MICROBIAL BIOFILM

By:
Name : Vio Indah Budiarti
SID : B1B015009
Section : II
Group :1
Assistant : Mufti Rahayu

PRACTICAL REPORT OF MICROBIOLOGY ENVIRONMENT

MINISTRY OF RESEARCH, TECHNOLOGY AND HIGHER EDUCATION

JENDRAL SOEDIRMAN UNIVERSITY
FACULTY OF BIOLOGY
PURWOKERTO
2017
 I. INTRODUCTION

A. Background

A biofilm is an assemblage of microbial cells that is irreversibly associated (not

removed by gentle rinsing) with a surface and enclosed in a matrix of primarily
polysaccharide material. Noncellular materials such as mineral crystals, corrosion
particles, clay or silt particles, or blood components, depending on the environment
in which the biofilm has developed, may also be found in the biofilm matrix.
Biofilm-associated organisms also differ from their planktonic (freely suspended)
counterparts with respect to the genes that are transcribed. Biofilms may form on a
wide variety of surfaces, including living tissues, indwelling medical devices,
industrial or potable water system piping, or natural aquatic systems. The variable
nature of biofilms can be illustrated from scanning electron micrographs of biofilms
from an industrial water system and a medical device, respectively (Donlan, 2002)
Biolms are viscoelastic materials comprising bacteria and secreted
macromolecules. By embedding themselves into a biopolymer matrix, the bacteria
are protected from harsh environmental conditions. Biolms formed by the model
bacterium Bacillus subtilis resist liquid wetting up to 80% ethanol,26 a mechanism
which severely limits its antibacterial efciency. The amphiphilic protein Bacillus
surface layer protein A (BslA) has been shown to contribute to the water repellency
of B. subtilis biolms by forming a hydrophobic surface layer and increasing the
microroughness of the biolm surface (Werb et al.,2017).
The solid-liquid interface between a surface and an aqueous medium (e.g., water,
blood) provides an ideal environment for the attachment and growth of
microorganisms. A clear picture of attachment cannot be obtained without
considering the effects of the substratum, conditioning films forming on the
substratum, hydrodynamics of the aqueous medium, characteristics of the medium,
and various properties of the cell surface. Each of these factors will be considered in
detail (Donlan, 2002)
Biofilms consist of microbial cells and extracellular polymeric substance (EPS).
EPS can cover 50% to 90% of the total biofilm organic carbon and can be considered
the primary biofilm matrix material. EPS can be different chemical and physical
properties, but mainly consists of polysaccharides. Some polysaccharides are neutral
or polyanionic, such as Gram negative EPS bacteria. The presence of uronic acid,
such as Dglucuronate, D-galacturonic, and thickened manuronic acid or pyruvate
into anionic material that causes divalent cation associations such as calcium and
magnesium, has been shown to react cross with polymer yarns and provide greater
binding strength in biofilm formation. In some Gram positive bacteria, such as
Staphylococci, the chemical composition of EPS may be very different and mainly
cationic finds that the coagulase-negative bacterial mucus is made up of teikoic acid
mixed with small amounts of protein. EPS is also highly hydrated because it can
combine large amounts of water into structures with hydrogen bonds (Homenta,
2016)
.
B. Purpose
Student can learn the diversity of microbial biofilm constituent in an aquatic
environment.

II. MATERIAL AND METHODE

A. Materials

The material that used in this practical are rocks, pieces of pipe, Nutrient agar
media, PDA media, dye of gram staining, river water, pond water, alcohol 70% and
aquades.
The tools that used in this practical are petri dish, reaction tube, wrapper, tissue,
pipette, aquarium, ose needle, cotton bud, tweezers, microscope.
 B. Methods
2.1. Observation in the aquatic environment
1. Things that will be used as a form of biofilm attachment water dipper, rocks
and pieces of pipe sterilize first.
2. Object that have been soaked in the sterile glass beaker which was filled river
water 250 mL then pour to the aquarium and wait for 2 weeks. These two
objects are also soaked in sterile distilled water that serves as a control.
3. After two weeks of surfaces for review on glass object and then observed the
microbes by platting and count the density using TPC method
2.2. Total Plate Count
1. Biofilm-forming microbes was lawn with sterile cotton bud on the surface of
the object.
2. Dilute to dilution series from 10 -1 until 10-5 and spread plate into petri dish
which has been contained NA media and PDA media.
3. Incubated for 2x24 hours for NA media and 7x24 hours for PDA media.
4. The bacteria colonies is counted from NA media and fungi colonies is counted
from PDA media.
2.3. Observation Macromorphology
1. The glass object is prepared, and take the water sample from biofilm-forming
and drop on the object glass
2. Observed under the microscope and identified.
2.4. Gram Staining
1. The glass object is prepared, and one colony from NA medium is taken using ose
needle.
2. Swab on the object glass and added 1-2 drops aquadest.
3. Fixation by burner bunsen 2-3 times.
4. The gram A staining is added 1-2 drops and wait until 60 second and washed
with distilled water and dry it.
5. The gram B staining is added 1-2 drops and wait until 60 second and washed
with distilled water and dry it.
6. The gram c staining is added until clean and washed with distilled water and dry
it.
7. The gram D staining is added 1-2 drops and wait until 45 second and washed
with distilled water and dry it.
 III. RESULT AND DISCUSSION

Table 3.1 Observation of Biofilm Formation

3.1.1 Observation of Biofilm Formation Day-7
Gro Water Substr Day-7 Gram S Microalgae
up sample ate NA PDA

I/1 River Rock 2,195.107 4.106 Negative Chlamydosmous

sp
Roya
I/2 Bathroo Pipe 1.735.107 5.105 Negative -
m water
II/1 River Roof 1,9.107 41.106 Positive Synedra sp,
Diniflapis
ascuminata,
Coelastrum sp.
II/2 Bathroo Water 1,165.107 TFTC Negative Choelosphaerium
m water dipper minuttissimum
III/1 River Water 4,6.106 2,5.105 Positive Botrydiopsis sp
dipper
III/2 Faucet Slippe Spreader 0,5.106 Negative Eurostrum
water r +Positive vernocosum,
Duraliella jalina
gata, Duraliella
salina

3.1.2 Observation of Biofilm Formation Day-14

Gro Water Substr Day-7 Gram S Microalgae
up sample ate NA PDA

I/1 River Rock 4,48.108 3,7..106 Positive Chlamylomones

sp

I/2 Bathroo Pipe 2,45.106 5,05.10 Positive Asteroticus

6
m water limneticus
II/1 River Roof 12.105 5,5.105 Negative Microspora sp,
II/2 Bathroo Water 28,6.105 1.105 Negative Actinophrys sol,
m water dipper Mallimuna sp.
III/1 River Water 57.106 5,7.106 Negative -
dipper
III/2 Faucet Slippe 166.105 126,5.1 Negative M radiosa,
water r 05 Clatopfiora sp,
Euglena cus.

3.1.3 Observation of Biofilm Formation Day-21

Gro Water Substr Day-7 Gram S Microalgae
up sample ate NA PDA

I/1 River Rock 1,163.108 6,17.10 Negative Chlamylomones

7
sp

I/2 Bathroo Pipe Spreader 7,5.105 Negative -

m water
II/1 River Roof 1866.106 5.105 Negative Goniochtori sp,
Anaboema
uccrainica,
Chaetropeltis sp
II/2 Bathroo Water 3,15.106 TFTC Positive Hyalothena
 m water dipper dissileus,
III/1 River Water 3,65.106 5,7.10 6
Negative Callothrix sp,
dipper Chaetophora sp,
III/2 Faucet Slippe 8.105 126,5.1 Negative Schizoclamys
water r 05 gelatin,
Bubochaete sp

Based on the table 3.1 the result the abundant of NA and PDA media of group
II/1 is higher than group II/2. The substrate that used for group 1 is roof and group 2
is water pipe according to Karatan (2009) stated the type of substrate material is so
small that it does not even have any effect on biofilm formation has not been found
microbes that are not able to form biofilm in pipe material of any kind. Microbes
have the same adhesion capability on all substrate types, such as stainless steel,
Teflon, PVC and PVDF (Kynar). Therefore, the ability of bacteria to produce
different types of enzymes, in this case ecto-enzymes and external enzymes, is a very
important factor in initiating the formation of interactions between cells and
substrates .
The biofilm structure is heterogeneous both in space and time, and is
constantly changing due to external and internal processes. Examines the role of cell
motility in the biofilm structure in the cell stream by examining the interactions of P.
aeruginosa and P. putida with a confocal laser microscope. Both of these bacteria are
added to the cell flow system, then each bacterium will form a micro-colony. Over
time, the colonies will be mixed showing the migration of cells from one micro-
colony to another. The micro-colonial structure changes from a compact structure to
a loose structure, this may occur because cells in the micro-colony become motile, in
which the spread cell comes from the biofilm micro-colony. The structure of the
biofilm can also be affected by the interaction of non-microbial particle components,
ie the host or environment, eg erythrocytes and fibrin accumulated in biofilms.
Biofilms in the heart valve provide a clear example of this type of interaction, in
which bacterial micro-colony bacterial biofilms thrive on platelet, fibrin, and EPS
matrices (Stoodley et al., 2004)
 Medium PDA after incubation

Picture 3.1 Medium NA and PDA after incubation

Criteria or certain characteristics that can considered to explain common
biofilms, including base film which are thin, ranging from sticking a single layer of
cells into multiple film layers which contains thick water channels. research shows
thickening of biofilm can be due to amount components of bacteria. pure biofilm
culture K. pneumoniae or P. aeruginosa in laboratory reactor produces the formation
of a thinner biofilm with sizes of 15 m and 30 m, respectively, in which the
biofilm contains both bacterial species are formed thicker with thickness of 40 m.14
this is due to the bacteria of one species increase stability of other bacterial species
on formation of biofilms (Homenta, 2016)

(+) (-) (-)

Picture 3.2 Gram Staining Each Week Along Three Weeks
Picture 3.32 showed kind of bacteria, and the result was found is bacteria
gram positive because the bacteria color become purple and bacteria gram negative
because the bacteria color become red its accordance Lay (1994), stated that negative
staining is staining directed against bacteria that are difficult to be colored, wherein
the bacteria are not dyed but its background, the method of negative staining is a
method of general coloring, used in the dye solution that does not soak into the
bacterial cells but the background so it looks or appears as empty forms colorless
(negative).

Coelastrum sp Dinophysis acuminata

Synedra sp
Picture 3.3 Microalgae of Biofilm Formation Using Microscope First Week.
Groups of this type of Bacillariophyceae class can be found in virtually every
aquatic environment with sufficient sunlight to sustain activity. Synedra sp has a high
abundance and can be found in various habitats such as wetlands, rock walls, cliffs,
peat, and bark. It can also be seen as a yellow froth over the mud on. Synedra sp.
Also known to have the ability to withstand changes in unfavorable environmental
conditions. This is possible because Synedra sp. Has a diatomous shape, so it has a
layered wrapping cell. In addition Synedra sp. Also able to survive in a low-nutrient
(oligotrific) environment with low nitrogen and phosphate concentrations. This is
because Synedra sp. Able to accumulate nutrients and store them as food reserves in
the form of unsolved polymers (Istianah et al., 2015).
Several species of Dinophysis have been recorded during the monitoring
period; however, three species are defined as the main species of Dinophysis in
Fldevigen Bay and along the Skagerrak coast. These three main species are: D.
acuminata, D. norvegica and D. acuta; these are the focus of this study. The species
show large variations in cell concentrations in the bay. A large variability in cell
concentrations of Dinophysis was observed both inter-annually and on time-scales as
short as a few days (Naustvoll et al., 2012)

Picture 3.4 Microalgae of

Biofilm Formation Using Microscope Second Week
Based on the picture 3.4 the microalgae of biofilm formation showed species
of Microspra sp. The characteristics of Microspora sp is the filaments of the colony
are unbranched, at a young age (embedded in the substrate) .Microspores are found
in many freshwater ponds, unbranched colony filaments. The cell wall is shaped like
the letter H so the protoplasm is in the "H" link. These cell walls are from cellulose,
but the outer layers in the filaments are made up of pectin. In cell division occurs the
formation of a thin cellulose coat enveloping the protoplasm of children followed by
the addition of hands of H letters that also from cellulose. Single-core cells are often
in the cells of too much starch for food reserves making it difficult to determine the
shape of the chloroplasts. In young cells, the form of chloroplasts is an irregular
stretching like a webbing. Chloroplasts do not have a pythonoid (Prasetyo, 1987).

Anabaena ucrainica Chaetropeltis sp Geniochlori sp

Picture 3.5 Microalgae of Biofilm Formation Using Microscope Third Week
 Anabaena is a genus of filamentous cyanobacteria that exist as plankton.
They are known for nitrogen-fixing abilities, and they form symbiotic relationships
with certain plants, such as the mosquito fern. They are one of four genera of
cyanobacteria that produce neurotoxins, which are harmful to local wildlife, as well
as farm animals and pets. Production of these neurotoxins is assumed to be an input
into its symbiotic relationships, protecting the plant from grazing pressure (Yogesh et
al., 2009 ).

IV CONCLUSION AND SUGGESTION

A. Conclusion

Based on the result and discussion it can conclude that substrate which able to
make biofilm formation is roof, rocks, stainless steel, Teflon, PVC and PVDF. The
microalgae that found in observation of biofilm consist of diverse organism such as
Synedra sp, Microspora sp, Anabaena ucrainica , Chaetropeltis sp , Geniochlori sp
Coelastrum sp and Dinophysis acuminata

B. Suggestion
 Student should be careful and keep aseptic condition to prevent contaminant
from microorganism.

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