- Biofilms are groups of microorganisms that attach to each other and often surfaces, and are found nearly everywhere in nature. They can include bacteria, fungi, and protists. Common examples are dental plaque and pond scum. - Biofilm formation is a complex, multi-step process involving initial attachment, microcolony formation within an extracellular matrix, maturation, and eventual dispersal or detachment of cells. Dispersal allows biofilms to spread and colonize new surfaces. - Biofilms grow in many habitats and can cause problems like infections, corrosion, and difficulties with sanitation. They are implicated in about 80% of all infections and can involve both gram-positive and gram-negative bacteria as

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BIOFILMS
PROJECT

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BIOFILMS
A biofilm is any group of microorganisms in which cells stick to each other and often also to a
surface. Biofilms are a collective of one or more types of microorganisms that can grow on many
different surfaces. Microorganisms that form biofilms include bacteria, fungi and protists.
One common example of a biofilm is dental plaque, a slimy buildup of bacteria that forms on the
surfaces of teeth. Pond scum is another example. Biofilms have been found growing on minerals and
metals. They have been found underwater, underground and above the ground. Biofilms are found in
hydrothermal environments such as hot springs and deep-sea vents.
HISTORICAL BACKGROUND:
Bacterial cell exhibits two types of growth mode i.e. planktonic cell and sessile aggregate which is
known as the biofilm. Biofilm is an association of micro-organisms in which cells stick to each other on
a surface encased within matrix of extracellular polymeric substance produced by bacteria themselves. A
Dutch researcher, Antoni van Leeuwenhoek, for the first time observed ‘animalcule’ on surfaces of tooth
by using a simple microscope and this was considered as the microbial biofilm discovery. Biofilms are
present everywhere in nature and can be found in industrial places, hotels, waste water channels,
bathrooms, labs, hospital settings and commonly occur on hard surfaces submerged in or exposed to an
aqueous solution. Its formation can occur on both living and non-living surfaces
COMPOSITION OF BIOFILMS:
Biofilms are group or micro-organisms in which microbes produced an extracellular polymeric
substances (EPS) such as proteins (including enzymes), DNA ,polysaccharides and RNA ,and in
addition to these components, water (up to 97%) is the major part of biofilm which is responsible for the
flow of nutrients inside biofilm matrix .The architecture of biofilm consists of two main components i.e.
water channel for nutrients transport and a region of densely packed cells having no prominent pores in
it.The microbial cells with in biofilms are arranged in way with significant different physiology and
physical properties. Bacterial biofilms are normally beyond the access of antibiotics and human immune
system.
HOW BIOFILMS ARE FORMED?
Biofilm formation is a highly complex process, in which microorganism cells transform from planktonic
to sessile mode of growth. It has also been suggested that biofilm formation is dependent on the
expression of specific genes that guide the establishment of biofilm. The process of biofilm formation
occurs through a series of events leading to adaptation under diverse nutritional and environmental
conditions.This is a multi-step process in which the microorganisms undergo certain changes after
adhering to a surface. Biofilm formation has following important steps (a) attachment initially to a
surface (b) formation of micro-colony (c) three dimensional structure formation (d) biofilm formation,
maturation and detachment (dispersal)
 Attachment:
When a bacterium cell reaches to near some surface/support so close that its motion is very slow down,
it make a reversible connection with the surface and /or already adhered other microbe to the surface.
For biofilm formation, a system of solid– liquid interface can provide an ideal environment for micro-
organism to attach and grow (e.g. blood, water). Presence of locomotor structures on cell surfaces such
as flagella, pili, fimbriae, proteins or polysaccharides are also important and may possibly provide an
advantage in biofilm formation when there are mixed community.

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 Micro-Colony Formation:
Micro-colony formation takes place after bacteria adhered to the physical surface/biological tissue and
this binding then becomes stable which results in formation of micro-colony. Multiplication of bacteria
in the biofilm starts as a result of chemical signals. They excrete extracellular polymeric substances
(EPS), so cells aggregate which finally result in micro-colony formation.
 Three-Dimensional Structure Formation and Maturation:
After micro-colony formation stage of biofilm, expression of certain biofilm related genes take place.
These gene products are needed for the EPS which is the main structure material of biofilm. It is
reported that bacterial attachment by itself can trigger formation of extracellular matrix. Matrix
formation is followed by water-filled channels formation for transport of nutrients within the biofilm.
Researcher have proposed that these water channels are like a circulatory systems, distributing different
nutrients to and removing waste materials from the communities in the micro-colonies of the biofilm.
 Detachment:
After biofilm formation, the researchers have often noticed that bacteria leave the biofilms itself on
regular basis. By doing this the bacteria can undergo rapid multiplication and dispersal. Detachment of
planktonic bacterial cells from the biofilm is a programmed detachment, having a natural pattern.
Sometime occasionally due to some mechanical stress bacteria are detached from the colony into the
surrounding. But in most cases some bacteria stop EPS production and are detached into environment.
Dispersing of biofilm cells occur either by detachment of new formed cells from growing cells. In
biofilm of cells are removed due to an enzyme action that causes alginate digestion. Phenotypic
characters of organisms are apparently affected by the mode of biofilm dispersion. Dispersed cells from
the biofilm have the ability to retain certain properties of biofilm, such as antibiotic in-sensitivity.
DISPERSAL OF BIOFILMS
Dispersal of cells from the biofilm colony is an essential stage of the biofilm life cycle. Dispersal
enables biofilms to spread and colonize new surfaces. Enzymes that degrade the biofilm extracellular
matrix, such as dispersin-B and deoxyribonuclease, may play a role in biofilm dispersal. Biofilm matrix
degrading enzymes may be useful as anti-biofilm agents. Recent evidence has shown that a fatty acid
messenger, cis-2-decenoic acid, is capable of inducing dispersion and inhibiting growth
of biofilm colonies. Secreted by Pseudomonas aeruginosa, this compound induces cyclo-heteromorphic
cells in several species of bacteria and the yeast Candida albicans Nitric oxide has also been shown to
trigger the dispersal of biofilms of several bacteria species at sub-toxic concentrations. Nitric oxide has
the potential for the treatment of patients that suffer from chronic infections caused by biofilms
It is generally assumed that cells dispersed from biofilms immediately go into the planktonic growth
phase. However, recent studies have shown that the physiology of dispersed cells from Pseudomonas
aeruginosa biofilms is highly different from those of planktonic and biofilm cells. Hence, the dispersal
process is a unique stage during the transition from biofilm to planktonic lifestyle in bacteria. Dispersed
cells are found to be highly virulent against macrophages and Caenorhabditis elegans, but highly
sensitive towards iron stress, as compared with planktonic cells.

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MECHANISMS OF BIOFILM DISPERSAL
Bacterial biofilm dispersal can be divided into three distinct phases: (i) detachment of cells from the
biofilm colony; (ii) translocation of the cells to a new location; and (iii) attachment of the cells to a
substrate in the new location. Thus, S. mutans cells that detach from dental plaque can be transported to
the saliva of an infant by direct contact or by means of a vector such as a shared spoon, and then attach
to the tooth surface and initiate colonization of the new host. Similarly, cells that detach from
a Legionella biofilm growing in a cooling tower can be transported by means of air-borne water droplets
to the lungs of a susceptible host, where they can attach to alveolar macrophages and initiate infection.
In the literature and in this review, the terms ‘detachment’, ‘dispersal’, and ‘dispersion’ are used
interchangeably to refer to the cell-detachment phase of the dispersal process. Studies on the movement
of detached cells to a new location fall mostly under the discipline of disease transmission.
In general, mechanisms of biofilm dispersal can be divided into two broad categories: active and
passive. Active dispersal refers to mechanisms that are initiated by the bacteria themselves, whereas
passive dispersal refers to biofilm cell detachment that is mediated by external forces such as fluid shear,
abrasion (collision of solid particles with the biofilm), predator grazing, and human intervention
(Lawrence et al., 2002; Choi and Morgenroth, 2003; Ymele-Leki and Ross, 2007). In a complex
community such as dental plaque, close relationships between species based on competition, mutualism,
predation, or parasitism are likely to have resulted in the evolution of various other passive dispersal
mechanisms. These may include interspecific antimicrobial compounds, quorum-sensing signals, or
matrix-degrading enzymes. Phagocytosis, a form of predator grazing, may also contribute to the passive
dispersal of oral biofilms (Erard et al., 1989).
At least three distinct modes of biofilm dispersal have been identified: erosion, sloughing, and seeding.
Erosion refers to the continuous release of single cells or small clusters of cells from a biofilm at low
levels over the course of biofilm formation. Sloughing refers to the sudden detachment of large portions
of the biofilm, usually during the later stages of biofilm formation (Marshall, 1988; Lappin-Scott and
Bass, 2001; Stoodley et al., 2001; Wilson et al., 2004). Seeding dispersal, also known as central
hollowing, refers to the rapid release of a large number of single cells or small clusters of cells from
hollow cavities that form inside the biofilm colony (Boles et al., 2005; Ma et al., 2009). Erosion and
sloughing can be either active or passive processes, whereas seeding dispersal is always an active
process. The following sections describe some of the mechanisms of active biofilm dispersal that have
been described to date.
PROPERTIES OF BIOFILMS
Biofilms are colonies that are formed in environments that are exposed to flowing liquid. They can be
formed from one single bacterial type, but they are more often collections of several different species.
The process of biofilm formation is very complicated, and depends on the type of bacterium that is
colonizing the surface. However, there are several distinct steps in the life cycle of a biofilm .The
initialization stage, where the biofilm is actually formed, must be viewed from the scale of a bacterium.
Initially there is a clean surface, often referred to as the substratum, which is exposed to water that has
some population of planktonic bacteria. A bacterium may attach to the substratum using either flagella
or cilia or both. This attachment is not strong, but suffices to allow the bacterium to adhere to the
substratum long enough to undergo a phenotypic change. This change illustrates an interesting property
of bacteria. Bacteria are able to sense and adapt to their surroundings in some very complex ways. The
first adaptation that takes place is the adaptation to the presence of a substratum.

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The change, which was demonstrated by Davies, is due to chemicals that are produced by the bacterium,
which build up on the interior of the cell wall. The concentration builds up on the interior because the
diffusion of the chemical is reduced due to the presence of the substratum. This is the mechanism by
which different genes are expressed, causing the bacterium to convert to a biofilm forming bacterium.
This process of regulation has been termed auto-induction and is sometimes referred to as quorum-
sensing and cell-cell communication. There are several situations where auto-induction has been shown
to play a role .Auto-induction takes place when the diffusion of bacterial products on the exterior of the
cell is reduced. The first signal that a bacterium receives indicates that it would be beneficial to form a
biofilm. If bacteria has attached to the substratum and has sufficient nutrients around, it would like to
stay there. To do this a planktonic bacterium will change phenotypes to a biofilm forming bacterium.
The conversion from planktonic bacteria to a biofilm forming bacteria demarcates the end of the
initialization stage.
Once the initialization stage is over, the biofilm begins to develop. The primary way to differentiate
between planktonic and biofilm bacteria of the same species is the production of an exo-polymeric
substance (EPS) by biofilm bacteria. This EPS consists of long strands of polymer which serve several
purposes. One of the main purposes of EPS is to lock the bacteria to the surface as well as to other
bacteria that join the biofilm from the bulk fluid, and which are produced within the colony by cellular
reproduction. The polymer, which generally makes up the majority of the biomass, is otherwise passive.
Once a bacterium has converted to a biofilm forming bacterium, it becomes well anchored to the
surface, and tends to stay in one place. Biofilm forming bacteria reproduce in a manner that is not
dissimilar to planktonic bacteria. The major difference is that the daughter cells are of the biofilm type
and also tend to remain attached to the substratum, or other nearby bacteria. Thus, a biofilm is made up
of bacteria from the bulk fluid that have attached and been converted, and their offspring. At a larger
scale, the biofilm begins to display many different properties.
HABITATS
Biofilms are ubiquitous in organic life. Nearly every species of microorganism have mechanisms by
which they can adhere to surfaces and to each other. Biofilms will form on virtually every non-shedding
surface in non-sterile aqueous or humid environments. Biofilms can grow in the most extreme
environments: from, for example, the extremely hot, briny waters of hot springs ranging from very
acidic to very alkaline, to frozen glaciers.
Biofilms can be found on rocks and pebbles at the bottom of most streams or rivers and often form on
the surface of stagnant pools of water. In fact, biofilms are important components of food chains in
rivers and streams and are grazed by the aquatic invertebrates upon which many fish feed. Biofilms are
found on the surface of and inside plants. They can either contribute to crop disease or, as in the case of
nitrogen-fixing Rhizobium on roots, exist symbiotically with the plant. Examples of crop diseases
related to biofilms include Citrus Canker, Pierce's Disease of grapes, and Bacterial Spot of plants such
as peppers and tomatoes.
In the human environment, biofilms can grow in showers very easily since they provide a moist and
warm environment for the biofilm to thrive. Biofilms can form inside water and sewage pipes and cause
clogging and corrosion. Biofilms on floors and counters can make sanitation difficult in food preparation
areas. Biofilm in soil can cause bioclogging. Biofilms in cooling- or heating-water systems are known to
reduce heat transfer. Biofilms in marine engineering systems, such as pipelines of the offshore oil and

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gas industry, can lead to substantial corrosion problems. Corrosion is mainly due to abiotic factors;
however, at least 20% of corrosion is caused by microorganisms that are attached to the metal
subsurface (i.e., microbially influenced corrosion).
TAXONOMIC DIVERSITY
Many different bacteria form biofilms, including gram-positive (e.g. Bacillus spp, Listeria
monocytogenes, Staphylococcus spp, and lactic acid bacteria, including Lactobacillus plantarum and
Lactococcuslactis) and gram-negative species (e.g. Escherichia coli, or Pseudomonas aeruginosa).
Cyanobacteria also form biofilms in aquatic environments.
Biofilms are formed by bacteria that colonize plants, e.g. Pseudomonas putida, Pseudomonas
fluorescens, and related pseudomonads which are common plant-associated bacteria found on leaves,
roots, and in the soil, and the majority of their natural isolates form biofilms. Several nitrogen-fixing
symbionts of legumes such as Rhizobium leguminosarum and Sinorhizobiummeliloti form biofilms on
legume roots and other inert surfaces.
Along with bacteria, biofilms are also generated by archaea and by a range of eukaryotic organisms,
including fungi e.g. Cryptococcus laurentii and microalgae. Among microalgae, one of the main
progenitors of biofilms are diatoms, which colonise both fresh and marine environments worldwide.
INFECTIOUS DISEASES CAUSED BY BIOFILMS
Biofilms have been found to be involved in a wide variety of microbial infections in the body, by one
estimate 80% of all infections. Infectious processes in which biofilms have been implicated include
common problems such as bacterial vaginosis, urinary tract infections, catheter infections, middle-ear
infections, formation of dental plaque, gingivitis, coating contact lenses, and less common but more
lethal processes such as endocarditis, infections in cystic fibrosis, and infections of permanent
indwelling devices such as joint prostheses, heart valves, and intervertebral disc. More recently it has
been noted that bacterial biofilms may impair cutaneous wound healing and reduce topical antibacterial
efficiency in healing or treating infected skin wounds. Early detection of biofilms in wounds is crucial to
successful chronic wound management. Although many techniques have developed to identify
planktonic bacteria in viable wounds, few have been able to quickly and accurately identify bacterial
biofilms. Future studies are needed to find means of identifying and monitoring biofilm colonization at
the bedside to permit timely initiation of treatment.
Pseudomonas aeruginosa
P. aeruginosa represents a commonly used biofilm model organism since it is involved in different types
of biofilm-associated infections. Examples of such infections include chronic wounds, chronic otitis
media, chronic prostatitis and chronic lung infections in cystic fibrosis (CF) patients. About 80 % of CF
patients have chronic lung infection, caused mainly by P. aeruginosa growing in a non-surface attached
biofilms surround by PMN (polymorpho nuclear leukocytes). The infection remains present despite
aggressive antibiotic therapy and is a common cause of death in CF patients due to constant
inflammatory damage to the lungs.
Streptococcus pneumoniae

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S. pneumoniae is the main cause of community-acquired pneumonia and meningitis in children and the
elderly, and of septicemia in HIV-infected persons. When S. pneumonia grows in biofilms, genes are
specifically expressed that respond to oxidative stress and induce competence.
It has been proposed that competence development and biofilm formation is an adaptation of S.
pneumoniae to survive the defenses of the host. In particular, the host’s poly-morph-nuclear leukocytes
produce an oxidative burst to defend against the invading bacteria, and this response can kill bacteria by
damaging their DNA. Competent S. pneumoniae in a biofilm have the survival advantage that they can
more easily take up transforming DNA from nearby cells in the biofilm to use for recombination repair
of oxidative damages in their DNA. Competent S. pneumoniae can also secrete an enzyme (murein
hydrolase) that destroys non-competent cells (fratricide) causing DNA to be released into the
surrounding medium for potential use by the competent cells.
IMPACTS OF BIOFILMS
In Industry
Biofilms can also be harnessed for constructive purposes. For example, many sewage treatment plants
include a secondary treatment stage in which waste water passes over biofilms grown on filters, which
extract and digest organic compounds. In such biofilms, bacteria are mainly responsible for removal of
organic matter (BOD), while protozoa and rotifers are mainly responsible for removal of suspended
solids (SS), including pathogens and other microorganisms. Biofilms can help eliminate petroleum oil
from contaminated oceans or marine systems. The oil is eliminated by the hydrocarbon-degrading
activities of microbial communities. Biofilms are used in microbial fuel cells (MFCs) to generate
electricity from a variety of starting materials, including complex organic waste and renewable biomass.
Food Industry
Biofilms have become problematic in several food industries due to the ability to form on plants and
during industrial processes. Bacteria can survive long periods of time in water, animal manure, and soil,
causing biofilm formation on plants or in the processing equipment. The buildup of biofilms can affect
the heat flow across a surface and increase surface corrosion and frictional resistance of fluids. These
can lead to a loss of energy in a system and overall loss of products. Along with economic problems
biofilm formation on food poses a health risk to consumers due to the ability to make the food more
resistant to disinfectants As a result, from 1996 to 2010 the Center for Disease Control and Prevention
estimated 48 million foodborne illnesses per year. Biofilms have been connected to about 80% of
bacterial infections in the United States.
In produce, microorganisms attach to the surfaces and biofilms develop internally. During the washing
process, biofilms resist sanitization and allow bacteria to spread across the produce. This problem is also
found in ready to eat foods because the foods go through limited cleaning procedures before
consumption Due to the perishability of dairy products and limitations in cleaning procedures, resulting
in the buildup of bacteria, dairy is susceptible to biofilm formation and contamination. The bacteria can
spoil the products more readily and contaminated products pose a health risk to consumers. One
bacterium that can be found in various industries and is a major cause of foodborne disease is
Salmonella. Large amounts of salmonella contamination can be found in the poultry processing industry
as about 50% of salmonella strains can produce biofilms on poultry farms. Salmonella increases the risk
of foodborne illnesses when the poultry products are not cleaned and cooked correctly. Salmonella is
also found in the seafood industry where biofilms form from seafood borne pathogens on the seafood

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itself as well as in water. Shrimp products are commonly affected by salmonella because of unhygienic
processing and handling techniques. The preparation practices of shrimp and other seafood products can
allow for bacteria buildup on the products.
New forms of cleaning procedures are being tested in order to reduce biofilm formation in these
processes which will lead to safer and more productive food processing industries. These new forms of
cleaning procedures also have a profound effect on the environment, often releasing toxic gases into the
groundwater reservoirs.
In Aquaculture
In shellfish and algae farms, biofouling species tend to block nets and cages and ultimately out-compete
the farmed species for space and food. Bacterial biofilms start the colonization process by creating
microenvironments that more favorable for biofouling species. In the marine environment, biofilms
could reduce the hydrodynamic efficiency of ships and propellers, lead to pipeline blockage and sensor
malfunction, and increase the weight of appliances deployed in seawater. Numerous studies have shown
that biofilm can be a reservoir for potentially pathogenic bacteria in freshwater aquaculture. As
mentioned previously, biofilms can be difficult to eliminate even when antibiotics or chemicals are used
in high doses. The role that biofilm plays as reservoirs of bacterial fish regarding pathogens has not been
explored in detail but it certainly deserves to be studied.
BIOFILM DETECTION
Various methods are currently used in medical areas for the detection of biofilm production, including
visual assessment by electron microscopy using different types of microscopes. The most versatile and
effective nondestructive approach for studying biofilms is confocal laser scanning microscopy (CLSM).
CLSM markedly reduces the need for pretreatments such as disruption and fixation, which reduce or
eliminate the evidence of microbial relationships, complex structures and biofilm organization, without
the limitations encountered with scanning electron microscopes. The authors reported that the combined
use of microplates and confocal imaging proved to be a good alternative to other high throughput
methods commonly used since it permits the direct, in situ qualitative and quantitative characterization
of biofilm architecture.
CLSM combines high-resolution optical imaging with depth selectivity which allows us to do optical
sectioning. This means that we can view visual sections of tiny structures that would be difficult to
physically section (e.g. embryos) and construct 3-D structures from the obtained images.
Microscopic Examination
1. Collecting Biofilm. Carefully scrape the biofilm from the surface of a recently collected stream rock
and transfer 5 to 10 ml of stream water into a small container to form biofilm ‘slurry’. You can do this
‘in the field’.
2. Preparation of the microscope. Rotate the scanning power (x4) objective lens into position, and using
the coarse focus adjustment knob, position the objective approximately 1 cm from the lens.

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3. Prepare the microscope slide. Stir the biofilm slurry gently and then transfer 3-4 drops of this solution
onto a clean microscope slide. Gently place a cover slip on top. If you don’t have a cover-slip – don’t
worry! It will still work.
4. Switch on the light source. Then adjust to about ¾ intensity using the light adjuster dial.
5. Position the slide. Place the slide into position on the microscope stage and center the biofilm sample
under the x4 objective lens using the stage adjuster.
6. Focus on the sample. Bring the slide onto focus by moving the objective lens away from the slide
using the coarse focus knob. The specimen can then be brought into sharp focus using the fine focus
knob and the illuminance adjusted with the iris diaphragm to provide the best view.
7. Try using a greater magnification. To view the specimen at a higher magnification, rotate the higher
power (10X or 40X) objective lens into the viewing position while watching from the side to ensure that
the objective does not touch the slide. The specimen can now be brought into sharp focus using the fine
focus knob. This procedure can then be repeated to view samples under progressively higher power
lenses.
DETECTION METHODS
Selection Of The Isolates
Organisms can be selected on the following criteria: those isolated from the pus, intravenous and urinary
catheter tips, urine, and naso-bronchial lavage specimens, and those showing increased resistance to
commonly available antibiotics. Urinary catheter tips, intravenous catheter tips, naso-bronchial lavage
specimens and few of the pus specimens were related to medical devices. All of the specimens were
received from patients with infections admitted to the hospital. Reference strain of positive biofilm
producer Staphylococcus epidermidis ATCC 35984, Staphylococcus aureus ATCC 35556,
Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 35218 and Staphylococcus epidermidis
ATCC 12228 (non-slime producer) can be used as control. Biofilm detection can be done by the
following methods:
Tube Method
10 ml of Trypticase soy broth with 1% glucose was inoculated with a loopfull of test organism from
overnight culture on nutrient agar individually. Broths were incubated at 37 c for 24 hours. The cultures
were decanted and tubes were washed with phosphate buffer saline pH7.3.The tubes were dried and
stained with 0.1% crystal violet. Excess stain was washed with deionized water. Tubes were dried in
inverted position and observed for biofilm formation. Biofilm Production was considered positive when
a visible film lined the wall and bottom of the tube. Ring formation at the liquid interface was not
indicative of biofilm formation. Tubes were examined and amount of biofilm formation was scored as 0-
absent, 1-weak, 2-moderate, 3-strong.
Congo Red Agar Method
The medium composed of Brain heart infusion broth (37 gm/l), sucrose (5gm/l), agar number 1 (10
gm/l) and Congo red dye (0.8 gm/l). Congo red stain was prepared as concentrated aqueous solution and
autoclaved at 121 C for 15 minutes. Then it was added to autoclaved Brain heart infusion agar with
sucrose at 55 C. Plates were inoculated with test organism and incubated at 37 C for 24 to 48 hours

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aerobically. Black colonies with a dry crystalline consistency indicated biofilm production; weak
producers usually remained pink, though occasional darkening at the center of colonies was observed.
Modified Congo RedAgar Method
In the MCRA the CRA is modified in the form of changing the concentration of Congo red dye and
sucrose, omission of glucose, replacement of BHI Agar by an alternative agar, that is, Blood Base Agar.
The MCRA plate was inoculated with organisms from a fresh plate with overnight growth, and then it
was incubated for 48 h at 37°C and subsequently 2–4 days at room temperature. Positive result was
indicated by black colonies with a dry crystalline consistency. The experiment was performed in
triplicate.
Tissue Culture Plate Method
Overnight culture of the isolate from nutrient agar plate is inoculated into Trypticase soy broth (TSB).
The primary inoculums are then inoculated in TSB with 1% glucose prepared in Different dilutions
(1:20, 1:40, 1:80, and 1:100) and loaded into 96 wells flat bottom microtiter plate. Plates are covered
and incubated at 37ºc for 24 hours in aerobic condition, the well are then decanted and washed three
times with Phosphate buffer saline (PBS). Then the wells are decanted and stained with crystal violet for
20 minutes. The wells are again decanted and washed with distilled water. Finally 33% glacial acetic
acid is added to the wells to extract the stain and adherence of the stained cells to the wells. Optical
density of each well is measured at 490 nm using an automated ELISA plate reader.
RESULTS
The TCP method was considered the gold- standard for this study and compared with data from TM and
CRA methods. In the TCP method, the number of isolates showing biofilm formation was 83 (83%), and
non or weak biofilm producers were 17 (17%).Tube method detected 57%isolates as biofilm producers
and 43% as non-biofilm producers where CRA only detected 20% as biofilm producer and 80% as non
or weak biofilm producer. On the other hand other parameters like sensitivity, specificity, false negative
value, false positive value and accuracy were calculated. True positives were biofilm producers by TCP,
TM and CRA method. False positive were biofilm producers by TM and CRA method and not by TCP
method. False negative were the isolates which were non-biofilm producers by TM and CRA but were
producing biofilm by TCP method. True negatives are those which were non biofilm producers by all
the three methods.
CONCLUSION
Biofilm can be composed of a single or multiple organisms on various biotic and abiotic surfaces. There
is association between biofilm production with persistent infection and antibiotic failure. Hence, in
infection caused by biofilm producing staphylococci, the differentiation with respect to its biofilm
phenotype might help to modify the antibiotic therapy and to prevent infection related to biomedical
devices. A suitable and reproducible method is necessary for screening of biofilm producers in any
healthcare setup and this TCP method can be recommended.