Gary Evans, 1939- ON TIIE MICROBIOLOGY OF …...73-5276 SECHLER, Gary Evans, 1939- ON TIIE...
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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
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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
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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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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
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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
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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
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FIGURES (CONTINUED)ix
PAGE
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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
10111213141516171819202122232425262728
~53132333435363738394041424344
ig474849505152
•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••
••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••..........
•••••••••••••••••••••••••••• 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 ..
••••••••••••••••••••••••••••••••••••• ••••
••••••••••••••••••••••••••••••••
.....I••••••••••••••••••••••••••••••••••••••••
.....
123456789
10111213141516171819202122232425262728293031323334353637383940414243444546474849505152
.... ..............
....•••••••••••••••••••••••••••••• ••••••••
........
••••••••••••••••••••••••••...................••••••••••••••••
...........
....
....
............
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
•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••
....•••••••••••••••••••••••••••••••••••••••••••••
•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••
........................................••••••••••••••••••••••••••••••••••••••••••
.......
.......
....
••••••••••••••••••
,........
.......n ..........................1
.....................
....
..................................
...
•••••••••••••••••••...............................................
••••••••••••••••••••••
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••••••••••••••••••••••••••••••....
•••••••••••••••••••••••
....
....
••••••••••••••••••••.......
.......
................................................................................
...................................
........................
...................
••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• •••••••••••••••••••••••• ••••
............................
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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