chirag PPT presn 1st julyedit

8/7/2019 chirag PPT presn 1st julyedit

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Development of Mixed

Culture Biofilms

By: - Chirag S. Shah, 16661787

Supervised by: Dr. Michael Phillips


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Outline of Seminar

Introduction and Background

Aims and objectives

Methodology

Results

Future studies recommended

References


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Why is it important to study biofilm?

Biofilms affect many parts of our every day life, which is whybiofilm research is becoming so important and gaining in

popularity.

Everyone may not be familiar with the term "biofilm," but must

have certainly encountered biofilm on a regular basis.- The plaque that forms on your teeth and causes tooth decay is a

type of bacterial biofilm.

- The "gunk" that clogs your drains is also biofilm.

- If you have ever walked in a stream or river, you may have

slipped on the biofilm-coated rocks


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Simple example of biofilm type

- Dental plaques i.e. is a sticky, soft and colorless film of bacteria thatconstantly builds up on the of teeth and gums. If not removed regularly will

cause tooth decay or dental cavities and forms a yellowish color.

- Over 500 different microorganisms have been found in typical dental plaque. Such

microorganisms are all around us, on us, and in us.


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What is a Biofilm?

Biofilms are composed of microbial communities that are attached

to an environmental surface. These microorganisms usually

encase themselves in an extracellular polysaccharide or slime

matrix.

Biofilms grow just about anywhere. All it takes is

-Microorganisms

-Moisture

-Nutrients-Surfaces

To form a biofilm, microorganisms must "glue" themselves to a

surface, form colonies, and reproduce.


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Biofilm Development

Five stages of biofilm development:1. Initial attachment

2. Irreversible attachment

3. Maturation I

4. Maturation II5. Dispersion

Benefits ofBiofilm formation to Bacteria

Stationary growth in a hospitable environment

Resistance to antibiotics, anti-fouling agents etc. (limited toxin penetration)

Synergism between species and metabolisms

Domination of immediate environment


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Infectious DiseaseBiofilms

- Cystic Fibrosis

- Dental plaques / dental diseases

- Endocarditis

- Urinary catheter- Biomedical implants

Problems caused by Biofilm Formation

- Damage to industrial equipment

- Contamination of food, pharmaceutical and medical products

- Medical infections and antibiotic resistance

Health andEconomic problems with Biofilms


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Aim: -

To develop mixed culture biofilms and to determine how co-

culture of bacteria interact in a biofilm formation.

Objectives: -

Defining conditions that encourages the formation of biofilms inpure cultures ofE.coli o157, E.coli K 12, Pseudomonas aeruginosa,

and Bacillus subtilis.

To study the formation of biofilm in co-cultures and determining

whether biofilms are composed of one or more species and howthese organisms are distributed.

Study the stability of mixed biofilms in terms of the viability of

each bacterial type.


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Characteristics of Organisms to be tested

Escherichia coli: -

- Family Enterobacteriaceae , Gram-negative, facultativeanaerobic and non-sporulating.

- Cells are typically rod-shaped- Optimal growth ofE.coli occurs at 37C, but some laboratorystrains can multiply at a temperature of up to 49oC.

- Strains that possess flagella can swim and are motile.

- The flagella ofE.

coli have a peritrichous arrangement.


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Characteristics of Organisms to be tested.

Pseudomonas aeruginosa: -

- Family Pseudomonadaceae, Gram-negative rod

- Almost all strains are motile having a single polar flagellum

- Metabolism is respiratory and never fermentative.

- Optimum temperature for growth is 37oC, but also grow at42oC.

Bacillus Species: -

- Family Bacillaceae, Gram-positive, form endospores- Grow in the presence ofO2- The trivial name assigned to them is aerobic sporeformers which

mostly are motile by means of peritrichous flagella.


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Methodology

Slides are placed in Coplin jars in which a colony of organism is inoculated intoTryptone Soya broth.

Incubate at 370C overnight at 20rpm (24-48 hrs)

One slide is taken for staining followed by studying characteristics of biofilm

by visualising under the confocal microscope.

Second slide is used for scrapping of biofilm after which different dilutions are

made which then are spread plated.

Number of viable cells determined for each organisms using spread plate

technique on differential media such as MacConkey Agar or Chromogenic

media


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Results

Viablecount: -

1) E.coli o157 In 10-3 dilution I got 52 colonies on average from 3 plates.

So total number of colonies on the slide are

521010320 = 1.04107

2) E.coli K 12 - In 10-1 dilution I got 84 colonies on average from 3 plates.


84101020 = 1.68105


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E.coli o157(biofilmarea)

E.coli o157(away from biofilmarea)


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E.coli K 12 (biofilmarea)

E.coli K 12 (away from biofilmarea)


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3) P.aeruginosa In 10-4 dilution I got 81 colonies on average from 3 plates.


811010420 = 1.6108


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P.aeruginosa(areaaway from biofilm)

P.aeruginosa(biofilmarea)


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4)Mix culture (P.aeruginosaandE.coli o157)

Spread plating in Nutrient Agar Media(for P.aeruginosa)

In 10-3 dilution I got 116 colonies on average from 3 plates.


1161010320 = 2.3107

In Chromogenic Media(for E.coli o157)

In 10-1 dilution I got 100 colonies on average from 3 plates.


100101020 = 2105


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Mixculture(P.aeruginosa & E.coli o157)

[areaaway from biofilm]

Mixculture(P.aeruginosa & E.coli o157)

[biofilmarea]


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P.aeruginosa (colorless colonies)

E.coli o157(Pink colonies)


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B.subtilis (biofilmarea)

B.subtilis (areaaway from biofilm)


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Conclusion

It is seen that P.aeruginosa shows 10 times more growth then

E.coli o157 in pure cultures.

Pure culture ofE.coli o157however, shows 100 times more

growth then pure culture ofE.coli K 12

In Mix culture ofP.aeruginosa and E.coli o157it is seen that

the former outgrows the later by almost 100 times. This shows

that P.aeruginosa dominates in a mix culture with that ofE.coli o157.


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Future studies recommended

To get the viable count results forBacillus subtilis.

Repeating all the results in order to check the

consistency

Mixing two or maybe three different organisms and

observing the results.

Performing semi-quantitative analysis for the current

and future results.


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Acknowledgment

I would sincerely like to thank Dr. Michael, all the

laboratory staff members of School of Natural Sciences

and to Dr. Narsimha for all their support and vital

suggestions.


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References

Allison, D., 2000, Community Structure and Co-Operation in Biofilms. Cambridge:Cambridge University Press,

Costerton, J. W.,&Donlan, R. M., 2002., Biofilms: Survival Mechanisms ofClinically Relevant Microorganisms. J Clinical Microbiology Reviews, 15: page-

172-175,

Todar, K, PhD.2009, Todars online textbook of Bacteriology, University ofWisconsin-Madison Department of Bacteriology,

Banning,N.,Toze, S. & Mee,B. J., 2003., Persistence of biofilm-associated

Escherichia coli and Pseudomonas aeruginosa in groundwater and treated effluentin a laboratory model system, J of Microbiology, 149, page- 47.


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