Evaluation of Antiseptics by Filter Paper Disk Method-Analytical Chemistry-Project Reports, Projects for Analytical Chemistry. Acharya Nagarjuna University

Analytical Chemistry

Description: This project report is for Analytical Chemistry course. It was submitted to Nandini Sarabhai at Acharya Nagarjuna University. It includes: Chemical, Agents, Control, Ecaluation, Antiseptics, Filter, Paper, Disk, Method, Staphylococus
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A

PROJECT REPORT

ON

“Chemical Agents of Control: Evaluation of Antiseptics by Filter paper disk Method”

CERTIFICATE

This is certify that Project entitled” Chemical Agents of Control: Evaluation

of Antiseptics by Filter paper disk Method”

Submitted by:

Isha Sharma

In the partial submission for the degree of Bachelor of Science in Biotechology.

This project has been prepared under our supervision and guidance. It is also

certified that.

The project is the result of the candidate “Own work” & is of sufficient high

standard to warrant its presentation for the examination.

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Submitted to: Mrs Jagriti Project Guide (Project guide) Lect. Biotech Deptt. Lect. Biotech Deptt.

Department of Biotechnology

Hindu Girls College – Jagadhri Session – 2006-07

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ACKNOWLEDGMENT

I express my sincere thanks to my Biotech teachers Mrs. Sheetal & Mrs. Jagriti for

their guidance & inspiration throughout my project work & in the preparation of

this report.

I also thanks them for solving my queries & catching the minute mistakes that

hindered the arrival of results.

I am also grateful to my lab assistant Mr. Gulshan for providing the materials & for

immediately getting the instruments repaired whenever some fault occurs so that

our project could not suffer.

I am also grateful to our library staff for providing us with essential books and

journals. At last I express my deep gratitude to all those also were involved either

directly or indirectly in conceptualizing & conceiving the project work & report.

Isha Sharma

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Contents

 AIM

 Introduction

 Isolation of Staphylococcus from Skin

 Sub culturing to obtain pure broth culture.

 Filter paper Disc method

 Bibliography

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AIM : To test the antimicrobial activity of different Antiseptics.

Introduction

Many microbes grow abundantly , both on surface & inside the normal body &

those that are more or less regularly present without causing disease constitutes its

normal flora while those that are present only for few days or months form its

transient flora. However under certain condition, some member of normal flora

become parasites, these organisms are then known as opportunistic pathogens.

The normal flora of skin comprises Micrococcus spp, Staphylococcus sp.

Propionibacterium etc. Perspiration and sebum serves as source of nutrition for

these microbes. Staphylococcus aureus is the part of normal flora, but when injury

occurs,it is able to penetrate the deeper tissues & causes infection.

In order to study the antimicrobial activity of antiseptics , S. aureus is chosen as

test organism because of its abundance on human skin & capability of becoming

opportunistic pathogen also.

Originally the term antiseptic was applied to any that prevented sepsis or

putrefaction which is caused by growing microorganisms. The preparations of

chemicals that are meant to be applied to skin or other living tissues to decrease

the number of microbes are called antiseptics. Ethanol (ethyl alcohol) and

isopropanol (isopropyl alcohol) are alcohols used to reduce the number of

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microbes on skin sites designation for hypodermic infections or for the withdrawal

of blood. Ethyl alcohol is occasionally used to disinfect electrodes, face masks, and

thermometers, which are first cleaned and then soaked in alcohol for 15-20

minutes. Dettol, savlon and alcohol are good antiseptics with bactericidal and

sporicidal property and are used for skin antisepsis.

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Isolation of Staphylococcus strain from Skin

Principle : Mannitol salt agar medium is used for selective isolation of pathogenic

Staphylococci since most other bacteria are inhibited by high salt concentration.

The colonies are surrounded by yellow halo indicating mannitol fermentation. The

acid produced by fermentation changes the colour of indicator phenol red to

yellow.

Requirement –

 Mannitol salt agar plates

 Autoclaved cotton swab

 Autoclaved saline (10ml. 0.85%)

Preparation of Materials :

(i) Mannitol Salt agar (pH 7.4)

Mannitol 400mg

Peptone 400mg

NaCl 3g

Beef extract 40mg

Phenol red pinch

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Agar 600mg

Distilled water 40ml

(ii)0.85%saline

Dissolve 85mg NaCl in 10ml d/w

Procedure –

1. Moisten a sterile cotton swab in saline aseptically.

2. Remove excess saline by pushing the swab against test tube wall.

3. Rub the swab over the skin surface of nose, elbow or any other area.

4. Spread the swab uniformly on the mannitol salt agar plates

5. Incubate the plates for 48 - 60 hrs at 37°C.

Observation –

Large opaque colonies surrounded by yellow halos are observed. These are

colonies of Staphylococcus.

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Subculturing (or picking off) technique

To obtain pure broth cultures

After incubation has been completed in streak-plate, pour-plate or spread

plate techniques & appearance of discrete well-separated colonies has

been examined, the next step is to subculture some of the cells from one of

the colonies to separate agar plates or nutrient broth.

Each of this new culture represents the growth of single species & is called

pure or stock culture.

Sub culturing term is used to describe the procedure of transferring of

microbes from their parent growth source to fresh medium. When the

transfer is from solid medium (agar) to liquid medium (broth) the term

picking off is used.

Requirement

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 Sterile test – tubes

 Nutrient broth (pH – 7. 0)

Peptone 250mg

Beef extract 150mg

NaCl 250mg

D/w 50ml

 Sterile loops

Procedure

1. Loopful of culture of Staphylococcus is transferred from mannitol

salt agar plate to nutrient broth tube.

2. The tube was incubated overnight at 37°C.

Observation –

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Turbidity appears in the tube due to growth of culture.

The culture obtained is pure culture of Staphylococcus.

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Testing Antimicrobial activity of different Antiseptics

By Filter-Paper Disc Method

Principle-

Filter paper disc method (or zone of inhibition method) is employed to

evaluate effectiveness of antimicrobial agents against selected test

organism.

A sterile filter paper disc is impregnated with an antiseptic & placed on a

fresh heavily inoculated agar plate of test organism. Following incubation

the agar plate is looked for zone of inhibition. The presence of clear zone of

inhibition surrounding disc is indicative of inhibitory activity against microbe.

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The diameter of zone of inhibition is directly proportional to the

antimicrobial activity.

Requirement

 Nutrient agar plates

 Sterile spreader

 Sterile pipette

 Sterile filter paper discs

 Solution of different antiseptics.

Antiseptics

1. 1/5 Dettol

2. 1/10 Dettol

3. 1/5 Savlon

4. 1/10 Savlon

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5. 100% Alcohol

6. 70% Alcohol

7. 50% Alchol

8. 30% Alcohol

 Sterile test tubes

 Sterile d/w

 Cotton Swab

Procedure –

1. About 0.2ml of Staphylococcus broth culture is spread on surface

of respective nutrient agar plates by sterile spreader.

2. Filter paper discs are dipped in different soap solutions & placed

on Staphylococcus inoculated plats.

3. The plates are incubated overnight at 37°C.

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4. Observe & measure the diameter of zone of inhibition around each

disc.

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Observations –

The diameters of zone of inhibition of different solutions are as follows :

Soaps

1. Dettol 4.1 cm

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2. Dettol 3.1 cm

3. Savlon 3.2 cm

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4. Savlon 2.6 cm

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Testing Antimicrobial activity of Alcohol Using Cotton Swab.

Procedure:-

1. About 0.2 ml of staphylococcus broth culture is spread on surface of

respective nutrient agar plates by sterile spreader.

2. Cotton Swabs are dipped in different concentration of alcohol

solutions and move on agar plate divided in four quadrants.

3. The plates are incubated overnight at 370C.

4. Observe the Result.

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Result –

 Solutions 1/5 Dettol has highest antimicrobial.

 Among alcohol solutions 10% alcohol has highest antimicrobial

activity.

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Bibliography

 Experiments in Microbiology by K.R. Aneja – New Age International

Publication.

 Textbook of Microbiology by Pelczar

 Eco Science Journal of Science & Technology – Yamunanagar

 www.google.com

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