Conglomeration Of Different Microbes Biology Essay

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Biofilms, as we know, are a conglomeration of different microbes forming microcolonies in a matrix of Extra Polymeric when a surface is available to attach in an aqueous environment. Research on biofilms is not a new concept and millions of dollars are being spent each year in industries to remove biofilms. Our aim in this project was to study the the biocidal effect of a biocide, namely chlorine dioxide, on two model organisms-Pseudomonas aeruginosa(PAO1) and Staphylococcus aureus. The initial phase of the project included optimization of a method to generate chlorine dioxide (ClO2).We performed experiments on planktonic cells as well as biofilms of the two microorganisms in two different dosing media. Microtitre plate assay and flow cell experiments were performed to study the effect of ClO2 on biofilms. Whereas, spread plating was performed to study the same in planktonic cells. Complete killing was observed for both S.aureus and PAO1 after 4-6h of treatment with 1ppm ClO2. The effect of ClO2 on planktonic cells was studied either by varying time intervals or by varying concentrations. Due to the reactivity of chlorine dioxide with organics, the Total Residual Oxidant in Tris glucose minimal media and Normal saline was also estimated to predict the actual volume to be dosed in order to achieve the desired concentration. To conclude, chlorine dioxide was very effective in checking the growth of biofilms of P.aeruginosa and S.aureus as well as removing preformed biofilms.



 Life on earth has its genesis with the evolution of micro sized particles which are today known as microorganisms or micobes. Microorganisms, in today's world, usually occur in aggregates of differing forms and shapes in various environments. This introduces us to phenomena grouped under the general term "Biofilms"[2]. Biofilms can be defined as matrix-enclosed microbial accretions that adhere to biological or non-biological surfaces[1,3]. Alternatively, they can be defined as a functional consortia made up of microorganisms and inorganic and organic solids. A more comprehensive definition of biofilms could be "a heterogenous conglomeration of a diverse species of microbes, enclosed in a slimy matrix, attached to a living or non-living surface, with obviously distinct features when compared to their free living counterparts."

History of biofilms

The development of the biofilm concept originated with the advent of microscopy. Anton van Leeuwenhoek was the first scientist to observe a surface-associated multicellular structure of bacterial cells in the 17th century. Since then, we have come to understand that microbes can be found on surfaces also. The first observation concerning biofilms is accredited to Henrici for concluding, in 1933, that water bacteria are not free floating instead, grow upon submerged surfaces [6, 8]. Historically, examination of biofilm growth at the level of single cells began with the application of the electron microscope to the field of microbiology. Microbiologists, especially those belonging to the Scandinavian schools, are known to have examined biofilms formed on tooth surfaces. However, the electron microscope could not provide significant information on the three dimensional architecture of different bacterial species within the biofilm.

With the invention of immunofluorescence microscopy and FISH, some of the basic questions regarding the formation, development and three-dimensional architecture of biofilms were answered. Over the years, with the creation of even more sophisticated devices, techniques and softwares it has become possible for researchers and scientists all over the world to study the behaviour of microbes on surfaces in a more detailed manner. Certainly, science and technology have revolutionised research on biofilms and, day by day, we are inching closer to unravel the mystery surrounding the behaviour of biofilms.

Due to their ubiquitous presence, biofilms are nowadays, being used as morphological markers to study the evidence of life in the oldest terrestrial rocks and even, extraterrestrial rocks. Some of the Early Archaean biofilms show that biofilms were preserved in carbonate, phosphate and silica matrices and occur in a variety of environments. This also brings us to a startling conclusion that some of the biofilms on earth can even survive in climatic conditions similar to that of Mars. As for the environments suitable for biofilm growth, they can grow in environments ranging from hot to cold deserts, evaporitic environments, hot springs, littoral and marine environments.

Some of the well known biofilm samples include: The hot spring deposits at Yellowstone National park, Oligocene(25m.y.) volcanic lake deposits in Central Germany, Early Proterozoic(20m.y.)  silicified  stomatolites  from the Gunflint  formation, etc. Biofilms have also been detected on meteorites and chondrites [7]. These findings show that biofilms originated millions of years ago and may provide answers to many of the unresolved mysteries of the earth.

Stages of Biofilm formation

Biofilm development involves a series of complex but discrete and well-regulated steps. The exact mechanisms differ from organism to organism, but the stages of biofilm development are similar across a wide range of micro-organisms. The stages contributing to biofilm development are as follows [10]:

1) Attachment/adhesion of a single cell by means of flagellum and outer membrane components (for surface signal interaction)

2) Proliferation toward monolayered coverage,

3) Propagation to aggregated microcolony by means of Type-IV pili and autoaggregation factors (like Type-I fimbriae or Ag43); an irreversible attachment,

4) Maturation to 3-dimensional structure (equipped with EPS and water-filled channels and characterized by differential expression of genes) ,

5) Subsequent detachment and dispersal of microbes.

  The different stages of biofilm formation are known for Gram negative bacteria, however, less is known about the biofilm forming processes for Gram positive bacteria (Davey and O'oole, 2000). This is of particular interest, as the majority of implant-related infections are associated with Gram-positive strains.

Factors Influencing Biofilm Formation

Many factors are known to influence the formation of biofilms. Environmental factors, including nutrient sources and local conditions such as pH, osmolarity, temperature, oxygen, surface properties and hydrodynamic conditions, greatly influence the species that colonize to form biofilms and also the biofilm thickness and density (Fletcher and Pringle, 1986; van Loosdrect et al.(1995); Stoodley et al.(1999) )[9]. It is expected that diverse conditions are required for different organisms and even, different strains of the same species. This is believed to be due to the different genetic pathways adopted by microbes in different environmental conditions.

As far as hydrodynamic conditions are concerned, shear forces and liquid forces play important roles in biofilm formation. The scale of surface roughness indicates the effect of physical forces on biofilm development. The influence of surface roughness is found to be more significant than the role of surface free energy. Apart from this, hydrophobicity also contributes to the process of biofilm formation like for example; hydrophobic surfaces allow only hydrophobic cells to attach at the time of contact.

In addition to the environmental factors, genetic requirements also play a vital role. For example, the primary colonizers are able to attach to pellicle coated surfaces through specific receptor-mediated binding and this precedes the coadhesion of the bridging organisms that mediate binding of primary and late colonizers (Kolenbrander 2000; Marsh and Bowden 2000). The mature biofilm undergoes reorganization in response to nutrient availability and production of essential growth factors.


A list of factors considered being responsible for biofilm resistance in papers and recent reviews on the subject include restricted penetration of antimicrobials into a biofilm, decreased growth rate, and expression of possible resistance genes.

Control of Biofilms[13]

Oxidizing biocides

Oxidizing biocides, due to their oxidizing nature, are found to destroy the cell structure of the organism. This destructive nature does not allow organisms to develop any sort of resistance against these oxidizing biocides within a short span of time. Oxidizing biocides usually are cost effective due to their low unit cost, rapid effect on the target organism and low effective dosage. Unfortunately, oxidizing biocides have some drawbacks. For example, some can decrease cooling water pH in an uncontrolled manner (gas chlorine and chlorine dioxide generators). Most oxidizing biocides increase the corrosive nature of the cooling water, and some, such as chlorine, produce undesirable byproducts. Moreover, oxidizing biocides are found to react with organics and ammonia present in water. This reduces the actual concentration of the biocide being dosed in the medium. Hence, the effective dosage of the biocide in the medium needs to be calculated each time the dosing is done.

Some of the commonly used oxidising biocides are Chlorine dioxide, chlorine, hydrogen peroxide, ozone, bromine,etc.

Table 1: Role of some oxidizing agents as biocides

Non-oxidizing biocides.

Non-oxidizing biocides, on the other hand, function by interfering with the metabolism of the organism. This nature allows the organisms to easily develop resistance against these biocides. Subsequently, a single dosage of the biocide is not enough to kill the microbes and multiple doses may have to be administered. This can indirectly affect humans if the permissible limit of dosage is crossed.

Non-oxidizing biocides can be costly because of the high effective dosage required, long contact times and often high unit cost. However, most non-oxidizing biocides do not appear to corrode metal surfaces and produce harmful by-products. These biocides are also found to remove the dead microorganisms after killing.

Some of the commonly used non-oxidizing biocides are Isothiazoline, Carbamates, Glutaraldehyde, Methylene bisthiocyanate, DBNPA, etc.

Table2: Effects of some non-oxidizing biocides

Other Methods being developed for the eradication of biofilms include [12]:

1. Combination of an antibiotic and an inhibitor that inhibits the resistance to antibiotics.

2. Drugs to disable persistor phenotype.

3. Induction of biofilm self-destruction pathway by inducing synthesis of enzymes like lyase.

4. Cyclical antibiotic regimen to remove microbes that persist.

5. Physical methods like use of EM field, Ultrasound and super high magnetic fields.

6. Use of bacteriophages.

7. Surface modification.

Impact of biofilm on the environment

Formation of biofilms is a natural phenomenon. Their complexity in structure allow biofilms to survive in a wide variety of environments like hot springs, hulls of ships, ocean floors, pipelines, dental plaque, etc. This ubiquity of biofilms in nature can have negative as well as positive implications on humans. The beneficial aspects of biofilms allow humans to use them in extraction of metals, wastewater treatment, etc. But, the negative impacts of biofilms outnumber the positive impacts. Some of the positive and negative impacts on humans are listed below:

The benefits of biofilms to humans are:

In water and wastewater treatment plant, biofilms are preferred to planktonic cells for treatment of water.

Biofilms can also be used in the remediation of contaminated soil and groundwater.

Another application of biofilms is in the paper processing industry. The solid waste discarded each day contributes to a lot of pollution. Biofilms could solve this problem by converting these toxic wastes to a less toxic form.

Nowadays, various other applications of biofilms are being considered. Researchers are working on the practical application of biofilms in microbial leaching, microbial fuel cells, microbial canaries and biofilm traps.

They are known to increase productivity and stability of fermentation process. Moreover, they find application in production of industrial products like acetic acid, ethanol, etc [21].

The disadvantages of biofilms are:

Microorganisms in biofilms contribute to a phenomenon called biocorrosion. Equipments and machinery used in industries become corroded easily as a result of the metabolic changes in these microbes.

On the industrial scale, biofilms are known to block pipelines connected to machines and other equipments. This can be a contingency as far as industries are concerned.

Moreover, biofilms can be a source of contamination in the processed products.

The biofilms of organisms like Pseudomonas aeruginosa are also being cited as the cause for diseases like cystic fibrosis.

Methods to study biofilms

Laboratory methods to grow biofilms are as follows:

The commonly used methods to study biofilms are Microtitre plate assay and Flow Cell method.

Another method of growing biofilms is to use removable biofilm growth surfaces (coupons) which enable easy analysis and sampling.

Other in vitro methods for growing and studying biofilms are :

a) Simple batch/ static systems

b) Batch systems with introduced shears

c) Perfused biofilm fermenters

d) Systems operated under continuous flow conditions such as

e) Rotating-disc reactor

f) The modified Robbins device

g) The annular reactor

Staining Techniques used to study biofilms

The commonly used staining techniques are Gram staining and Acridine orange staining. In the Gram staining method, crystal violet is used to differentiate between Gram Positive and Gram Negative bacteria. Crystal Violet, which is the dye used in Gram staining, is a type of Methyl violet dye. The more methylated the dye is, the deeper blue the colour will be. Methyl Violet dyes are mixtures of tetramethyl, pentamethyl and hexamethyl pararosanilins. When the methyl violet dye is hexamethylated, it is known as Methyl violet 10B or Crystal violet.

Another staining technique used to stain biofilms is Acridine orange staining. Acridine orange (AO) is a nucleic acid selective fluorescent cationic dye. It is prepared from creosote oil and coal tar. It binds to the nucleic acids present in the nucleus and this allows us to selectively determine whether the cells are in active state or inactive state.

AO is potentially superior to the Gram stain in the direct microscopic examination of clinical specimens because it gives striking differential staining between organisms and background cells and debris. The lack of sensitivity of crystal violet for detecting microorganisms and the poor differential staining of Gram negative organisms can be overcome with AO stain.

Confocal Laser Scanning Microscope (CLSM)

The confocal microscope was invented by Marvin Minsky [16]. The purpose of using this microscope is to attain high-resolution three dimensional images of specimens. The principle behind the working of this microscope is as follows:

A laser light flashed on the specimen serves as the source of excitation. The excited light is passed through a dichroic mirror which reflects the light to two mirrors fixed on motors. From the moving mirrors, the light is then allowed to pass through the specimen. This allows scanning of the specimen. The fluorescent dye present in the specimen fluoresces the sample. Light of higher wavelength is emitted as a result of fluorescence and traverses the same path followed by the incident light. The reflected light is then focused on a pinhole which provides a high resolution image of the object. The pinhole prevents emitted light which are out-of-focus from reaching the eye piece. This is the reason for the high intensity, blur-free, high resolution images obtained by the confocal laser scanning microscope.

Advantages of using this microscope are its non-invasive nature and high resolution of images produced. Live cells can also be differentiated from dead cells by this technique.

Fig. 2 Schematic Diagram of CLSM working principle.


The main objective of this study was to study the effect of chlorine dioxide on planktonic cells as well as biofilms of Pseudomonas aeruginosa and Staphylococcus aureus. The specific objectives were:

To optimize the production of chlorine dioxide using any of the laboratory methods.

To determine the Total Residual Oxidant(TRO) of chlorine dioxide in the dosing media-Tris glucose and 0.85% NaCl solution.

To study the survival curve of the two model organisms in both the dosing media using ClO2 as a biocide.

To determine the effect of increasing concentrations of ClO2 on the planktonic cells of P.aeruginosa and S.aureus.

To study the effect of ClO2 on P.aeruginosa and S.aureus biofilms by microtitre plate assay and confocal microscopy.

To study the effect of ClO2 on preformed biofilms of P.aeruginosa and S.aureus by microtitre plate assay, glass slide studies and confocal microscopy.


Chemicals Used Company Name

Sodium Chlorite Sigma-Aldrich

Hydrochloric Acid MerckR

DPD No.1 tablets Lovibond

Luria Agar Himedia

Sodium Chloride Himedia

Glucose/Dextrose Rankem

All other chemicals used were from Himedia and Rankem.

Composition of Tris Minimal Media

a) Solution 17

Chemicals Used Weight (in g/100ml)

Tris free base 6.05

Sodium chloride 4.67

Potassium chloride 1.5

Ammonium chloride 1.06

Sodium sulphate 0.42

Magnesium chloride 0.233

Calcium chloride 0.03

b) Solution 8

Only 0.004 g/l of Sodium dihyrogen phosphate dihydrate is used.

c) Solution of Trace Elements

Trace Elements Weight (in g/l)

Zinc Sulphate (heptahydrate) 143.77

Magnesium chloride (tetrahydrate) 98.96

Boric acid 61.83

Cobalt chloride 190.34

Copper chloride 17.05

Nickel chloride 23.77

Sodium molybdate 26.29

Preparation of Tris Minimal Media

100ml of Solution 17, 4ml of Solution 8 and 100µl of Trace elements stock solution was added and made upto a volume of 900ml with MilliQ water. The pH of this solution was then set to 7 using Hydrochloric acid. After sterilization of Tris medium, 50ml of 1% glucose solution was added to make the final volume to 1000ml.

Note: A small volume of MilliQ water can be poured before the addition of the three solutions to avoid precipitation of the salts present. Also, glucose should not be added to the Tris medium before sterilization so as to avoid charring of glucose at high temperatures.

1. Optimization of Chlorine Dioxide generation

Three methods were tested to optimize the production of chlorine dioxide. Due to the dwindling concentration of chlorine dioxide the most suitable method was chosen.

1.1 Generation of Chlorine dioxide

Chlorine dioxide (ClO2) was generated by the reaction of Sodium chlorite and Hydrochloric acid. This reaction was carried out in a dark bottle due to the light sensitive nature of ClO2. The stoichiometric equation is as follows:

NaClO2 + 4HCl à 4ClO2 + 5NaCl + 2H2O.

1.2 Methodology

0.75g of sodium chlorite and 0.76 ml of HCl were taken.

Both were made upto 10ml with MilliQ water in measuring cylinders.

The solutions were then transferred to a dark bottle and incubated at 4 C for 15mts.

At the end of 15mts, 200µl of the stock solution was made upto 100ml with MilliQ water.

20ml of the working solution was taken in a vial. To it, 400µl of 10% glycine solution was added and a DPD tablet was dissolved.

20ml of MilliQ water + 400µl of 10% glycine solution and 1 DPD tablet comprised the blank.

The absorbance was then checked with a 76 stored program of ClO2 at 530nm using a spectrophotometer.

Note: Care must be taken to not expose sodium chlorite outside for long as it is highly hygroscopic.

1.3 Estimation of ClO2

The concentration of chlorine dioxide was estimated by the standard DPD methodology (Palin, 1957). DPD, also known as N, N-diethyl-p-phenylenediamine, oxidizes chlorine dioxide to form a pink coloured complex which can absorb light at a wavelength of 530nm. The intensity of the colour formed is directly proportional to the concentration of the oxidant. Thus, chlorine dioxide concentration can be indirectly measured by this method. The concentration obtained was expressed as ppm( mg/l) chlorine dioxide. To avoid interference of free chlorine in the estimation of chlorine dioxide concentration, glycine solution is added. The chemical reaction involved in the estimation is as follows:

DPD amine + ClO2 à Wruster dye (a semi-quinoid cationic compound)

Estimation of ClO2 Total Residual Oxidant (TRO) in 0.85% NaCl and Tris Glucose.

This test was performed to find out the effectual dosage of ClO2 in a microbial culture.


Concentrations of 0.1, 0.2, 0.5 and 1 ppm of chlorine dioxide were prepared in both N. saline and Tris glucose medium.

The final volume of each solution was 20ml.

Then the actual concentrations of chlorine dioxide were checked at 0 h and 2 h by dissolving 1 tablet of DPD in each vial.

With this, the demand of ClO2 in the individual dosing media was found out.

Planktonic studies

Effect of ClO2 (1ppm) on planktonic cells of P.aeruginosa & S.aureus at different time intervals in two different dosing media viz., 0.85% NaCl and Tris-Glucose media.

Single colony of S.aureus and P.aeruginosa was inoculated in 10ml Luria broth and incubated for 14hrs at 30 C.

The culture, taken in 15ml centrifuge tubes, was centrifuged at 8000rpm for 5mts, washed twice with saline and resuspended in Tris-G or 0.85% saline. The absorbance of each organism was set to 0.2 and the dosing mixture was prepared as below.

Table 3: Experimental design of Expt 3.1

Culture (set to 0.2 abs, 600nm)

(in ml)

Dosing medium

ClO2 (ppm)

Volume of ClO2 (1ppm) from the stock

(in ml)

Make up volume with N.saline/Tris-glucose


0.85% NaCl





Tris-glucose medium


0.8 + 0.4


The culture was then spread plated on Luria agar plates and incubated for 24hrs at 30C. The count of bacterial cells was then taken in CFU/ml.

Effect of different concentrations of ClO2 on planktonic cells of Pseudomonas aeruginosa and Staphylococcus aureus at fixed time intervals.

Single colony of PAO1 and S.aureus were inoculated in Luria broth (LB) and incubated overnight at 30 C.

The culture was centrifuged, washed twice with saline and resuspended in Tris-glucose or N.saline. The abs was set to 0.2 and the dosing mixture was prepared as below.

Table 4: Experimental design of Expt.3.2

The demand for Tris glucose solution which was found to be 0.4ppm was included in the dosing volume.

Culture (set to 0.2 abs nm)

Volume (in ml)

ClO2 (ppm)

Volume of the ClO2 (1ppm) from the stock

Make up volume with N.saline or Tris glucose

Dosing medium

Dosing medium

0.85% NaCl


Tris Glucose media (ml)

0.85% NaCl


Tris glucose media











0.11 + 0.4






0.22 + 0.4






0.55 + 0.4






1.11 + 0.4



Note: The average concentration of the stock solution of ClO2 was 1.8 ppm.

All calculations for demand in the above table are based on this.

The final volume of the microbial suspension used for plating was 2ml.

The tubes were incubated for 120mts at 30 C. After incubation, the cells were washed with normal saline and the cell pellet was suspended in normal saline or Tris glucose. This was followed by spread plating on Luria agar plates. The plates were incubated at 30 C for 24 h for CFU measurements after growth.

4) Biofilm studies

Effect of ClO2 on P.aeruginosa and S.aureus biofilm development: microtitre plate study

Single colony of PAO1 and S.aureus were inoculated in Luria broth (LB) and incubated overnight at 30 C.

The culture was centrifuged, washed twice with saline and resuspended in Tris-glucose or N.saline. The abs was set to 0.2.

The dosing mixture was prepared as per the table shown below. From each tube, 200ul of mixture was added in the microtitre plate (LaxbroR) , followed by incubation at 30 C for 48h.

After 48h, the plates were washed with MilliQ water thrice. Thereafter, 250ul 0.1% crystal violet in methanol was added in each well and the plates were incubated at room temperature for 15mts. After 15mts, stain was decanted followed by washing the unbound stain with MilliQ water thrice. The plates were allowed to dry at room temperature for 2-3 h followed by addition of 250ul ethanol to solubilise the bound stains in the wells. The optical density was measured at 570nm using Micro Titre Reader (Multiskan, Thermo Labsystems).

Flow Cell studies

The flow cell consists of a base made of plastic or metal and a square glass tube of optical quality glass through which microorganisms and nutrient will be pumped.

There are four components of a basic flow cell reactor system.These are:

the nutrient supply

an access port

the flow cell

the waste disposal container

5.1) Effect of ClO2 on P.aeruginosa and S.aureus biofilm development

Single colony of PAO1 and S.aureus were inoculated in Luria broth (LB) and incubated overnight at 30 C.

The culture was centrifuged, washed twice with saline and resuspended in Tris-glucose or N.saline. The abs was set to 0.2.

The flow cell apparatus was set up as shown below.


The inlet bottle containing the media alone was named 'Control' and the second inlet bottle containing 1ppm ClO2 along with media was labeled 'Experimental'.

The media was made to flow through the set-up and 1ml of culture was injected into the media laden flow cell. Prior to injection, the two ends of the flow cell were tightened and the flow was stopped to allow cells to attach.

After attachment for 2h, the flow was started and the flow cell was left undisturbed for another 4-6 h.

At the end of 6h, CLSM images of the architecture of biofilms in the flow cell were taken for the 'control' and 'experimental' channels.

5.2) Effect of ClO2 on preformed biofilms of P.aeruginosa and S.aureus

Single colony of PAO1 and S.aureus were inoculated in Luria broth (LB) and incubated overnight at 30 C.

The culture was centrifuged, washed twice with saline and resuspended in Tris-glucose or N.saline. The abs was set to 0.2.

The flow cell apparatus was created as shown in the above diagram.

Media was made to flow through the set-up and 1ml of culture was injected into the media laden flow cell. Prior to injection, the two ends of the flow cell were tightened and the flow was stopped to allow cells to attach.

After attachment for 2h, the flow was started and the flow cell was left undisturbed for 4 and a half days.

On the 5th day, 1ppm chlorine dioxide was dosed in one of the bottles and allowed to flow through the respective channel to which the tubing was connected.

At the end of 6h, the flow cell was stained with acridine orange for 1 min and washed off.

After staining, CLSM images of the architecture of biofilms in the flow cell were taken for the 'control' and 'experimental' channels.

6) Glass Slide studies

Single colony of PAO1 and S.aureus were inoculated in Luria broth (LB) and incubated overnight at 30 C.

The culture was centrifuged, washed twice with saline and resuspended in Tris-glucose or N.saline. The abs was set to 0.2.

A set of two sterile petriplates were taken for each organism. A sterile glass slide was placed in each petri plate.

1ml culture and 1ml Tris glucose media were added to each petriplate.

The petriplates were incubated for 5days at 30C.

On the 5th day, the glass slides were transferred (using alcohol-wiped forceps) to new petriplates.

To one petriplate, 2ml N.saline was added. This petriplate represented the 'Control'. To the second petriplate, N.saline and ClO2 solution were added.

After 6h incubation, the glass slides were stained with acridine orange stain followed by washing with MilliQ water.

CLSM images of the biofilms formed on the surface of the petriplates were taken to study the effect of ClO2 on preformed biofilms.


Optimization of ClO2 Generation

The strength of original stock solution, prepared from sodium chlorite and HCl, was back calculated and found to be 1000ppm. The working standard solution was prepared from the 1000ppm stock solution. The concentration was found to be in the range of 1.8-2.4 ppm using the DPD method. Fig. 3 shows the ClO2 concentration of the working standard solution prepared on each day.

Estimation of ClO2 TRO (Total Residual Oxidant) in 0.85% NaCl and 1% Tris glucose

The strength of ClO2 was found to remain almost constant in 0.85% NaCl. Only a slight reduction from 1ppm to 0.98ppm was observed in the N. saline medium. On the other hand, a considerable decrease in the concentration of ClO2 was observed in Tris glucose media. The demand factor was found to be 0.4ppm in Tris glucose media due to a reduction of concentration from 1ppm to 0.6ppm.

Planktonic studies

Effect of ClO2 (1ppm) on planktonic cells of P.aeruginosa & S.aureus at different time intervals in two different dosing media viz., 0.85% NaCl and Tris-Glucose media.

Survival curves of P.aeruginosa and S.aureus were studied by dosing 1.0ppm ClO2 to 105 CFU/ml of each organism in two different dosing media. The dosing media used were 0.85% NaCl and Tris glucose media. As shown in fig. 4A and B, a reduction of 1 log unit of S.aureus was observed in N.saline medium, whereas, it took 60mts to kill the same number of cells in Tris glucose medium. As expected, the complete killing of cells was faster in N.saline medium when compared to the killing in Tris Glucose medium. Complete killing was achieved at 4h and 6h of treatment with ClO2 in N. saline and Tris glucose medium respectively.

Similarly, survival curves were obtained for P.aeruginosa in both media. Complete killing of PAO1 was observed at 6h of treatment with ClO2 in both dosing media. At each time interval taken for plating the killing of cells was observed to be the same.

Figure 4: Effect of 1ppm ClO2 on PAO1 and S.aureus cells for different time intervals. A. S.aureus in NaCl; B. S.aureus in Tris glucose; C. P.aeruginosa in Tris-G; D. P.aeruginosa in 0.85% NaCl.

3.2) Effect of different concentrations of ClO2 on planktonic cells of Pseudomonas aeruginosa and Staphylococcus aureus at fixed time interval.

Different concentrations of ClO2 were used ranging from 0.1ppm to 1ppm. The effect of these concentrations on 105 CFU/ml of S.aureus and P.aeruginosa was studied. As shown in fig. 5, 0.1ppm ClO2 had no effect on both the microorganisms. At 0.2ppm, the number of cells reduced by 1 log unit. Finally, at 1ppm, the cell count reduced from 105 CFU/ml to 101 CFU/ml in both the organisms. The effect of ClO2 on the microbes was observed to be the same in both the dosing media.

Figure5: Effect of increasing concentrations of ClO2 on PAO1 and S.aureus cells.

A. PAO1 in Tris Glucose; B.PAO1 in NaCl; C. S.aureus in Tris Glucose;

D. S.aureus in NaCl.

4) Biofilm Studies

Effect of ClO2 on P.aeruginosa and S.aureus biofilm development: microtitre plate assay

The biocidal effect of increasing concentrations of ClO2 on the development of biofilms of S.aureus and PAO1 was studied. As shown in fig.6, the optical density of both the cultures without administration of ClO2 was observed to be 0.65. The bioicidal effect of the gas on the microbes was observed even at a low concentration of 0.1ppm. There was a 2.25 fold reduction for both the microbes. Finally, a 13-fold reduction of growth of both the cultures was observed when a concentration of 1ppm ClO2 was dosed. As observed in the experiment on planktonic cells, there was no significant difference in the effect of ClO2 on both the microorganisms in the biofilm studies also.

Figure 6: Effect of different concentration of ClO2 (0.1-1.0ppm) on the development of P.aeruginosa and S.aureus biofilm after 48h of incubation by Microtiter plate assay.

Table 6: Results of Microtitre plate assay

5) Flow Cell studies

5.1) Effect of ClO2 on PAO1 and S.aureus biofilms development

The effect of ClO2 on the biofilm development of S.aureus and PAO1 was studied using a flow cell apparatus. Fig.4A and 4C show the density of biofilm formed by S.aureus and PAO1 respectively. The thickness of PAO1 biofilm was found to be 20microns.Whereas, the thickness of S.aureus biofilm was 15microns. The concentration of ClO2 dosed in Tris glucose medium was 0.3ppm. As biofilms form best in minimal growth conditions, Tris glucose was chosen as the dosing medium. As shown in fig. 4B and 4D, only few cells of PAO1 and S.aureus were observed after ClO2 was dosed for 48h.

Fig.7 :CLSM images of P.aeruginosa and S.aureus biofilms after 48h of incubation. A) S.aureus biofilm; B) S.aureus biofilm + continuous dosing of 0.3ppm ClO2;C) P.aeruginosa biofilm; D) P.aeruginosa biofilm + continuous dosing of 0.3ppm ClO2.

5.2) Effect of ClO2 on preformed biofilms of PAO1

The effect of ClO2 on the removal of PAO1 biofilms which were already formed was studied using a flow cell apparatus.


Chlorine dioxide, as we know, is slowly becoming an alternative to the widely used biocide-chlorine due to its inherent advantages. Chlorine dioxide is insensitive to pH, does not form harmful by-products and has a higher oxidizing capacity when compared to chlorine. Our aim in this project was to study the fundamentals of chlorine dioxide action on biofilms formed by two model organisms-P.aeruginosa and S.aureus. In the first phase of our project, our aim was to find out a reliable method to optimize the production of ClO2. Once chlorine dioxide production was optimized, experiments were carried out on planktonic cells as well as biofilms of the model organisms. We concluded from our experiments that a dosage of 1ppm ClO2 for 4-6h is enough to achieve complete killing of microbial cells. As our work included dynamic studies as well as static studies on biofilms, we affirm the fact that chlorine dioxide is an effective biocide on biofilms.