(iii) In Situ Attachment of Macroorganisms to Immersed Test Materials

Wood (1950) examined the attachment of animal larvae (fouling

organisms) to ship bottoms and other non-silica based materials.

Although bacteria per se were not essential for the attachment of the

larvae, other microorganisms were found to stimulate colonization. In

particular, Wood and Allen (1958) showed that diatom communities

constituted a physical and chemical stimulus for the rapid growth of

the fouling organisms.

Similarly, Edmondson and Ingram (1939) comprehensively studied

the attachment of invertebrates to a wide variety of test materials

immersed in Hawaiian marine waters. Initially (Edmondson and Ingram,

1939) no importance was attributed to the primary film. Subsequently,

however, Edmondson (1944) proposed that slime layers we~e of great

(potential) importance for the attraction and initial colonization of

the immersed material by sessile macroorganisms.

(iv) The Development of Primary Films on Immersed Glass Slides:

Laboratory Studies

From the in situ studies cited above it is certain that a primary

film or slime layer serves as a stimulus to (and possibly an actual

precursor for) the attraction and attachment of macroorganisms to

immersed materials.

5

A laboratory investigation by Miller, Rapean, and Whedon (1948)

demonstrated that slime layers generated by microorganisms facilitated

the attachment of bryozooan test larvae to immersed glass slides. These

slime layers were not absolutely essential for attachment, however, and

formation of a slime layer did not insure colonization. In a similar

study, Meadows and Williams (1963) observed that larvae of Spirorbis

borealis attach with a greater-frequency to immersed slides covered

with bacteria-diatom containing primary films.

In an analogous laboratory study (though not using glass slides)

Meadows (1964) found that Corophium preferentially colonized natural

marine sand rather than sand treated to remove indigenous bacterial and

other organic components of the natural slime layer. Furthermore,

Meadows (1965) established that not only the number but also the

variety of bacteria in a slime layer influenced the composition of the

population of the colonizing macroorganisms.

(v) The Importance of Primary Films in the Marine Ecosystem

The investigations cited above establish that the formation of

a primary film or slime layer is important, though perhaps not essential,

to the development of the zoological community on immersed glass slides.

I shall assume, therefore, that the composition of the primary film that

forms on any immersed material is equally important not only for the

immediate biotic succession which follows, but ultimately for the total

marine ecosystem.

In this context it is significant that the only major studies to

date have investigated the microorganisms found in the primary films

formed on immersed glass slides. In this thesis I shall examine the

6

primary films generated in situ on various chemically distinct materials

immersed in a Hawaiian marine environment. In order to carry out these

studies, new sampling techniques had to be devised to circumvent the

problem inherent in any attempt to examine any opaque material with a

light microscope.

Assuming that the primary film is important for the subsequent

development of a zoological community on the immersed material, a

description of the communities of microorganisms in the primary film on

each material is an essential starting point for any comparison between

the resulting biotic successions on different materials. In particular,

characterization of the rather simple primary films on various materials

could then be correlated with the rates of formation and composition of

the eventually resulting complex zoological communities.

(vi) Formation of a Primary Film: the Role of the Material

The immersed material upon which the slime layer forms is

potentially of great importance. Negatively charged surfaces (for

example, glass and galvanically active metals in aqueous media) adsorb

charged organic and inorganic substances (Tyler and Marshall, 1967).

Presumably these adsorbed substances can initiate chemotactic responses

[Adler, 1969; see section (vii)] in bacteria in the immediate region of

the immersed material. In addition, bacteria characteristically behave

as negatively charged colloids in an aqueous medium (Reddick, 1961;

Daniels and Kempe, 1966). Unless the negatively charged sites on the

immersed materials are neutralized or masked, bacteria (or any other

negatively charged microorganisms) are electrostatically repelled.

7

(vii) Formation of a Primary Film: Attraction of Microorganisms

Bacteria (and presumably other microorganisms) contact an

adsorbing surface not only by random collisions (Brownian motion, etc.)

but also by specific attraction mechanisms. The chemotactic response

in bacteria [recently the subject of an extensive review by Adler

(1969)] has been used to establish that bacteria possess chemoreceptors

for specific chemical substances. The chemotactic mechanism directs

the bacterium (propelled by flagellar action) toward the source of the

chemical stimulus. It is of interest that this chemical stimulus need

not be of direct nutritional value.

(viii) Formation of a Primary Film: Attachment of Microorganisms

Following attraction, bacteria may attach to the immersed

material at specific points on the bacterial cell surface. Meadows

(1968), Jones, Roth and Sanders (1969), and Marshall, Stout and

Mitchell (197la) have observed the attachment of bacteria to glass

surfaces in both a polar and a longitudinal orientation. In addition,

Meadows (1968) and Floodgate (1965) report that bacteria attach,

detach, and reattach to glass surfaces numerous times before firm

attachment occurs. ZoBell (1943) and Marshall, Stout and Mitchell

(197la) have studied the attachment of bacteria to an immersed surface

following attraction. Firm attachment of the bacteria requires a

period of several hours; this suggests that it is necessary for the

bacteria to synthesize sufficient amounts of extracellular adhesive

material prior to firm attachment.

Marshall, Stout and Mitchell (197la) have recently emphasized

the concepts of "reversible" and "irreversible" adsorption of bacteria

8

to immersed glass slides. "Reversibly" adsorbed bacteria are held

weakly near the surface of the glass slide by electrostatic forces,

and are easily eluted with 2.5% sodium chloride solution. In addition,

"reversible" adsorption occurred with the bacterium in either a polar

or a longitudinal orientation; as a consequence flagellar activity

easily displaced the adsorbed organisms. In contrast, "irreversible"

adsorption depended on the release of extracellular polysaccharide

material. This secretion occurred only in media low in assimilable

carbon (7 mg glucose/ml). Following "irreversible" adsorption,

bacteria could not be washed off with 2.5% sodium chloride solution and

no bacterial motion or displacement was observed.

One interpretation of these results is that nutritionally

deficient bacteria are induced to synthesize polysaccharide attachment

material (Marshall, Stout and Mitchell, 1971a). The survival value

(and ecological significance) of such a mechanism to attach

physiologically defective cells to potentially nutrient rich surfaces

is obvious. In a similar study, Seki (1964) found that extracellular

slime layer production in marine bacteria increased with a decrease in

the concentration of assimilable nitrogen in the medium.

The production of an extracellular polysaccharide adhesive

apparently is a necessary accompaniment to attachment. Thus, a common

primary film forming pseudomonad produces large quantities of

extracellular acid polysaccharide (Corpe, 1970a; 1970b). Electron

microscopic studies of a slime layer (Jones, Roth and Sanders, 1969)

showed that actively growing sessile organisms were enmeshed in, and

attached to, the glass surface by a common, weblike network of

9

polysaccharide strands. A similar study of bacterial aggregation

(Tenney and Stumm, 1965; Busch and Stumm, 1968) demonstrated the

involvement and interaction of polysaccharide material exposed on the

bacterial surfaces.

Direct confirmation of a fibrillar polysaccharide attachment

mechanism for a marine pseudomonad was obtained by Marshal, Stout and

Mitchell (197la). Using a combination of chemical and electron

microscope techniques, they proved that direct attachment existed. To

emphasize their point: the attachment was so tenacious that surfaces

from which bacteria were mechanically sheared retained observable

polysaccharide fibrils directly attached to the surface.

In sum, these studies suggest that primary films (slime layers)

are formed as a result of the actions of microorganisms. Following

attraction, successful bacterial attachment depends on the concomitant

synthesis of extracellular slime material. Bacteria (and other

microorganisms) that do not either synthesize extracellular adhesive

material or do not become enmeshed in the slime layer are probably only

transient members of the surface microcosm. As a consequence, this

thesis concentrates on a description of the microbiology of primary

films.

(ix) Interaction of Primary Films with Test Panels

The biological i~teractions are complex, and act at the

immediate surface level to modify the chemical properties determining

the rate of corrosion of the test panels (Appendix 1). The significance

of adsorbed organic matter and the resulting microbial influence for

the formation of the primary film were discussed above.

10

Of extreme importance are the local chemical changes occurring

in the slime layer. They may alter not only the corrosion rate of the

metal, but the proliferation of other microorganisms. As a result of

either bacterial secretion or lysis, amino acids, carbohydrates and

vitamins could induce high diatom populations. According to Droop and

Elston (1966) and Sieburth (1968), many diatoms exhibit antibacterial

activity proximally to the diatom cell wall.

In daylight, photosynthesis by the attached diatoms, green, and

blue-green algae saturates the film with oxygen and enhances the

formation of metal oxides. Heterotrophic activity (both in dark and in

light) rapidly utilizes the oxygen and also produces local changes in

pH due to the accumulation of catabolic products, the formation of CO 2

and a variety of intermediate metabolites.

Development of sessile invertebrates with calcareous bases

typically depletes the oxygen level beneath the points of attachment

CLaQue, 1969). This causes a change in electrochemical potential

relative to the surface, generating a battery effect. An increased

emigration of metal ions from the "anodic" point beneath the organism

is the net result.

Entrapment and subsequent multiplication of sulfate-reducing

bacteria in this reduced-oxygen environment would increase the dissolu­

tion of metal ions further due to the production and concentration of

acidic metabolites. Thus LaQue (1969) has reported pH levels as low as

3, with potential differences from the surface proper exceeding 500 mV

in corrosion "pits" on iron test panels. The metabolite hydrogen

sulfide would subsequently precipitate the solubilized iron ions as

11

iron sulfide in these pits. The iron might also serve directly as a

substrate for sessile iron bacteria, which oxidizes ferrous ions to

ferric ions as a means of obtaining energy.

Nonmetals

Nonmetals do not "corrode" per ~, but deteriorate due to

attached organisms which produce specific enzymes (Turner, 1967). Wood

is attacked by numerous cellulolytic bacteria and fungi, and by certain

invertebrates. Investigations by Coscarelli (1964) have shown that

many plastics, rubber products and natural fibers are similarly

susceptible to microbial attack in marine waters. Deterioration may

also occur (1) by the concentration of acidic and other metabolites in

the primary film, (2) by dissolving in the water itself (for example,

the aliphatic alcohols of wood), or (3) by chemical reactions (for

example, oxidations catalyzed by ultraviolet light penetrating the

water). Perry (1969) reports that glass is not detectably subject to

either biological or chemical deterioration in natural marine environ­

ments.

12II

METHODS AND MATERIALS

(i) Field Test Site

The test area was located on the west side of Coconut Island

(Mokuoloe), in the southwest portion of Kaneohe Bay, Oahu, Hawaii

(Figure 1). The site itself was a narrow channel 7 m wide and 4-4.5 m

deep, bounded by non-living coral and spanned by a narrow concrete

bridge. Suspension lines were anchored to the bridge during test

periods.

Although the water of Kaneohe Bay is substantially affected by

the urbanization of adjacent land areas (from water runoff, sewage

disposal. etc.) the water of the test site is generally clear and con­

tains ample fauna. The current through the narrow channel is negligible.

since only eddy action occurred during the semi-diurnal tidal exchange;

occasionally, a slight northward drift was noted.

Bathen (1968) gives the following physical and chemical

characteristics of waters near the test site. based on yearly averages:

Mean Tidal Amplitude (for 411 consecutive days): plus 29.8 cm (high)

to minus 32 cm (low), or a mean average tidal exchange of 62.6 em per

day.

Temperature (sea surface): 22.2 C (midwinter) to 27.4 C (late summer).

Salinity (sea surface): 32.30 0/00 (fall-winter rainy season) to

35.60 0/00 (late summer dry season).

Dissolved Oxygen: 3.5 - 5.6 ml/liter.

Reactive phosphate: 0.4 - 0.6 ug-at per liter.

MILES? 0.,5 1,

OAHU

1sofso'w

(lQ ~ HAWAII

Oahu ~

C>KANEOHE

13

Figure 1. Geographic location of the field sampling site. Testmaterials were immersed at the appropriate test depth in a narrowchannel off Mokuoloe Island in Kaneohe Bay, Oahu Island, Hawaii(indicated by circled dot). The inset shows the location of KaneoheBay relative to the major Hawaiian Islands.

14

Nitrite-nitrogen and nitrate-nitrogen at the test depth were

found to be 0.60 ug-at per liter and 1.44 ug-at per liter, respectively,

on 11/26/69 (test performed by c. W. MOuntain). Total organic carbon

in the sea water on the same date was 1.61 mg per liter (test performed

by Donald Gordon).

pH values determined during the present study ranged from 8.1

(February) to 8.3 (August).

Viable heterotrophic bacteria were assayed at a 3 m depth on

two separate occasions. The bacteria were collected by Mi11ipore

filtration and cultured by resuspending and plating the cells suspended

on the membrane. These assays showed 1.35x103 bacteria/m1 on 8/26/68,

and 3.40 x 102 bacteria1/m1 on 11/26/69.

No significant local precipitation or major storms occurred in

the Kaneohe Bay area during any of the three test periods between

March and August, 1968 and 1970, that would significantly alter the

properties of the test site as described.

(ii) Test Panels

Test panels of various materials (generally about 75 mm x 25 mm

x 1 mm) were immersed at the test depth (generally 3 meters) for

periods of time ranging up to 120 days. The materials used in the

test panels and their characteristics in a marine environment are

given below. A brief review of corrosion theory of the materials

invo1yed in this study is given in Appendix 1. The materials from

which test panels were cut are:

151Steel

Steel is heat-treated iron, containing variable amounts of

carbon and metallic trace impurities. It is highly susceptible to

corrosion in oxygenated water, characteristically forming a surface

scale (Le., "rust" layer) of hydrated ferrous oxide.

Stainless Steel 304

Stainless steel 304 is a steel alloy containing 18-20% nickel.

Because of the electrical passivity afforded by the oxide layers formed

at the surface, it is highly susceptible to attachment by marine

organisms. Since the attaching organisms often prevent maintenance of

the oxide coat, considerable electrochemical differences arise locally.

This results in unevenly distributed "pitting", a condition that often

persists until the panel is perforated.

Aluminum 5052

Aluminum 5052 is aluminum containing 2.5% magnesium and 0.45%

chromium. It is noted for its excellent corrosion resistance in sea

water and has found extensive marine applications. Although high on

the galvanic activity scale, aluminum alloys rapidly become protected

by a Al203

surface coating: maximal thickness is reached during the

first day of immersion.

Aluminum 7075

Alloy 7075 is aluminum containing 5.5% zinc, 2.5% magnesium,

1.5% copper and 0.3% chromium. It is not noted for its corrosion

lGeneral information on the characteristics of metals used in thesetests were obtained from Taylor Lyman, (ed.). 1961. In MetalsHandbook, Vol. 1. Reinhold Pub. Co., N.Y., 8th edition. A galvanicseries of all metals considered here is presented in Table I.

16 -

resistance in sea water.

Zinc

Zinc is high on the galvanic activity scale and is commonly

used as a protective layer on the surfaces of other metals, most

notably "galvanized" iron. It rapidly forms a surface oxide which

exfoliates at a relatively constant rate for many months. Zinc has

high surface electronegativity when initially immersed in the sea.

This potential declines for several days, then remains stable for many

months. Galvanized steel was used in these experiments as the zinc

test surface.

Phosphor-bronze (A)

Phosphor-bronze (A) is copper containing 5% tin. Corrosion is

due to copper solubilization; this occurs at a lower rate than in pure

copper. Copper and high-copper alloys solubilize at relatively

constant rates for periods exceeding one year under normal circum­

stances. Free copper ions at the surface are potentially toxic.

The observed decrease in solubilization rate results from the

gradual accumulation of copper corrosion products on the surface; this

occurs during conditions of slow water flow past the immersed metal.

Phosphor-bronze (A) exhibits moderate galvanic activity. Adsorption

of organic matter also decreases solubilization (and hence the surface

toxicity) under conditions of decreased water flow.

Monel

Monel is electrochemically passive in oxygenated waters. Its

high nickel content (66%) results in the formation of a stable NiO

17

protective layer over the surface. The nickel is alloyed with copper

(3Z%) and small quantities of iron, manganese and silicon. The chemical

passivity of this alloy encourages biological attachment in calm waters.

When organisms attach, however, the protective oxide layer is damaged

and corrosion occurs as individual pits. This pitting is slow and

evenly distributed over the entire surface; the pits also tend to

"heal" and are characteristically shallow. A galvanic series for the

metals used in these field experiments is given in Table I.

In addition, the following non-metallic test panels (including

control glass slides) were used: Z

Glass

Common soda-lime silica glass microscope slides (Scientific

Products Co.) were used as test surfaces. The chemical composition of

this type of glass is approximately 73% SiOZ' 14% NaZO, 10% CaD and

Plexiglass

Plexiglass, (acrylic plastic), is the common name for

polymethylmethacrylate, a non-toxic, transparent, moderately

hydrophilic cast-plastic resin. It is chemically inert in sea water,

and is subject to heavy fouling by marine organisms.

Fiberglass, manufactured with a protective layer of

polymethylmethacrylate (Fi1on, Type 140) was used in some experiments

2Characteristics of the non-metalsobtained from A. A. Layne. 1970.Selector Issue) Vol. 7Z, Reinhold

presented in this section wereMaterials Engineering (MaterialsPublishing Co., N.Y.

18

TABLE I

Galvanic Series of Metals Used in Field Experiments

MetalPotential in Slow-Flowing Sea Water

(mV Versus Sat. Calomel at 25C)

Steel --1G30

Stainless Steel 304 (passive) 80

Stainless Steel 304 (pits) 530

Aluminum 5052 740

Aluminum 7075 780 (Approximately)

Zinc - 1030

Phosphor-Bronze (A) 300 (Approximately)

Monel 80

Potentials were taken from A. H. Tuthill and C. M. Schillmoller. 1966.Guidelines for the selection of marine materials, International NickelCompany, Inc., N.Y., and A. H. Tuthill and C. M. Schillmoller. 1969.Guidelines for the selection of marine materials, Nav. Eng. J. 81:66-89. Estimates for some alloys were made on the basis of thepotentials of related alloys.

19in place of pure acrylic. The results obtained with this product are

considered to be similar to those obtained with pure acrylic, and

thus reference is made only to "plexiglass" in these experiments.

Teflon

Teflon is the trade name for a synthetically-produced polymer

of tetrafluoroethylene, composed of repeating (-CF2-CF

2-) units. It

is chemically inert and extremely hydrophobic. Teflon was used in

these experiments as ultrathin (75 ~m) membranes 25 x 25 mm square,

with a 5 ~ pore size (Beckman Instruments Co., Part No. 77948). These

were applied tightly to various test panels in order to separate

adsorbed microorganisms from the underlying test surface.

Wood

Wood is a highly porous, soft-textured, chemically polar natural

product composed of cellulose (about 50%), lignin (20-30%), pentosans,

starch and pectic substances (Browning, 1963). The type of wood used

in these experiments was Douglas Fir, Grade A.

(iii) Test Panel Preparation

Holes were drilled 2 em from the top and bottom edges of all

panels for supporting and anchor lines. Metals and glass panels were

washed in acetone; 95% ethanol was used to clean plexiglass panels.

The wood panels were sanded to smoothness with fine-grained sandpaper.

All test panels were subsequently sterilized by ultraviolet

irradiation for 24 hours at a distance of 0.5 m from ultraviolet lamps

(Hanovia) emitting predominantly at 265 nm. In addition, an overhead

ultraviolet lamp at a distance of 1 m from the test panels was operated

20

continuously. The test materials were wrapped in heavy, presterilized

paper after irradiation and sealed for transport to the sampling site.

At the test site, the predrilled holes were exposed to attach

the nylon suspension and anchor lines. The test surfaces were then

fully unwrapped and quickly immersed to the test depth.

(iv) Post-Immersion Processing of Test Panels

At each sampling interval the panels to be analyzed were removed

from the channel and routinely rinsed with 500 ml of filter-sterilized

sea water. Specimens were then taken directly to the Hawaii Institute

of Marine Biology for initial processing, which varied according to the

test procedure. All materials were subsequently transported to the

Manoa Campus of the University of Hawaii (on the same day) for further

analysis.

Equipment

The sea water used in these experiments was collected at the

test site in 20-liter polyethylene carbuoys and stored at 5 C until

used. Before use, all sea water was filter-sterilized.

All filtration equipment used in these experiments was

manufactured by the Milliporc Corporation, Bedford, Massachusetts.

White, cellulose acetate filter membranes with 0.45)Uffi pores were_used

exclusively. These were mounted in a sterile pyrex (or polycarbonate)

filtering apparatus.

All microscopy was done using a Leitz Labolux trinocular phase

microscope equipped with a MIKAS photographic attachment. Test

surfaces and bacterial colonies on growth media were observed and

counted with a Unitron MSF 10-20x dissecting microscope with an oblique

21

light source.

Color photomicrographs were taken with a 35 rom Leica M2 camera

using Kodak Kodachrome II film. All black and white photography was

done with a 35 rom Asahi Pentax Spotmatic camera using Kodak Plus-X

film exclusively.

(v) Rationale

Three separate procedures were employed to follow the develop-

ment of primary films (slime layers) on the test materials: (1) the

surface swabbing technique; (2) the Teflon membrane technique; and

(3) the Parlodion filming technique.

The surface swabbing technique was used primarily as a control

procedure to characterize the bacteria fo~qd on the different test

panels.

The Teflon membrane and Parlodion filming techniques are

introduced in order to characterize the in situ growth patterns of

microorganisms on the different test panels. Obviously the swabbing

technique does not retain these in situ relationships.

(vi) Surface Swabbing Technique: Comparative Bacterial Numbers from

Chemically Diverse Surfaces

Test surfaces used in this experiment were metal sheets 1-2 rom

thick and 30 x 30 cm in area of steel, phosphor-bronze (A) and aluminum

5052; a 25 mm thick panel of wood was also used.

3 x 3 cm areas were marked on each panel with a sharp metal

2punch. A 3-4 cm border was left to the panel edges; each 9 cm area

was numbered.

22

After five days of immersion, fifteen ruled areas in panel were

swabbed, using six strokes for each of two cotton swabs per area. The

swabs were then immersed in 10 m1 of sterile (filtered) sea water in

test tubes, vigorously shaken for 30 second~, blotted on the side of

the tube and discarded. Each sample tube was then serially diluted

(1:10) with sterile sea water. Samples not immediately being processed

were stored at 5 C.

Media

From each sample suspension, three replicate portions (undiluted,

10-1 and 10-2 dilution) were plated on six different media:

Marine Agar:

The standard bacteriological culture medium used was Bacto­

Marine Agar (Difco) , prepared as directed from dehydrated form and

autoc1aved at 121 C for 15 minutes.

Iron-Marine Agar:

Sterilized discs of steel (22 mm in diameter) were overlaid with

Marine Agar in petri dishes to a point just covering the discs.

Copper-Marine Agar:·

Sterile phosphor-bronze discs (22 mm in diameter) were overlaid

with Marine Agar.

Aluminum-Marine Agar:

Sterile aluminum 5052 discs (22 mm in diameter) were overlaid

with Marine Agar.

Mineral Agar:

1.5% agar-agar in 75% sea water was made to 0.1% NH4N03 and

0.5% K2HP0

4• The pH was adjusted to 7.8 with IN NaOH.

23

Cellulose - Mineral Agar:

Mineral agar was overlaid with discs of sterile filter paper

(Whatman No.2) after plating.

After each agar was poured, the petri dishes were allowed to

remain at room temperature for 4 days before use. After less than 24

hours, orange and blue metal corrosion products diffused into the agar

from the steel and phosphor-bronze discs, respectively. No physical

changes were noted in the aluminum medium. At 48 hours, the steel and

phosphor-bronze media were almost entirely colored; visible physical

changes were not observed in the other media.

Qualitative tests were then made on all media for Fe+3 , Fe+2 ,

Cu+ and AI+3 ions, according to methods described by Fiegl (1947). The

sensitivity of these tests to the various ions was 0.05, 0.03, 0.02

d 0 65 . 1 Th . I . f F +3an • "ug respect~ve y. e test was cons~stent y negat~ve or e ;

tests for the remaining ions were strongly positive in their respective

media. Media without embedded discs were negative in all ion tests.

After incubating the plates at room temperature for 72 hours, visible

colonies were counted. The number of viable bacteria per square cm of

immersed test surface was calculated from the average of the colony

counts on three serial dilution plates.

(vii) Surface Swabbing Technique: Bacterial Succession Patterns on

Chemically Diverse Surfaces

Test surfaces of steel, aluminum 7075, zinc, plexiglass and

Douglas fir were used. All surfaces were of the same dimensions as in

Section (vi) except the plexiglass was a modified triangular panel of

pure acrylic 5 cm thick, with approximately 1200 cm2 of surface area

24

available on each face. All surfaces were marked off in 9 cm2 areas,

cleaned, irradiated and immersed at the test site.

At intervals ranging from 1 hour to 40 days, five areas on each

test panel were swabbed as in Section (vi). Duplicate swabs were then

placed in 9 ml of sea water, mixed, rinsed, blotted and transferred to

a second tube containing 1 ml of sea water. After similar treatment,

the swabs were discarded, and the contents of the second tube added to

the first giving a total volume of 10 mI. It was assumed that a second

rinse would provide a more accurate estimation of total fiable number

of bacteria per unit area. Three of the five samples from each test

panel were then used for bacterial quan~itation, while the other two

were saved for morphological studies.

Replicate serial dilutions were made (1:10) using sea water as

before. Four of the five dilutions were filtered, and each membrane

was placed on Marine Agar in 54 mm plastic petri dishes with wedge-fit

covers (Millipore Corp., Bedford, Mass.). After incubation at room

temperature for 48 hours, the resulting colonies were counted; three

samples of all four dilutions from each surface at each sampling

interval were included.

These plates were used to estimate the number (per cm2) of

viable, aerobic, heterotrophic bacteria on each test material. Colonies

of different size, morphology and pigmentation were also selected and

streaked on Marine Agar slants for subsequent identification. Gram­

positive isolates were given provisional generic designations based on

colony characteristics, motility and cellular morphology, following

subculturing. Gram-negative forms were tentatively classed at the

25

generic level using the criteria of Batonsingh and Anthony (1971) and

Pilkington and Fretter (1970), based on the scheme of Shewan (1963) and

Shewan et al. (1960a,b).

The contents of the two remaining sample tubes from each test

surface were filtered directly without dilution. l5x15 sections were

excised from the center of each filter and stained with Loeffler's

methylene blue, using the technique suggested by Millipore (1965).

Light microscope observations were then made for representative

microorganisms and typical fields were photographed.

Closeup views of identical areas on each test surface were also

photographed at various periods during the immersion interval to show

the macroscopic changes as a function of immersion time.

(viii) The Teflon Membrane Technique: Preliminary Laboratory Tests

Preliminary tests were conducted in the laboratory prior to field

studies to determine (1) if microorganisms could attach to Teflon

membranes and (2) if certain metal ions could pass through the 5}Iffi

pores in Teflon membranes bounded by sea water.

For the first test, Teflon membranes were attached to glass slides

by two methods: waterproof tape around the periphery, and silicone

lubricant spread beneath the membranes. Replicate preparations were

immersed with cleaned glass slides in a beaker of freshly-collected,

natural sea water for 24 hours. The membranes and glass slides were

then rinsed 30 seconds with filtered sea water and stained for five

minutes in Loeffler's methylene blue.

After drying, the membranes were mounted in immersion oil with a

cover slip and observed in the microscope. Counts over ten fields on

26

each type of preparation showed:

91 - 102 bacteria per field on glass slides

97 - 111 bacteria per field on taped Teflon membranes

64 - 79 bacteria per field on silicone-attached Teflon membranes

Several bacteria were seen within the pore spaces on all but silicone­

attached membranes; all cells were differentiated easily from the

non-staining Teflon background. Only individual bacteria were seen

and no microcolonies.

These results suggested that Teflon serves as an attachment

surface for bacteria in a capacity similar to that of glass (i.e., the

adsorbed surface components are easily differentiated due to trans­

parency). Silicone may inhibit attachment by blocking the pore sites

by retarding adsorption of dissolved organic matter on the glass

surface at these points (perhaps preventing a chemotactic stimulus).

This might be expected since silicone has been reported by Baier et al.

(1968) to possess hydrophobic properties which discourage permanent

adhesion due to its non-wettability.

In the second test, six Pyrex-Millipore filtration units with

20 ml funnels and sintered glass filter beds were assembled, using

Teflon membranes in place of filters. Each apparatus was inverted and

the bottom portion of each filter holder was filled to capacity with

filtered sea water. A 10x150 rom test tube previously filled with sea

water was quickly inverted over the spout of the filter holder and

taped securely in place against the rubber stopper.

The assemblage was then placed upright into a filter flask for

support. Five of the funnels were filled to approximately three-fourths

Sea water control:

27

capacity with 10 x 10 x 1 mm pieces of one of each of the following

metals: steel, aluminum 5052, phosphor-bronze (A), Monel and stainless

steel 304. Filtered sea water was then added to the 20 ml mark on the

funnel. A control unit was also assembled, containing only sea water

without metals. No air bubbles were present at the interface on either

side of the Teflon membranes, i.e., in the funnel portion or the

sintered glass filter bed.

After 72 hours at room temperature, the sea water on both sides

+H- ++ +of all membranes was checked qualitatively for Fe , Fe ,Cu and

Al+H- ions using the methods of Fiegl (1947). Tests on the non-metal

side of the membranes showed the following reactions:

Negative for all tests.

Iron- sea water: ++Positive only for Fe •

Aluminum - sea water: +H­Positive only for Al .

Copper - sea water:

Monel - sea water:

+Positive only for Cu •

+Weakly positive only for eu •

Stainless steel-sea water: Negative for all tests.

As a confirmatory test, the sea water solutions containing the metals

on the opposite sides of the membranes showed the same reactions as

above.

Thus Fe++, Cu+ and Al+++ ions pass through the 5)Um pores in

Teflon membranes in a sea water medium. On the basis of these

preliminary results, a field test was designed to assess the

applicability of Teflon membranes as removable surfaces to overlay

various materials immersed in sea water for in situ assays of micro-

organisms.

28

(ix) The Teflon Membrane Technique: Field Tests

Duplicate test panels 20x 20 x 1 mm of aluminum 5052, steel,

zinc, phosphor-bronze, glass and wood were placed 8 -10 mm from each

end of a glass slide and covered with autoclave-sterilized Teflon

membranes. The membranes were taped tightly over the surfaces at the

overhanging border, which also fastened the test panels securely to the

glass slides (which served as submounts). For glass test surfaces,

Teflon membranes were taped over acid-cleaned, ultraviolet irradiated

glass slides.

Duplicate submounts were made of each of the six test surfaces

and fastened onto one of two large acrylic slabs; these were immersed

for intervals of one and four days, respectively. After immersion,

sections 15 x 15 mm were then cut from the center of the Teflon membranes

overlying each test surface, stained in Loeffler's methylene blue for

five minutes, dried and mounted in immersion oil.

The numbers of bacteria and diatoms were counted in 10 micro-

scopic fields from the one and four-day preparations and converted to

2number per cm. Photomicrographs were taken of several fields during

routine observations.

(x) The Parlodion Filming Technique

A method referred to as the Parlodion filming technique was

developed during these studies (Sechler and Gundersen, 1971) to make

possible the in situ observation of surface microcosms. This permitted

observation of the microorganisms and abiotic particles on test

materials immersed in the sea, regardless of opacity. A description of

29this technique follows:

1. The immersed test surfaces of approximately standard

microscope slide size (i.e., 75 x 25 x 1 mm) are removed

from the test site, loosely covered with aluminum foil,

and allowed to dry for at least 1 day.

2. The surfaces are immersed into a solution of 10% Parlodion

(Mallinckrodt) in amyl acetate for at least 2 minutes. The

Parlodion vessel is a small jar or 50 ml beaker situated

inside a one-liter filming jar with a plastic screw cap

having a 2 x 4 cm hatch for inserting test surfaces. The

inside atmosphere of the filming chamber is saturated with

amyl acetate.

3. After filming, the test panels are raised from the Parlodion

solution with forceps, braced beneath the chamber cap to

drain for at least 1 minute, blotted at the draining end

and placed in a hood to dry for at least 24 hours.

4. Selected areas of the filmed surfaces are excised with a

scalpel, applying sufficient pressure to contact the test

surface proper. The Parlodion layer is freed by carefully

inserting the scalpel blade beneath one corner of the film

and gently peeling the entire portion away from the surface.

5. The extracted film is stained in Huncker's crystal violet

for approximately 30 seconds, dipped 4-5 times in tap water

to rinse, dried thoroughly in an air jet, and mounted in

Permount (Fisher Scientific). In mounting, the side

originally next to the test surface must face upward. A

30

No.1. thickness cover slip should be used to allow for oil

immersion.

6. The mount is allowed to dry for at least 24 hours before

microscopic examination.

(xi) The Parlodion Filming Technique: Field Studies

Field Studies

Test panels were glass, plexiglass, stainless steel 304, aluminum

5052, Monel and phosphor-bronze prepared as in Section (ix). Duplicate

panels of all materials were placed in each of 21 glass staining racks

(Wheaton Glass Co.).

The racks were strung by nylon monofilament line 5 cm from the

bottom of larger (100 x 50 xL 5 cm) polycarbonate lattice frames. The

glass racks were then immersed in 70% ethanol for 10 seconds, dipped in

acetone, and quickly wrapped with heavy, presterilized paper for

transport to the test site.

The panels were suspended at the test depth in a vertical

position and parallel to the course of the channel. The mounts were

supported in a manner which gave great stability to the test system.

Even when forcibly rotated the three polycarbonate mounts immediately

returned to their previous positions.

At 21 intervals ranging from 1- 120 days, sample racks were cut

loose from the larger mount, rinsed, and placed in a glass staining jar

(Wheaton Glass Co.) for transport. Drying was begun in the laboratory

within 1 hour, and the test surfaces were processed according to the

Parlodion filming technique.

31

Total cell and colony counts of bacteria and diatoms were made.

All extraneous, non-cellular staining particles greater than 5pm in

diameter (of probable organic origin) were also counted. Routinely 25

2microscopic fields, each with an area of 0.01 mm , were counted and

averaged for each test panel at each sampling interval. The number of

bacteria or diatoms per colony were also calculated from these data.

A test was made to determine whether the cell and particulate

distributions were random. The values obtained from the glass test

panel (as a representative surface) were compared to a set made using a

Table of Random Numbers (Interstate Commerce Commission, 1949) to

locate the vertical and horizontal microscopic coordinates. A three-way

chi-square test (summary, pooled and heterogeniety) was then performed

on the total number of bacteria, diatoms and extraneous particles per

25 microscopic fields at representative sampling intervals. Standard

deviations were subsequently calculated only for data within the limits

where randomness was affirmed at the 0.05 level determined by the chi-

square test.

Although no microscopic field could be considered "typical" in

all respects, an attempt was made to characterize each preparation by

time sequence. This was done by taking several photomicrographs of

each preparation and selecting the most typical view. The procedure

was also intended to illustrate the potential use of the Parlodion

filming technique to show the microbial morphology, while retaining the

in situ spatial relationships between the examined organisms. In this

way, visual comparisons were easily made on the microorganisms

occurring at each time interval on each test surface.

III

RESULTS

(i) The Interaction of Primary Films with Test Panels

32

Development of a primary film or slime layer on the surface of

an immersed material follows a sequence of events which is character-

istic of the underlying material. As a prelude to the study of the

microbiology of these developing primary films, a series of pictures

showing the appearance of slime layers after various periods of

immersion are presented.

In Figures 2 through 6 photographs of plexig1ass, aluminum,

steel, zinc, and wood test panels are shown.

From these pictures it is evident that the formation of primary

films or slime layers exhibits several characteristics which are

dependent upon the underlying material. In addition, each test

material has a characteristic response to the marine environment (rust,

pit formation, etc.) which may be reflected in the microbiological

composition of the resulting slime layer. In the following section I

shall examine the microorganisms found in the slime layers formed on

several test materials at various time intervals.

(ii) Surface Swabbing Technique

(a) Determination of the Average Bacterial Density on Immersed

Test Panels Following Colony Formation on Modified Growth

Media

The purpose of these experiments was to dete1~ine: (1) whether

Marine Agar can serve as an assay medium for bacteria recovered from

chemically diverse test panels immersed in the sea; (2) the effect of

33

any of three different metal ions present in Marine Agar media; (3) the

relative density of cellulolytic bacteria on each of the various test

surfaces compared to the total number of heterotrophic bacteria

developing on Marine Agar.

In Table II the calculated average density of viable ~~~teria on

each of four test panel surfaces tested are given after growth on the

indicated media.

Viable cell densities on the wood test panels were comparatively

high on Marine Agar, iron-Marine Agar and aluminum-Marine Agar. No

growth was observed during the first 15 days on any medium which had

been plated with swabbings from the phosphor-bronze test panel. No

growth was observed on copper-Marine Agar regardless of the initial

source of the plated material. On the mineral agar, the agar-digesting

bacteria appeared both as orange-pigmented and as small-indistinct

colonies within 5 mm craters in the agar. On mineral agar overlaid

with cellulose, bacterial colonies were yellow-pigmented and confluent.

These results suggest that bacteria developing on each test

surface by five days after immersion do not require specific cations

but grow to different densities depending on the test material.

A priori it might be expected that a larger proportion of the organisms

found on wood would be cellulolytic. However, no selective effect of

any test materials was demonstrated. In addition, I conclude that

simple Marine Agar is an adequate medium for bacteria isolated from

the primary films found on chemically-diverse materials.

34

(b) Bacterial Succession Patterns on Test Panels During a

40-Day Immersion Period

This experiment was designed (1) to examine the duration of any

chemical effect of each of the various materials on the composition of

the resulting bacterial population and (2) to show bacterial succession

patterns on each test material.

The density of viable bacteria on each test surface was found to

vary significantly for the first five days (Figure 7). A roughly

constant density appeared between 15-40 days on each test surface.

Of 102 bacterial isolates originally selected on the basis of

colonial characteristics, 52 were differentiated by subsequent testing.

These isolates were then grouped into seven provisional genera, each of

which fulfilled the criteria of Shewan (1963) and Shewan et a1.

(1960a,b). The genera and respective isolates are given in Table III.

The occurrence of these isolates on each of the five test

surfaces is reported in Figures 8 to 12. A vertical line indicates the

presence of at least one colony of that isolate on the test material at

that test interval.

Each isolate appeared in anyone of several patterns on each of

the various test surfaces. Some of these patterns were (1) from 1-24

days; (2) from 1 hour through the first few days; (3) from 1 hour

through the first 20-30 days; (4) after the first few days, for short

periods only; (5) sporadically or· for only one sampling period; and

(6) only after the first four weeks, usually remaining until termination

of the test at 40 days. These data also show that none of the isolates

originally present during the first few hours or days persisted through

35

the entire test period. In two cases, isolates "disappeared" and were

"rediscovered" at 40 days (Isolate numbers 29 and 39).

The number of isolates (species?) found on metallic panels

decreased rapidly after a maximum value was obtained within the first

18 hours (Figure 13). These data also suggest that the maximal number

of isolates recovered is proportional to the position of the respective

metal surface in the galvanic series.

The number of bacterial isolates on plexig1ass and wood test

surfaces never reached the higher values seen on metallic test panels.

At least three general conclusions about the appearance of

bacterial types are suggested by the data in Figure 13.

(1) A sharp increase occurs from 18-24 hours, followed by a

rapid decline which is especially pronounced on metallic panels.

(2) A "stabilization" period occurs between 2-20 days in which

the number of bacterial species shows little variation.

(3) A period of decrease in isolate numbers between 20-30 days,

followed by various responses, depending upon the test surface.

Species Diversity Ratios (Gorden et a1., 1969), which are a

numerical representation of the relationship between the total number

of isolates and the number of tentative species in each sample, are

given in Table IV. These data show comparatively high Diversity Ratios

for the first 18 hours. This is followed by a steady decrease, which

is paralleled by a decrease in the total number of isolates. A general

tendency toward similarity in the Diversity Ratio as a function of

36

increasing immersion time for all materials is shown by the similar

Diversity Ratios for each material at 40 days.

Figure 14 indicates the relationships between the numbers of

species observed and number of days of occurrence on one, two, three,

four or all five of the test panel surfaces. As shown here a small:

number of isolates comprise the bulk of stable populations of the test

surfaces while the majority of isolates are infrequent occurrences.

Thus, of the total time on all five surfaces the eight major isolates

present accounted for almost 54% while the top fifteen comprised almost

82%.

Twenty of the 52 isolates were recovered from only a single test

surface, and all of these except four (22, 32, 50 and 51) were found

during only one sampling; of these four, three were recovered only

twice. Only one (22) persisted through several sampling intervals.

During routine sampling procedures to determine the bacterial

density in the water at the test site, only 9 of the 52 isolates were

recovered (i.e., Nos. 1,2,3,7,8, 22,28,36 and 37). All of these

except 7 and 22 were consistently present on most test surfaces (at

least during the initial stages of immersion).

Sample micrographs from stained Millipore membranes are presented

in Figure 15. These are shown principally to demonstrate the type of

results obtained with the Millipore method. In general, the bacterial

and diatom staining was so poor that these organisms were not readily

discernible. Faint outlines of diatoms could be seen, but bacteria

were resolved only by darkfield microscopy. It should be noted that

the bacteria were difficult to distinguish from extraneous particles.

37

Morphological damage "from swabbing, mixing and filtering was extensive

using the Millipore membrane-staining technique. Another difficulty

was the presence of overlapping residues (such as that seen in Figure

15) which obscured the organisms and further restricted identification.

In view of these results, I shall discuss the reasons why

surface swabbing techniques are unsuitable for studying the microbiology

of primary films.

In a review of techniques for determining viable surface

bacterial counts, Favero et al. (1968) recommended the swab method as

the most reliable for field studies. pfaender and Swatek (1970) have

recently used this technique to successfully identify and enumerate

bacterial and fungal densities on a variety of metallic and

non-metallic surfaces.

However, the results of the current study indicate that the

chemical nature of the test surface may significantly influence the

numbers and varieties of bacteria that attach and develop. The results

also confirm my earlier conclusion that population levels may vary

according to surface chemical composition.

Most notable are the bacterial populations found on wood test

panels, which were consistently higher than those found on all other

test surfaces during the first five days. Since the same culture medium

was used to assay for all bacteria, and since the data show essentially

the same types of bacteria on wood as the other surfaces, abnormally

high cellulolytic populations must be ruled out. O'Neill and Wilcox

(1971) have also shown a significantly higher bacterial population on

wood as compared to steel, plexiglass and glass test panels.

38

The initially high populations observed on wood test panels

suggests that bacterial adsorption is rapid compared to the other

surfaces. This may be attributed to two factors: (1) wood possesses

no galvanic activity and thus does not repel bacteria; and (2) wood is

extremely "wettable". According to Baier et al. (1968), good

"wettability" is a prime criterion for the stable adhesion of bacterial

cells to solid surfaces. Hydrogen bonding between the cell wall amino

acids and external polysaccharides and the adsorbed water layer on the

wood surface may be the determining factor.

The adsorption of organic matter to metallic surfaces necessarily

precedes bacterial attachment. In addition, substantial surface

corrosion occurs altering the electrostatic repelling effect. The data

also indicate that the relative galvanic activities of metals may

determine the adsorption rate of organic matter. Bacterial densities

at 3 days were proportional to the galvanic activity of the metal.

The dependence of bacterial adsorption to immersed surfaces on

the concentration of available dissolved organic matter has been

previously emphasized by ZoBell (1943), Kriss and Makianovich (1954),

Gorbenko (1969) and others. In our case the concentration of dissolved

organic matter at the test site was determined to be many times that

observed in pelagic sea water.

Plexiglass, unlike wood, is only moderately wettable and has

little polarity. The sequence of events in bacterial adsorption to the

plexiglass test panel may nonetheless be similar to adsorption to the

wood panels, but considerably retarded due to slow initial adsorption.

Stable population levels on plexiglass at three days were lower than

39those on all other test surfaces except steel.

A tendency was observed for bacterial populations after ten days

to assume similar densities on all test materials. This strongly

indicates that surface galvanic and direct chemical effects had

decreased and that the materials approached homogeneity on the surface.

Marshall et ale (197Ib) have recently emphasized that modifications of

the surface properties may make materials more amenable to colonization.

The observations on steel test panels, however, indicate that sloughing

of exfoliation products apparently retards surface passivity until at

least fifteen days after immersion.

The somewhat higher population levels observed on aluminum test

panels at 30 or 40 days may be attributed to the presence of a greater

number of well-adapted bacteria. Figure 13 shows a similar trend in

the number of isolates present for all materials (with the exception of

aluminum) at 40 days.

Since so few of the isolates comprised most of the primary

surface populations, it must be concluded that most strains are not

well-adapted for vigorous competition in surface microcosms. Many

isolates appeared transiently; almost one half were recovered from only

one surface during a single sampling interval. This response indicates

that many varieties are present at very low concentrations in the water

and are unable to compete successfully with established populations.

Further evidence to support this view is given by Marshall et a1.

(1971a), who found "irreversible" adsorption characteristics only in

certain groups of marine bacteria.

The (tentative) bacterial genera recovered were identical to

those reported on glass, acrylic plastic and wood by O'Neill and Wilcox

(1971).

40

The Woods Hole report (1952)3 and ZoBell (1946) also note

that these same genera are among the most common from species isolated

in marine waters.

Active proliferation on all test surfaces is indicated by the

rapid decline in species coupled with concomitant. steep, population

increases within hours after immersion. According to Tyler and

Marshall (1967), this constitutes a "take over" by certain bacterial

species which multiply on the surface at rates in excess of their rate

of loss from the microcommunity. This opinion is supported by other

workers who report that the number of species in isolated ecosystems

tend to lessen with time, while the total number of organisms increases

rapidly (Gause, 1936; ZOBell, 1943; Gorden et al., 1969).

Gorden et ale (1969) also believe that a microcosm exhibits the

characteristics of the larger, better studied natural ecosystems. In

this consideration, Patrick (1963) has reported that numerical

dominance of diatom communities is held by only a very few species.

She also notes that an extremely large number of organisms must be

observed in order to expose all varieties. These tendencies are shown

in the current study in two primary ways:

(1) Only 9 of the 52 isolates were identified from a routine

sampling of the sea water at the test site. (The sea water may be

considered a stabilized ecosystem).

3Reference will hereafter be made in this manner to Contribution No.580, Marine Fouling and Its Prevention, 1952. Prepared for theBureau of Ships, Navy Department, by Woods Hole OceanographicInstitution, Copyright U.S. Naval Institute, Annapolis, Md., GeorgeBanta Pub. Co., Menasha, Wis.

41

o

Figure 2. Appearance of the "plexiglass" test panel after variousperiods of immersion. The number of days at the test depth isindicated on each photograph. Note the complete opacity of theplexiglass test panel by 7 days after immersion and the appearance ofa prominent slime layer by 25 days. Young oysters are visible amongthe filamentous algae. At 42 days the oyster morphology is typical asis the finely-granular surface coating. The same test panel area isdepicted at each time interval.

42

Figure 3. Appearance of the aluminum test panel after various periodsof immersion at the test depth. A thick slime layer is prominent at15 days, with long, filamentous algal fronds. By 25 days the slimelayer has greatly diminished, and a young barnacle is easilyrecognizable. Foraminiferans are also present, appearing as granulessurrounding the barnacle. Considerable slime has again formed by 45days. The same area is depicted at each time interval.

43

Figure 4. The appearance of a steel test panel after immersion at thetest depth for the indicated number of days. A thick rust layer ispresent by 7 days, and the primary film is clearly visible by 15 days.Considerable sloughing of rust has occurred by 25 days. Also visibleare some attached sand grains. At 35 days, however, a thick, greenalgal mat has completely covered the test surface. By 45 dayssloughing again has removed considerable portions of the surface asshown by the obvious patches. The same test panel area is depicted ateach time interval.

44

Figure 5. Appearance of the zinc test panel after various· periods ofimmersion at the test depth. Purely chemical reactions are present atthe macroscopic level at all time periods. The pits formed near thecorrosive focal points are obvious over the entire surface after 5days. The process appears to be initiated by the formation of small.metallic bubbles (day 7). which later slough off. The same area isdepicted at each time interval as can be seen by the easilyrecognizable design during most time intervals.

45

Figure 6. Appearance of a wood test panel after various periods ofimmersion at the test depth. A very thick slime layer is evident at5 days. However, the panel only appears darkened at 7 days. Again by15 days, the heavy slime layer has returned. This conditioneventually is succeed~J by a thick algal mat (from 25 days on).Circular holes characteristic of the wood-boring isopod Limnoria areseen at 45 days throughout the thick algal mat. The same area is shownat each time interval.

TABLE II

THE GROWTH OF BACTERIA ON TEST PANELSAFTER PLATING ON VARIOUS MEDIA

a Diffusegrowth

.02 ± .01 Diffuse growth+agar-diges ters

0.03 ± 0.01Diffuse growth+agar-digesters

a a

Iron Aluminum Cuprous

MEDIAMarine Marine Marine Marine

Agar Agar Agar Agar

TEST PANEL MATERIAL"4(All values x 10 )

Hood ~ 67 ~ 67 ..? 67 a

Steel 2.5 ± .07 1.9 ± .07 1.5 ± 0.08 0

Aluminum 0.18 ±0.02 0.24 ±0.17 0.15±0.02 0

P-Bronze a a a a

MineralAgar

CelluloseMineral

Agar

Average cell densities were determined by the surface swabbing procedure described on pp. 24-25 Methodsand Materials, section (vi). The immersion period was five days.

"'"0\

SURFACEWood •Aluminum-'-'-'-'-eZinc-- AP'eliJalall •••••••••••••••••,DSteel-- • - • - . - • - 0

(Data basedon'3 :~ ~- l:r Ireplicates)~ '"'2 ,/.1 ,-,-,-~

J /'f --!of ,.:=,--,,~.(,r -----. - ..... tlF.7AU--'...:....-

I '~__'-'-'-', ;1' -,,",:: . ~~;; ...... 00;;:.

l I •• ~.,.,.'.oJ:) ..~ ..~ :p. ,.'},: .. , .... ~

i /,. )' 1:. ·-·+· .....Y·. :1 I'· _.--rt T/-, \ .1£ \ .'

1"

o.-C1o...

N~U....00(

GI:&II...U00(IllI

Figure 7. Developmental pattern of heterotrophic surface bacterial populations on eachof several chemically-diverse materials during a 40-day immersion period. Densitieswere determined using the swabbing-filtration-culturation techniques described inmethods and materials. Range limits for the three replicate samples are indicated.

.po....,

GENERA

TABLE III

BACTERIAL GENERA ISOLATED BY SWABBING METHODS

ISOLATE NO.

48

TOTAL NO.

Achromobacter 1, 2, 4, 6, 7, 8, 9, 19, 22, 30, 32, 38, 45 13

Flavobacterium 21, 23, 27, 39, 44, 46, 47, 48, 50, 51 10

Micrococcus 3, 18, 28, 31, 33, 35, 31, 52 8

Vibrio 13, 14, 24, 25, 26, 43 6

Pseudomonas 12, 20, 34, 40, 41, 42 6

Bacillus 5, 11, 15, 16, 17, 36 6

Sarcina 10, 29, 49 3

40

SAMPLING PERIODS PRESENTISOLATE~~;;;;;';;;;";;';;p.;;~...;;..;;;,;,;,;;;",;,;;~~~-- __

Hours DaysNO. 1 3 6 18 1 2 3 5 10 15 20 30

1 ••• • .2 111 1

3 11 .4 , .5 ,67 ...8 .9

101112 ,1314 ....151617 ...1819 ....20 ,•••••••••••••••••••••••••111••111111•••

21222324252627 ...2829 ••••••••••3031 •••••••••••••••••••••••••••••323334 ..3536 •••••••••••37 •••••••••••38 .39 • .40414243 ....444546 , .47 I .

48 .49505152

49

Figure 8. Occurrence of various bacterial isolates at each samplinginterval on plexiglass test panels. Each bar indicates the observa­tion of an isolate in the test interval.

40

I SOLATE t-......_S_AMPL,.I_N_G......P_E_RI__O_D~S......P_RE.;;..S.;;;E;;;,N;.;;T;....__

N Hours Dayso.1 3 6 18 1 2 3 5 10 15 20 30

50

123456789

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~53132333435363738394041424344

ig474849505152

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••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••..........

•••••••••••••••••••••••••••• 1

.u..........................,

, ...................,...,.

••••••••••••••••••••••

.....•••••••••••••••••••••••••••••••••••••••••••••••••.....uu..........i ....

....1 I"".I""...............

••••••••••••••••......... .....•••••••••••••••••••.... ....,..... ................

Figure 9. Occurrence of various bacterial isolates at each samplinginterval on aluminum 7075 test panels.

SAMPLING PERIODS PRESENTISOLATE ....~......OiiiiioI...............iiiioiiioO.....~...................._--Hours Days

NO. 1 3 6 18 1 2 3 5 10 15 20 30 40

51

0 ..

••••••••••••••••••••••••••••••••••••• ••••

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10111213141516171819202122232425262728293031323334353637383940414243444546474849505152

.... ..............

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••••••••••••••••••••••••••...................••••••••••••••••

...........

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....

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Figure 10. Occurrence of various bacterial isolates at each samplinginterval on steel test panels.

ISOLATENO.

SAMPLING PERIODS PRESENTHours Days

1 3 6 18 1 2 3 5 lU 15 20 30 40

52

•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••

....•••••••••••••••••••••••••••••••••••••••••••••

•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••

........................................••••••••••••••••••••••••••••••••••••••••••

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123456789

10111213141516171819202122232425262728293031323334353637383940414243444546474849505152

Figure 11. Occurrence of various bacterial isolates at each samplinginterval on zinc test panels.

40

ISOLATE .........,;S;,;AMPL.I..N..G....P_E..R.-.IO..D....S.....P,;,;RE....S..E,;,;N.,;,T _Hours DaysNO.

1 3 6 18 1 2 3 5 10 15 20 30

53

••••••••••••10••••••••••••••••••••••••••••••....

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............................

123456789

10111213141516171819202122232425262728293031323334353637383940414243444546474849505152

Figure 12. Occurrence of various bacterial isolates at each samplinginterval on wood test panels.

SURFACE

Wood •Aluminum •Zinc API••iglass 0Sfeel 0

A

A

•

0

• •• • •

0 A •0 • • • A • •0 •

0 • 0 • 0 •0

0 •A

0

013 b 12 18 14 2 3 4 5 - 10 15 20 25 30 35 40

~Hours---l Days IIMMERSION TIME

VIIII...C..I

0VI-010loi 141- •...et-... 12"'0%10loial:W 10~.

"""....0

""0al:10loiaI~::;)

%

Figure 13. Number of bacterial isolates present at each test interval on the surfaceof each test material during a 40-day immersion period.

VI~

55

TABLE IV

DIVERSITY RATIOS*CALCULATED FROM SURFACE

BACTERIAL SUCCESSION PATTERNS

Diversity Ratios

Time

Plexiglass Aluminum Steel Zinc Wood

1 hour 13.95 10.02 8.10 7.70 5.303 hours 18.72 10.08 7.35 9.20 4.086 hours 8.85 10.07 4.94 8.72 3.98

18 hours 2.33 6.47 5.00 12.65 1.821 day 2.36 4.24 2.06 3.582 days 1.67 1. 79 3.00 1.59 1.083 days 1.77 0.91 1.46 1. 23 1.315 days 1. 29 0.89 1. 67 1. 22 1.37

10 days 0.84 1.34 1.35 1.25 1.1615 days 0.89 1.03 0.89 1.54 0.8620 days 0.81 1.53 0.81 1.37 1. 2030 days 0.51 0.71 0.17 0.39 0.8640 days 0.55 0.87 0.74 0.65 0.60

*From an original concept, D = S/log(N), by Gorden et al. (1969), whereD = Species Diversity, S = total numbers of species and N = totalnumbers of bacteria present in the same water sample. It is hereinapplied to represent the relationship between the numbers of differen­tiated isolates (S) and the total numbers of bacteria (N) per unitarea for each test material per time interval. Higher valuesrepresent greater numbers of species and/or fewer total numbers ofbacteria, while lower "D" values indicate fewer numbers of isolatesand/or greater total numbers of bacteria per unit of surface area.

No. ofIsolates

8

3 Ach.2 Pseu.2 Bac.1 Sar.

TotalDays

Present

7

GROUPS CITED

Ach. - AchromobacterFlav. - FlavobacteriumPseu.- PseudomonasMic.- MicrococcusBac. - Baci lIusSar. - SarcinaVib. - Vibrio

10

3 Ach.3 Vib.2 Bac.1 Pseu.1 Flav.

20

7 Flav.3 Ach.3 Pseu.3 Mic.1 Vib.1 Bac.1 Sar.

Figure 14. Number of days present by the various isolates recovered by swabbing methodson 1- 5 test surfaces. Note that only a few appeared to constitute the dominant surfacecolonizers, ~, only eight isolates colonized all five surfaces, while 20 were foundon only one surface for approximately one day on the average.

IJI0\

57

Figure 15. Typical microscopic fields of microorganisms removed fromvarious test surfaces by surface swabbing and prepared by theMillipore filtration technique as described in methods and materials.The sample fields are:

(A) Wood, darkfield, 40 days after immersion, showing diatoms, blue­green algae and refractile particles (bacteria?).

(B) Plexiglass, 5 days after immersion showing diatoms associatedwith large particles of extraneous matter. Light microscopy doesnot discern diatoms clearly due to low stain retention.

(C) Zin~ 20 days after immersion. One small algal fragment may bepresent.

(D) Steel, 10 days after immersion. The structure shown resembles afungal sporangium.

(E) Aluminum 7075, 10 days after immersion. Large, thick algalfronds are prominent.

58

(2) A significant decline in number of isolates (species?)

occurred on the test surfaces (i.e., in isolated ecosystems), which

coincided with stabilizing bacterial population levels.

In summary, the Millipore staining technique was ineffective

for morphological analysis of the microorganisms recovered from the

test surfaces by the surface swabbing technique. Furthermore, it was

impossible to determine total numbers of microorganisms using direct

microscopy on stained filter membranes. In part this was due to the

fact that spatial relationships between microorganisms were not

preserved since the attached material was removed non-specifically.

(iii) Teflon Membrane Technique Field Study

The purpose of these experiments was to determine whether

microorganisms could successfully attach to Teflon membranes and

whether a selective effect from underlying test surface would be

manifested through the membrane pores.

At I and 4 days following immersion, no conclusive differences

were observed among the bacterial and diatom populations (Table V) on

any of the test panels.

Active bacterial growth on the Teflon membrane surface was

strongly suggested by these microscope studies. Figures 10 and 17 show

bacterial colonies or chains of cells at day 4 on all materials except

the phosphor-bronze test panel. In contrast, there was little evidence

of diatom growth after 4 days of immersion on any panel.

This study showed that a number of morphologically distinct

microorganisms could attach to and develop on Teflon membranes in

59

marine waters. Since these microorganisms had firmly attached during

the first day on most surfaces, a biphasic surface layer was probably

present.

Aside from the obvious toxicity of the phosphor-bronze test

panels, the data do not confirm nutritional or galvanic responses by

the attaching bacteria.

As noted by Baier et al. (1968), the hydrophobicity of Teflon

poses a threat to potentially adsorbing microorganisms by prohibiting

a firm bond to the surface. However, these workers suggested that a

good attachment could be effected by intermediate chemical layers.

Proteins were believed to be most beneficial since their non-polar

moieties could align next to the Teflon surface. Polar groups on the

outside could then form hydrogen bonds either with the amino acids in

bacterial cell walls or with the enveloping bacterial slime layer.

The subsequent development of the attached microorganisms

(clearly shown at four days) depends upon the adsorption of organic

nutrients in quantities sufficient to promote growth. Subsequent

bacterial growth would then provide growth factors and assimilable

organic matter for the attaching diatoms.

The effect of metal ions diffusing through the Teflon membrane

pores was shown explicitly by the toxicity of the copper ions towards

bacteria. The diatoms seen on phosphor-bronze test panels appeared

consistently associated with fragments of detritus, indicating that

particulate matter may have served as a vehicle fv= incidental

attachment. Viability of these diatoms is subject to doubt. Baier

et ale (1968) and Marshall (1971), have previously noted that

60

Figure 16. Stained Teflon membrane preparations from certain materialsafter 1 and 4 days of immersion. Note sparcity of bacteria at day 1 onglass (G), steel (S) and aluminum 5052 (A), and the subsequent proli­feration at 4 days. Substantial numbers of diatoms are also present onglass and aluminum at 4 days, as opposed to steel. Large extraneousparticles are visible on aluminum at 4 days. All magnifications x 2000except (A-I), x 1300.

61

Figure 17. Stained Teflon membrane preparations from certain materialsafter 1 and 4 days of immersion. Note sparcity of bacteria at day 1 onzinc (Z), phosphor-bronze (B) and wood (W). Considerable bacterialdevelopment is obvious at 4 days on all but phosphor-bronze. DiatomsappeAr sparingly on all materials. Extraneous particles are prominenton all surfaces at 4 days, and to a lesser degree at day 1. Allmagnifications x 2000 except (Z-l), x 1300.

TABLE V

BACTERIAL AND DIATOM DENSITIES AT TWO SAMPLING INTERVALSON TEFLON MEMBRANES OVERLYING VARIOUS TEST SURFACES

BACTERIA / CM2* (x10-4) DIATOMS / CM2* (x10-4)

SURFACE Day 1 Day 4 Day 1 Day 4

Glass .95 ± .90 7.5 ± 2.0 .15 ± .18 2.8 ± 1.4

Al 5052 2.7 ± .90 5.7 ± 1.6 .25 ± .35 1.6 ± .95

Steel 2.9 ± 1.1 9.7 ± 2.1 0 0

Zinc 1.9 ± 1.0 5.6 ± 1.4 0 .20 ± .25

P-Bronze 0 1.8 ± 0.9 0 .50 ± .60

Wood 2.8 ± 1.0 8.0 ± 2.1 1.8 ± 1.3 .90 ± .80

*Based on a total of 10 microscopic fields counted.

(j\N

~

adsorption of microorganisms to surfaces does not necessarily predicate

viability.

Very few diatoms were also seen on the zinc test panel by four

days after immersion. This may be due to a specific sensitivity of

diatoms to this ion, since bacteria appeared unaffected, and evinced

active growth. The sparsity of diatoms in steel cannot be fully explained.

The Teflon membrane technique makes in situ observations on

chemically diverse surfaces feasible. Examination of the attached

microorganisms and associated particulate matter is facilitated by the

non-staining Teflon background resulting in a high degree of

resolution for all attached particles. This was especially evident

when lightly-staining particles of (apparently) inorganic matter were

observed (Figures 16 and 17, A-4, Z-4 and W-4). Considerable

crystalline material (primarily calcite) was found by Christie and

Floodgate (1966) to comprise a significant part of the primary film

forming in natural marine waters.

Nevertheless, this method minimizes contact with the test

surface per~. It is thus possible that many forms of sessile

bacteria, algae and protozoa could be adversely affected by the physical

nature of Teflon regardless of the biphasic adsorption layer.

In situ analyses of microorganisms attached to opaque materials

require an analytical method which retains the clear perspective of

Teflon, yet allows a complete response to the physical and chemical

effects of the test surface per ~.

(iv) Parlodion Filming Technique

The purpose of these experiments was to investigate the total

64number of bacteria, diatoms and extraneous particles adsorbing on

chemically-diverse test surfaces immersed for various periods in the

sea. A new assay method, the Parlodion filming technique, was used.

A chi-squarp. test of samples taken by the Parlodion filming

technique (as shown in Table VI showed that bacteria counts could be

considered random at the 0.05 significance level only during the first

10 days, diatoms during the first 24 days and extraneous particles

during the first 75 days. In addition, the 24-day diatom count was

itself significant, during the first as an individual chi-square test.

Representative microscopic fields from Parlodion film

preparations of all surface materials per each time interval are shown

in Figures 18 to 24. Considerable diversity in morphology and relative

numbers of diatoms and particulate matter at each sampling is easily

detectable. Bacteria are seen more clearly -under higher magnification,

as shown by Sechler and Gundersen (1971), and only occasionally

resolved in the figures at the magnifications shown.

Bacteria

Bacteria densities on all test panel surfaces are given for 120

days immersion in Figure 25. Values (with standard deviations) during

the first 10 days are presented in Table VII. Assuming that two

standard deviations from the mean account for 95% of the data, only

five scatter differences in bacterial deviates were seen on all test

surfaces. Except for these deviations all other values overlapped at

at least one common point.

65

The data in Figure 26 suggests that most sporadic increases in

surface population after 3 days immersion were due to active,proliferation rather than to subsequent attachment. The number of

bacteria per colony at day 1 also appeared to be inversely proportional

to the galvanic activity of the materials. For example, on Monel panels

moderately-sized microcolonies were present; on aluminum panels

virtually no active growth was observed.

Diatoms

Diatom densities on all materials during the 120 immersion test

panels are presented in Figure 27, while values (with standard

deviations) through 24 days are given in Table VIII. Using the two

standard deviation criteria for all counts during the first 24 days,

only eight isolated instances were found to be significantly different

from those on the other surfaces. In general, diatom populations on

phosphor-bronze and Monel remained relatively low during the entire

24-day immersion test period.

Figure 28 indicates that diatom proliferation was absent during

the first 5 days, and retarded as long as 45 days on phosphor-bronze

test panels. During the first 24 days, sporadic colonization occurred

on glass, stainless steel, and, to a lesser extent, on plexiglass test

panels.

Extraneous Particles

As indicated in Figure 29 there is great uniformity in numbers

of extraneous particle densities on all surfaces. Using two standard

deviations from the mean as the criterion (in Table IX, no surface has

a significantly different number of particles per unit area. Thus all

66

Figure 18. Micrographs of the primary film or slime layer formed onimmersed glass slides examined by the Parlodion filming technique. Theimmersion time in days is indicated for each preparation.' Allmagnifications x 1600.

67

Figure 19. P1exig1ass test panel. Microbiotic changes occurring as afunction of time of immersion, in days, from Par1odion mounts. Allmagnifications x 1600.

68

, I' ~:,~

Figure 20. Stainless Steel 304 test panel. Surface microbiotic changesoccurring as a function of time of immersion, in days, from Parlodionmounts. All magnifications x 1600.

69

Figure 21. Aluminum 5052 test panel. Surfacz microbiotic changesoccurring as a function of time of immersion, in days, from Parlodionmounts. All magnifications x 1600.

70

".".",'.4 '.<:.',

';i~i;::,;{{'

Figure 22. Monel test panel. Surface microbiotic changes occurring asa function of time of immersion, in days, from Par1odion mounts. Allmagnifications x 1600.

Figure 23. Phosphor-Bronze test panel. Surface microbiotic changesoccurring as a function of time of immersion, in days, from Parlodionmounts. All magnifications x 1600.

71

72

Figure 24. Some atypical or unusual observations on Parlodionmicroscopic mounts, including members of many phyla not quantitatedin these studies. These organisms observed on glass (G), plexiglass(P), stainless steel (S), aluminum (A), monel (M) and phosphor-bronze(B) test panels are presented mainly to illustrate the applicabilityof the Parlodion filming technique. See text for discussion. Allmagnifications x 1600.

TABLE VI

DETERMINATION OF THE RANDOMNESS OF MICROSCOPIC BACTERIAL COUNTS(CHI-SQUARE TESTS)

BACTERIA DIATOMS EXT. PARTICLES

Ave. Ave. X2 Ave. Ave. X2 Ave. Ave. x2

DAYS No. : No. : Total d = No. : No. : Total d No. : No. : Total d =(A) * (B) *. 4d2/N (A)* (B) ** 4d

2/N (A)* (B) ** 4d2/N

1 37 51 88 7 2.23 8 5 13 1.5 0.46 24 26 50 1 0.082 83 108 191 12.5 3.37 13 11 24 1.0 0.17 18 38 56 10 7.143 476 455 931 10.5 0.47 49 39 88 5.0 1.14 - - - - -8 - - - - - 461 415 876 23.0 2.41 84 93 177 4.5 0.46

10 1970 2088 4158 68.5 3.43 - - - - - 45 58 103 6.5 1.6413 (407) (475) (882) ( 34.0) (s.20) - - - - - 110 104 214 3.0 0.1716 - - - - - 519 561 1080 21.0 1.63 69 85 154 8.0 1.6624 - - - - - (263) (331) ( 594) (34.0) (7.78) 69 62 131 3.5 0.3775 - - - - - - - - - - 37 56 93 9 .. 5 3.88

120 - - - - - - - - - - (46) (23) (69) 2.5 (7.67)

Type Max. Values Type Max. Values Type Max. ValuesOf Accept. To 10 Of Accept. To 24 Of Accept. To 75

Chi-Square Value Days Chi-Square Value Days Chi-Square Value DaysTest (0.05) (0.05) Test (0.05) (0.05) Test (0.05) (0.05)

Total 9.49 9.40 Total 13.50 13.59 Total 15.51 15.40Pooled 3.84 3.51 Pooled 3.84 3.67 Pooled 3.84 4.45Hetero. 7.82 5.89 Hetero. 11. 30 9.92 Hetero. 14.07 10.95

*r1icroscopic fields selected in a statistically non-random fashion.**Microscopic fields selected using a Table of Random Numbers to locate coordinates.

-...JW

•• I t • •• •c

I A i "; ~ ~ 0I I 0iI ! i i• i

A •A

• • •• Glasso Plexiglass• Aluminumo Stainless Steel• MonelA Phosphor' Bronze

""""'" ,~# I,. I , I , I Lo 1 2 3 4 5 6 7 8 9 10 15 t 20 30 45 60 75 90 105" 120

8

I • • •• • •

1 t :L~:8~ 5 e~.:···U·_ I A

: A ~ ~....u-e:CD

o,..C)

o-'

DAYS OF IMMERSION

Figure 25.120 days of

Total surface bacterial populations on chemically-diverse materials duringimmersion. Consult text for discussion.

".I:-

TABLE VII

VARIATION OF BACTERIAL DENSITY ON IMMERSED TEST PANELSASSAYED BY THE PARLODION FILMING TECHNIQUE

Number of Bacteria on Test Materials / em2 (x104)

Day Glass P1exiglass St. Steel Aluminum Monel P-Bronze

1 0.74 ± 0.90 5.7 ± 8.4 1.6± 2.1 0.92 ± 0.96 7.1 ± 16.0 0.18± 0.66

2 1.7 ± 1.5 12.0 ± 10.0 6.9 ± 3.7 140.0 ± 47.0 1.8 ± 1.6 9.2 ± 4.2

3 9.5 ± 6.7 4.8 ± 2.1 140.0 ± 76.0 3.6 ± 1.5 3.6 ± 1.5 5.0 ± 3.6

4 32.0 ± 1.4 12.0 ± 9.4 21.0 ± 20.0 87.0 ± 92.0 19.0 ± 15.0 9.5 ± 6.6

5 15.0 ± 8.1 65.0 ± 17.0 37.0 ± 22.0 1200.0 ± 610.0 78.0 ± 56.0 4.8 ± 1.9

6 19.0 ±10.0 24.0 ± 11. 0 24.0 ± 11.0 280.0 ± 530.0 400.0 ± 430.0 36.0 ± 16.0

7 24.0 ± 8.5 54.0 ± 2.6 23.0 ± 5.8 26.0 ± 6.0 100.0 ± 41.0 22.0 ± 9.2

8 24.0 ± 7.1 38.0 ± 20.0 25.0 ± 1.1 13.0 ± 9.9 20.0 ± 13.0 25.0 ± 20.0

9 56.0 ±13.0 27.0 ± 14.0 16.0 ± 5.0 920.0 ± 530.0 150.0 ± 80.0 37.0 ± 29.0

10 39.0 ±14.0 210.0 ± 74.0 120.0 ± 46.0 300.0 ± 110.0 110.0 ± 470.0 100.0 ± 47.0

-...J1I1

(275)

•(8.9)

(13.5)(6.5)(8.8) •• • •5

>- • GlassZ o Plexiglass0 • • Aluminum... o Stainless Steel0 • MonelU 4 6 Phosphor- Bronze

"CGI::I.&' I •to- • 0Uc(

3m - 0• •d I 0 • •Z• 0 •LA.!

2~ • 0 e~ • • •• 6

0 ~ • i 0 0• • • 0 • 0 •0• ~ 6 ~ r• I • L0 5 10 15 20 30 45

DAYS OF IMMERSION

Figure 26. Bacterial developmental pattern on various materials. Counts are based ontotal numbers present assayed by the Parlodion filming technique (see Methods andMaterials).

~

0\

7

6I

A A

0 8 i , 6

!A A ~A 0 • !5 ~ . iN AtI§ 006 i A

I • 01: • • • • • .' •u Ii • • • • 6

.... 4 • i 0 • • A•III o •1: 6

0 • 0 • 6... 3 • 6 IIce 6 6 6Q

60 2 AGlass.,.

o PlexivlassC) • AlumInum0 o Stainless Steel...1 • Monel

A Phosphor. Bronze

.u...J J, J, I # I Io 1 234 567 8 9 10 15 20 30 45 60 75 90 105 120

DAYS OF IMMERSION

Figure 27. Total surface diatom populations on test panels during 120 days of immersion.Consult text for discussion.

-..J-..J

TABLE VIII

VARIATION OF DIATOM DENSITY ON IMMERSED TEST PANELSASSAYED BY THE PARLODION FILMING TECHNIQUE

2 4Number of Diatoms on Test Materials / Cm (x10)

Glass P1exig1ass St. Steel Aluminum Monel P.Bronze

Day

1 0.16 ± 0.87 0.04 ± 0.15 0.12 ± 0.15 0.02 ± 0.10 0.12 ± 0.29 0.02 ± 0

2 0.26 ± 0.5 2.2 ± 8.1 0.02 ± 0.11 0.22 ± 0.40 1.8 ± 0.81,

3 0.98 ± 0.87 0.28 ± 0.78 - 0.10 ± 0.28 0.08 ± 0.21

4 5.7 ± 1.6 1.0 ± 0.93 2.7 ± 1.3 0.80 ± 1. 7 0.62 ± 0.72 0.46 ± 1.1

5 5.5 ± 2.7 4.8 ± 1.8 1.1 ± 1.3 2.6 ± 1.3 0.58 ± 0.59

6 4.4 ± 1.6 4.4 ± 1.3 3.9 ± 1.4 5.4 ± 3.9 2.1 ± 1.6 0.06 ± 0.31

7 9.5 ± 3.2 3.7 ± 1.3 5.4 ± 2.8 5.1 ± 1.8 8.1 ± 4.1 0.02 ± 0.1

8 9.2 ± 3.0 3.7 ±20.0 6.8 ± 2.8 1.1 ± 1.0 0.84 ± 0.79 0.04 ± 0.20

9 11.0 ± 5.7 5.0 ± 1. 7 9.6 ± 3.5 2.2 ± 1.3 0.20 ± 0.39 0.10 ± 0.38

10 7.5 ± 3.0 6.2 ± 2.1 6.3 ± 1.8 3.4 ± 1.8 0.08 ± 0.3

13 9.3 ± 2.2 12.0 ± 3.7 7.2 ± 2.3 1.6 ± 0.9 0.8 ± 1.1 0.40 ± 0.67

14 7.5 ± 3.5 10.0 ± 3.0 14.0 ± 4.5 6.9 ±3.6 2.0 ± 0.2 0.14 ± 0.33

16 10.0 ± 3.8 6.9 ± 2.1 7.0 ± 3.7 4.6 ± 2.1 1.1 ± 1.3 0.04 ± 0.15

24 5.3 ± 3.7 5.9 ± 2.1 9.1 ± 3.1 3.8 ± 2.5 5.3 ± 2.0 3.1 ± 7.8

-..J00

(8.0)•

5l-• • Glass

o Plexiglass

>- I• Aluminumo Stainless Steel

Z • Monel0 6 Phosphor-Bronze.... 40u-11'I

~ I 0 •0. ~

3t-~ 0 •Q •• •d

~~0 6

6Z 0 6

..... 0 • 80

~ • 0

• •0 • •• • •• • • 0 00

00.00 • a • •d 6 , • 0 • • j • • 6

0

0 5.. -- •• 12010 15 20 30 45 60 75 90 105

DAYS OF IMMERSION

Figure 28. Diatom development on immersed test panels. Counts are based on total numberpresent per colony assayed by the Parlodion filming technique.

"-.J\0

5C'l

I I I~ lillRo8 :' I I I i ~ iu.... 4 : • i ! I b. AI/) A A

"" o •• 0 0 ••-J o A b.U 3 b. b....

A GlassII:

< oPlexiglassQ. • Aluminum

2 o Stainless Steel0 • Monel...

b. Phosphor-BronzeC)

0 1-J

I I I I I I I I Io 1 234567 8 9 10 15 .- 20- 30- 45 60 75 90 105

DAYS OF IMMERSION

Figure 29. Extraneous particles deposited on test panels during 120 days immersion.Consult text for discussion.

00o

TABLE IX

VARIATIONS IN NUMBERS OF EXTRANEOUS PARTICLES ON CERXAIN MATERIALSAFTER IMMERSION AS ASSAYED BY THE PARLODION FILMING TECHNIQUE

2 4Nwnber of Particles on Test Materials / an (x10)

Glass P1exig1ass St. Steel A1wninum Monel P-BronzeDay

1 0.48 ± 0.36 0.12 ± 0.26 0.30 ± 0.10 0.46 ± 0.52 0.30 ± 0.35 0.02 ± 0

2 0.34 ± 0.37 0.16 ± 0.24 0.36 ± 0.24 1.1 ± 0.8 0.66 ± 0.59

3 0.56 ± 0.51 0.46 ± 0.33 - 0.30 ± 0.45 0.62 ± 0.53

4 0.92 ± 0.82 1.0 ± 0.93 1.2 ± 0.8 0.28 ± 0.40 1.2 ± 0.68 0.46 ± 1.1

5 0.80 ± 0.60 1.6 ± 0.93 0.36 ± 0.40 2.6 ± 1.3 1.6 ± 0.79

6 1.9 ± 1.1 1.9 ± 0.84 0.16 ± 0.28 1.7 ± 1.1 2.1 ± 1.0 0.16 ± 0.30

7 1.8 ± 0.82 2.3 ± 0.81 2.1 ± 0.91 1.5 ± 0.81 2.4 ± 1.6 0.20 ± 0.32

8 1.7 ± 0.54 1.4 ± 0.84 1.4 ± 1.1 1.9 ± 0.69 2.8 ± 1.3 0.10 ± 0.25

9 0.96 ± 0.45 1.7 ± 0.82 1.1 ± 0.48 0.76 ± 0.63 0.26 ± 0.32 0.30 ± 0.35

10 0.90 ± 0.50 2.4 ± 1.1 1.9 ± 0.98 0.84 ± 0.50 0.34 ± 0.50 0.50 ± 0.63

13 2.2 ± 0.59 2.4 ± 0.10 3.0 ± 1.0 0.80 ± 0.55 0.60 ± 0.60 0.10 ± 0.20

14 2.2 ± 0.9 1.8 ± 0.5 1.7 ± 0.7 1.2 ± 0.6 0.86 ± 0.69 1.4 ± 0.62

16 1.4 ± 0.6 2.7 ± 0.7 1.4 ± 0.8 2.5 ± 0.9 1.9 ± 0.9 3.0 ± 0.924 1.4 ± 0.6 3.5 ± 1.6 1.6 ± 1.0 1.3 ± 0.5 4.1 ± 1.2 2.5 ± 0.9

30 1.7 ± 0.5 2.6 ± 0.8 2.3 ± 1.3 1.6 ± 0.8 2.1 ± 1.0 1.1 ± 1.0

45 4.6 ± 1.5 4.6 ± 1.3 2.3 ± 1.0 1.6 ± 0.8 2.2 ± 1.4 1.6 ± 1.1

60 2.3 ± 1.2 3.8 ± 2.1 3.8 ± 1.6 3.1 ± 1.0 5.0 ± 1.4 2.2 ± 1.4 00t-'

75 0.74 ± 0.58 2.7 ± 1.1 2.3 ± 0.7 1.4 ± 0.7 2.3 ± 1.0 1.6 ± 0.7

82

test panel surfaces responded in a statistically homogeneous manner,

regardless of chemical composition.

The significance of the Parlodion filming technique is suggested

by the following analysis.

Few workers have investigated surface microcosms except on glass

slides because of the requirement of light microscopy for transparent

substrate. In a review of methodology for studying surface microcosms,

Cooke (1956) notes that microorganisms on the semi-transparent leaves

of Elodea have been observed directly. Meadows and Anderson (1966 and

1968) have also successfully examined the microflora resident on sand

grains by direct microscopy.

Margalef (1948) briefly reported a plastic-layering technique

based on a suggestion originally made in 1901. Although no quantitative

or illustrative data were presented, Margalef recognized bacteria,

fungi, diatoms, green and blue-green algae which originally had been

constituents of natural epilithic communities.r .;

Although similar in concept to the method described by Margalef

(1948), the Parlodion filming technique used here is more developed and

applicable to most opaque surfaces. Laboratory tests optimizing reagent

concentrations, timing, and specimen preparation have eliminated most

common sources of error.

The Validity of Direct Counting Methods

Several workers have reported total counts of microorganisms on

glass and transparent plastic materials immersed in the sea. None,

however, have presented conclusive statistical evidence to justify their

data. The greatest difficulty is that the distribution apparently is

83

uneven. According to Brock (1971), random distribution in natural

populations is rare; a species more frequently is patchy or clumped.

Marshall et al. (197lb) believe this is due primarily to specific areas

on the test panel surface which have been modified electrochemically

and nutritionally because of adsorption of organic and inorganic

matter. This makes it necessary not only to count a large number of

microscopic fields, but also to make sure that the fields selected for

counts are completely random. Brock (1971) showed the importance of

field size in making microscopic counts: randomness increases with

increase in field area. Thus, higher magnifications necessarily show

more non-randomness in microbial counts than lower magnification.

Skepticism of direct counting methods therefore appears to be justified.

The Woods Hole report (1952) presents only a simple graphical

representation of bacterial and diatom numbers on a test surface during

30 days of immersion. In a 48-hour study of bacterial population

development on immersed surfaces, Bott and Brock (1970) used the average

numbers obtained by counting 13 fields parallel to the short axis of the

slide at 8 evenly spaced intervals. The 13 fields selected could

therefore have been biased. O'Neill and Wilcox (1971) and Skerman

(1956) presented data on numbers of microorganisms obtained by a direct

counting method based on a circular smear made from a bacterial suspen­

sion. Marshall et al. (197la,b) report counting at least 10 "random"

fields routinely to obtain an average of the bacteria densities on glass

slides. In fact no standard method exists for direct determination of

bacterial densities. Important in this context is the recent statement

by Lorenzen (1970): "Statistical procedures operate on the average, and

84

one must realize that unusual events are easily missed when using

statistical averages."

Other workers have given no statistical data to accompany their

results on bacterial surface densities; only simple numerical averages

were presented. It is highly probable therefore, that their statistics

are not applicable since Brock (1971) has shown that the distribution

of organisms on a surface immersed in natural waters is not random.

High variation in bacterial densities are expected between microscopic

fields in the same preparation, especially when the fields are rela­

tively small. Similar conclusions were expressed earlier by Meadows

and Anderson (1966 and 1968) in their analyses of distribution of

microorganisms on sand grains recovered from natural waters. These

workers found bacteria, yeasts, diatoms, blue-green algae and early

stages of brown algae in localized patches rather than evenly

distributed over the surfaces. Brock (1971) supports this view by

noting that microbial niches are microscopic in nature.

Chi-Square Tests

Thus, as previously noted, it was found that purely random

counts of bacteria, diatoms and particulate matter in the Parlodion

preparations were not substantiated by chi-square methods past 10, 24

and 75 days respectively.

The most probable reason for bacterial density discrepancies

between the two counting methods for any time past 10 days was

obstruction by diatoms and overlying particulate matter. Using a

counting method in which microscopic coordinates were not located by

a Table of Random Numbers, interpolative estimates for bacterial

85

densities in partially-obstructed fields were made.

When diatom populations and extraneous particulate matter became

prominent, differences in bacterial counts between the two methods

(surface swabbing vs. Parlodion filming) became significant, as shown

by the chi-square tests. O'Neill and Wilcox (1971) similarly found

bacterial and diatom density measurements unfeasible after 8 days

immersion due to extremely high densities and too many extraneous

particles. Skerman (1956) found bacterial densities too high to count

conveniently after two days.

Diatom density measurements remained within the 0.05 confidence

level longer (24 days) than did bacterial measurements (10 days); this

was attributed to the ease with which diatoms are recognized in

microscopic fields. Only when massive colonies were encountered was

accurate counting difficult. Significant chi-square differences

between the two counting methods most probably arose after 24 days

because of an encounter with a large colony.

Due to the relatively low densities of extraneous, non-cellular,

particles present throughout the entire l20-day immersion interval,

randomness in counting was confirmed during the first 75 days.

In general, standard deviations calculated within each random

interval became less significant with respect to the mean as time of

immersion increased. This apparently is due to the greater number and

more even distribution of organisms and acellular particles over the

surfaces during the more extended periods.

Bacterial Densities

The experiments described here generally confirm other data based

86

on experiments using glass and transparent plastics: bacterial

densities show a steep logarithmic increase during the first 24-48

hours following immersion (Woods Hole report, 1952; Skerman, 1952;

Bott and Brock, 1970;and O'Neill and Wilcox, 1971).

to maximal population density of 106 - 107 organisms

A gradual increase

2per cm follows

within 2-10 days. After maximal densities are reached, the populations

show a tendency to decrease slightly, then remain stable thereafter.

Brock (1971) notes that a stable population does not necessarily

indicate a "steady state" in which growth is not occurring. He defines

a "steady state" in ecological terms, whereby cells are lost from the

population at the same rate at which they are added: there is no change

in population size even though cell division takes place.

According to Brock (1971), "microbial population" in the

calculation of growth rates is defined not as the cells of a single

colony or pure culture, but as the bacteria which compose a single

population in terms of location. Bott and Brock (1970) used this latter

method when identification of microcolonies on the basis of

morphological characteristics· was impossible.

In studies to determine the extent of in situ surface bacterial

growth, Bott and Brock (1970) found that the majority of organisms

appearing in surface populations arise from previously-attached cells

by active proliferation. They noted that growth (in terms of total

population) initially was exponential. After a colony reached a certain

size, however, growth ceased and the bacteria became less refractile

(indicating lysis). Subsequent attachment or migration of

microorganisms was found insignificant .as a contributing factor to the

87

total density after the exponential growth had begun.

Bott and Brock (1970) found that some colonies never developed

beyond the 2-4 cell stage, while others formed larger aggregates, after

which most of the cells lysed. Still other colonies "spread out" after

reaching the 16- to 32-cell stage, making identification of the colony

difficult. Only a few colonies developed to a large size (defined as at

least 128 cells).

From in situ studies on glass, Bott and Brock (1970) postulated

that many organisms which initiate colony formation are not well adapted

to a sessile existence. These bacteria presumably die off after

utilizing the adsorbed organic material in their immediate environment.

Marshall et al. (197lb) note that "swarmer" colonies of sessile bacteria

are common on immersed surfaces. Very few varieties were well-adapted

to permanent attachment using the criterion of "irreversible"

adsorption.

As indicated in Figure 26 most colonies on the test panels used

in my experiments averaged less than four individuals per colony on all

materials. In only one instance (on aluminum at 5 days) did the size

of the average colony become relatively large.

The importance of galvanic activity and relative electronegativity

of a test surface is also shown by the results in Figure 26. By day 1

following immersion bacteria have not only attached to Monel, but show

signs of active growth. On aluminum, however, the relatively high

electronegativity at the surface apparently repelled microorganisms

significantly. At the same time, it is likely that large amounts of

organic matter gradually were adsorbed to the aluminum surface,

88

eventually minimizing the electrostatic potential. Bacteria were then

able able to attach either passively (due to the absence of

electrostatic repulsion), or to be attracted chemotactically. This is

shown by the high bacterial densities on the aluminum test panel seen

after day 1.

These studies also showed that bacterial attachment was

negatively affected by surface chemical toxicity, as well as the relative

galvanic activity. In Figure 25 and Table VII the number of bacteria

adsorbed to phosphor-bronze are comparable to most of the non-toxic

metals. Figure 26, however, shows that little bacterial growth occurs

during the first 8 days (disregarding the slight increase at 24 hours).

This suggests that the attached bacteria were probably non-viable or

physiologically inhibited. This is supported by the fact that most

bacteria observed on the phosphor-bronze test panels during the first

two weeks compared to those adsorbed to the other test panel surfaces.

By 90 days, however, the phosphor-bronze surface was coated with

a thick, porous, green~hite salt which has provided suitable

insulation to protect attaching bacteria from the toxic but slowly

dissolving cuprous ions. A similar proposal about the insulating

effects of copper surface coating was expressed previously in the Woods

Hole report (1952).

Diatom Densities

Using the criterion of two standard deviations from the mean

(Table VIII) diatom populations were statistically similar on all test

panel surfaces during the first 5 days. From 6-16 days immersion time,

surface population differences were observed in isolated cases on

89

glass, plexiglass and to a lesser extent, stainless steel test panels.

These data contrast with bacterial density measurements made on

the same test panel surfaces, which indicated that the highest

populations were present on the most galvanically-active surface

(aluminum). The reason for this presumably lies in the ability of the

test material to adsorb bacterial nutrients. Conversely, diatoms attach

equally well on all but the toxic phosphor-bronze surfaces. Subsequent

bacterial growth probably influences diatom populations by limiting

available growth factors and easily assimilated organic matter.

The decreasing diatom densities on the phosphor-bronze test

panels observed after 4 days immersion indicate toxicity and possible

lysis of the organisms initially attaching. These results conflict with

those of Wood and Allen (1958), who report that diatoms have a

characteristically high resistance to copper and copper salts.

Extraneous Particle Densities

The distribution of extraneous particulate matter on the test

panel surfaces was shown to be random. This is in contrast to the

adsorption of microorganisms, which was nonrandom (presumably due to

asymetric growth). The homogeneous particle densities on all test

materials indicated that galvanic or other direct chemical influences

were not able to cause differences. This inadsorption also implies that

bacterial and diatom growth on galvanically-active metals were due

primarily to the selective adsorption of dissolved organic matter rather

than extraneous particles.

90

Some Observations on Other Microorganisms Found in Primary Films

Although only bacterial, diatom and particulate matter densities

were determined during this study, Figures 18-24 show a diversity of

other microorganisms common in these surface microcommunities.

Fungal hypae appear as dark, narrow, thread-like bands of

constant diameter. Occasionally, a distinctive reproductive structure

made fungal identification possible: Figure 18 at 10 days immersion

shows a Cladosporium sp. and in Figure 24 M-9, a Mucor sp. is

identifiable. Also in Figure 24, B-14 and 16, a Geotrichium sp. is

present, characterized by thick arthrospores. The same fungus is also

shown, but less clearly in Figure 23 at 8 and 45 days immersion. This

organism was found to infest the phosphor-bronze panels, with growth

indicated as early as 6 days following immersion. By 120 days only

isolated openings in the mycelia could be found. This observation

confirms the "fine, filamentous algae" reported by Edmondson and Ingraham

(1939) as the sole colonizer of suspended copper panels in Kaneohe Bay.

Also in Figure 24 at day 2, streptomycetes are seen as extremely

fine, barely perceptible, filamentous organisms with elongated terminal

spores. These organisms were observed frequently in phosphor-bronze

preparations during the first 14 days following immersion.

It was difficult to distinguish yeasts from the algae and other

microorganisms except in isolated instances. A budding yeast is

apparent in Figure 17 in an open space in cuprous exfoliation; and a

yeast colony is present in Figure 24 S-2.

Other organisms identifiable in Figure 24 are Chaetoceros sp.

(P-9), a centric diatom with filamentous appendages; a triradiate sponge

91

spicule (G-16); rotifers (A-12); the peritrichous flagellate,

Vorticella (M-lO); worm-like larvae or adult forms of unidentifiable

nematodes and annelids (B-16, A-45 and S-120); and a foraminiferan

(A-7). The anterior portion of Saggita, the Arrow Worm (M-1, S-7) was

also seen; in addition to a copepod with its body bent at a right angle

(S-90) and the Tardigrade "Water Bear", Echiniscus (P-13).

Diatom genera identifiable in this study were: Nitzschia

(usually the earliest diatom representative appearing within 1-3 days

after immersion). Navicula, Skeletonema, Cyclotella, Gyrosigma,

Lyncmorpha, Bacillaria, Astrionella. Amphora, Fragilaria, Chaetoceros,

Grammatophora, Cocconeis and Melosira. There appeared to be no

preferences by these genera for any specific test material.

The only common identifiable blue-green alga was Arthrospira sp.

although another unidentified species was found with high frequency.

Ulothrix sp. was the only green alga identified in the preparations.

Invertebrates were found attached to panels during the latter

part of the immersion period. These included the barnacles Balanus

amphitrite and B. eburneus. oysters (either Ostrea or Crassostrea sp.)

hydroids and ascidians and at least two genera of bryozoans.

92IV

DISCUSSION

Three separate techniques were used to investigate the develop­

ment of microorganisms in primary films found on various materials

immersed in marine waters. These techniques included microbial viable

counts by the traditional swabbing method, and total visible counts by

two new direct microscopic observation techniques developed during this

study: the Teflon overlay technique and the Parlodion filming

technique. A variety of test materials were investigated, including

glass, plexiglass, wood, zinc, stainless steel, steel, Monel, aluminum,

and phosphor-bronze.

Surface Swabbing Techniques

In the studies based on classical swabbing techniques, only

bacteria were enumerated and (tentatively) classified at the generic

level, including 52 differentiated isolates. The materials used were

plexiglass, ,aluminum 7075, steel, zinc and wood. These were immersed

at the test site during intervals ranging from 1 hour to 40 days.

Although similar varieties of aerobic heterotrophic bacteria

were found on all surfaces regardless of their chemical nature, viable

population levels were characteristic of each test material during the

first few days following immersion.

The most polar material, wood, accumulated the greatest number

of bacteria in the shortest period of time (within 3 days). The maximal

number on plexiglass did not appear until 10 days after immersion. The

maximal number on the metal panels was not observed until 20-30 days

93

after immersion.

The most rapid increase in attachment and colonization on all

materials occurred between 6 hours and 2 days. A rapid decrease in the

number of bacterial isolates consistently occurred after 18-24 hours;

roughly 40% remained for a period of several weeks.

Nonmetal panels exhibited a more complex response with regard to

the number of isolates present in each sampling interval. These

results showed that a higher percentage of the organisms which attached

initially remained to form the bulk of the stable populations. This

indicates that a greater proportion of the initial population was well­

adapted for sessile growth. In addition, viable populations tended to

stabilize on all materials between 5-10 days following immersion.

The Diversity Ratios for all test panels became very similar at

the termination of the 40-day immersion period. This indicated an

increasing tendency toward microbial homogeneity and suggested that the

surface chemical effects present at the time of immersion (see Appendix

1) were minimized. Thus after the initial colonization by

microorganisms, and formation of the primary film, the function of the

surface is to provide foothold for sessile organisms with little direct

chemical influence from the original test material.

Direct light microscopy of stained Millipore membranes was

ineffective for determining the number of bacteria per cm2 on the test

surface. The total counts obtained from opaque surfaces in this manner

did not correlate well with the viable bacterial counts obtained by

cultural methods.

94

Teflon Membrane Overlay Methods

Bacteria were found to attach to Teflon membranes by 1 day and

to proliferate in situ by at least 4 days. The status of diatoms in

this respect is in doubt. No qualitative and quantitative differences

in adsorption of bacteria and diatoms to Teflon membranes overlying

glass, steel, aluminum 5052, zinc and wood were observed. The phosphor-

bronze panel, composed of an alloy which slowly released copper at its

surface, showed a general toxicity toward all microorganisms.

Diffusion of ferrous, aluminum and cuprous ions from metal

plates immersed in sea water through the Teflon membrane pores was

demonstrated by qualitative chemical methods. This suggested that the

electrochemical and toxic effects from the underlying test materials

were present on the Teflon membrane outer surface.

Parlodion Filming Techniques

The most extensive studies were performed using the Parlodion

filming technique. The greatest advantage of this technique is its

ready application to opaque materials, making possible precise

qualitative and quantitative observations of the microorganisms

attached to the opaque surfaces.

Using this method, it was found that the number of bacteria per

2cm of test panel could be accurately determined within the first 10

days after immersion. Thereafter, it became increasingly difficult to

recognize all the organisms in each microscopic field. Diatom counts

were accurate for 24 days, while extraneous particle counts were valid

at least 75 days.

95

Within these intervals few differences in the total number of

bacteria per unit area could be confirmed statistically between the

various test materials. The reason for this apparently was the uneven

bacterial distribution, a condition which became increasingly marked

during the first 30 days. The high variability between the microscopic

fields tended to mask possible biological differences among the test

panels. Few instances were seen in which comparative surface population

levels were confirmed (using a variability criterion of two standard

deviations). No basic differences in attachment or developmental

patterns due to surface chemical composition were observed.

Within the first few days the primary films containing bacteria

were stabilized with only a few prominent isolates present on each test

panel. Apparently these were well-adapted sessile forms which

proliferated to the maximal level allowed by local growth conditions.

Total microorganism populations generally stabilized after 2 or 3 days,

but further sporadic proliferation appeared periodically.

No stable diatom population was recognized on any test surface

until 6 days after immersion, indicating that bacterial growth may

prepare the test surface for the development of unicellular algae. The

more chemically passive test panels (glass, p1exiglass, and stainless

steel) consistently exhibited the highest diatom population level up to

16 days after im~ersion.

The lag in diatom development following bacterial colonization

of immersed surfaces previously was reported by Scheer (1945), ZoBel1

(1946), the Woods Hole report (1952), Aleem (1957), O'Neill and Wilcox

(1971) and others. O'Neill and Wilcox noted that homogeneous

96

populations of the same diatom species occurred unpredictably at

intervals on immersed glass slides. Similar observations were made

during the current study for the other five test materials.

These studies suggest that sessile marine microorganisms adsorb

to and colonize at certain unique sites on immersed surfaces. The

variability about these local colonization centers depends not only

upon the physiological nature of the adsorbing organism but also upon

the amount, distribution and nature of the nutrient substrate.

Thus, the chemical composition of an immersed surface appears to

influence the total number of and the varieties of bacteria and diatoms

that eventually attach. These attachment differences appear to lie in

the polarity and relative e1ectronegativity of the surface of the test

material. Increasing polarity is important to induce attachment. The

e1ectronegativity on the test panel surface is important directly (by

repelling the negatively charged bacteria) and indirectly (by the

adsorption of organic and inorganic material). These substances may

either attract microorganisms (chemotactically) or may provide polar

sites for attachment.

A general observation resulting from these studies is the marked

resemblance between the establishment of bacterial ecosystems in

primary films and the establishment of macroecosystems by higher

organisms. As with multicellular plants and animals, the formation of

stable ecosystems involves the loss of most of the original members-­

leaving a specialized few which successfully occupy an ecological niche.

Survival within any ecosystem depends on a proliferation rate

equal to (or greater than) the losses incurred through natural death

97and predation. In addition stable ecosystems are balanced so that the

components (i.e., populations) contain a fixed optimal number of

organisms per unit area or volume. Thus, ecosystems are dynamic in

scope, with each representative population exhibiting its own pattern

and rate of growth. When a foreign organism invades a stable ecosystem,

its ability as a competitor is tested against the established

population.

Data illustrating these characteristics for the microorganisms

found in primary films have been presented in the above studies. In

addition, these slime layer microecosystems are similar irrespective of

the chemical composition of the test panel.

Since these bacterial ecosystems resemble higher order

ecosystems, factors such as the relative time interval required for

colony formation measured in this thesis may become important to the

ecologist. A microecosystem is established within 5-10 days,

appreciably shortening the interval characteristic of higher organisms

and facilitating the study of the microecosystem.

In this thesis, therefore, I have characterized the development

of microorganisms in primary films (slime layers) formed on immersed

chemically diverse materials. This represents an extension of previous

description studies carried out on immersed glass slides. An~

technique, the Parlodion filming technique, was introduced to simplify

the observation of in situ developmental characteristics. The results

obtained for a wide variety of test materials significa~tly contribute

to our knowledge of the microbiology of primary films.

98

APPENDIX 1

CORROSION THEORY AND MATERIALS DETERIORATION

Corrosion is a state of gradual "wasting away" in metals, while

deterioration is a term normally used to describe the dissolution or

natural destruction of nonmetals •

.. __ Metals

When metals are immersed in water, cations emigrate from the

surface leaving free electrons and a negative surface potential (LaQue,

1969; West, 1965). The relative strength of the resulting electrical

potential may vary considerably, and is a function of the rate at which

metal ions leave. The potential developed may be determined by the

following factors:

1. Relative chemical activity of the metal in sea water, or its

position in the galvanic series

A brief galvanic series beginning with the most active metal is

(Tuthill and Schi11mo11er, 1969; Uhlig, 1963): Mg, Zn, Al, Fe, brasses

(Cu-Zn), Cu, bronzes (Cu-Sn), Ni, stainless steels (Fe-Cr-Ni), Monel

(Ni-Cu) and Pt (essentially inactive galvanically).

2. Oxygen concentration of the water

Oxygen dissolved in the water may significantly affect the

corrosion of various metals and alloys in different ways. On an iron

surface, oxygen is responsible for the recurrent formation of surface

rust (i.e., Fe203.x H20) while the scale periodically sloughs off from

its own weight. Conversely, oxygen forms a stable, protective metallic

oxide "barrier", 2.5 - 10 }.1m thick (Uhlig, 1963) on the surfaces of such

99

metals as stainless steel (Cr20

3,NiO), Monel (NiO) and aluminum

(Al20

3). The flow of water is important in maintaining a constant

supply of dissolved oxygen to the submerged surfaces. This is highly

influential in modifying the corrosion rate.

3. Ion concentration and chemical composition of sea water

A salinity (i.e .• the concentration of all dissolved salts) of

approximately 35 0/00 allows almost optimal electrical conductivity in

water (Uhlig. 1963). According to Harvey (1957), the major

constituents of sea water with a salinity of 35 0/00 are:

19.270

10.714

2.696

1.293

0.406

0.386

% of Total

55.05

30.61

3.69

1.16

1.10

Br

Org. C

Others

0.126

0.064

0.026

0.010

0.004

0.005

% of Total

0.36

0.18

0.07

0.03

0.01

0.04

Cl- is most important in terms of metallic corrosion. Its action is

to chemically break down or prevent the formation of protective oxide

layers (Uhlig, 1963).

Hydrogen ion concentration (pH) of surface sea water is

relatively stable in a given geographical location. pH range is

approximately 8.1 to 8.3, and at these values it is in equilibrium

with the CO2 in the atmosphere (Sverdrup ~ a1., 1942). Corrosion of

metals is virtually independent of pH in sea water in the range of

4-10 (Uhlig, 1963).

100

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