ISOLATION AND BIOCHEMICAL CHARACTERIZATION OF STAPHYLOCOCCUS AUREUS FROM SWEET SAMPLES OF

DIFFERENT AREAS

“Dissertation Report”Submitted to

GOVT. KAMLA RAJA GIRLS POST GRADUATE AUTONOMOUS

COLLEGE (JIWAJI UNIVERSITY) GWALIOR (M.P.)

PRESENTED BY :

CHANDRESH SENGAR

M.Sc IV sem

UNDER THE SUPERVISION OF

Asst. Prof. Ms. PRIYANKA KAMRA

BIMR COLLEGE OF PROFSSIONAL STUDIES,SURYA MANDIR ROAD, GWALIOR

Aim Of The Project

Isolation, Detection, Biochemical Characterization and Sensitivity Test on Staphylococcus aureus existing in variety of sweet samples in differents area.

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CONTENTS

Introduction

Classification

Morphology and Characteristics

Growth Requirements

Methods and Result

Conclusion

STAPHYLOCOCCUS AUREUS

Staphylococcus aureus was first observed by Von Recklinghausen but not isolated.Sir Alexander Ogston, a Scottish surgeon, first showed in 1880 that a number of human pyogenic diseases were associated with a cluster-forming micro-organism. He introduced the name staphylococcus aureus. 2

The word staphylococcus is a Greek language “staphyle means a bunch of grapes” and “kokkos means a round or granule”.Staphylococcus aureus is also known as golden staph.It is a common cause of foodborne illness, commonly called “Staph aureus”, this is produce a poison or toxin that cause the illness.Staphylococcus aureus is a common bacterium found on the skin and in the noses of up to 25% of healthy people and animals. 3

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It is important because it has ability to make seven different toxins that are frequently responsible for food posioning.Staphylococcal food poisoning is a gastrointestinal illness.It is caused by eating foods contaminated with toxins produced by Staphylococcus aureus.

CLASSIFICATION

Family - Staphylococcacea

Member - Firmicutes

Genus - Staphylococcus

Some species - S. saprophyticus S. epidermidis S.hemolyticus S.ludgunesis

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more than 36 species

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MORPHOLOGY AND CHARACTERISTICS

It is gram positive cocci arranged in grape like clusters forms.It is a non-motile, non-sporing or catalase positive bacterium.It is a single cells in pairs or short chains of three or four chains.Its individuals coccus size is 0.5 to 15 µm in diameter.

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GROWTH REQUIREMENTS

Its bacteria are able to grow in a wide range of optimum temperatures is 30 to 37°C.Its Ph – 7.0 to 7.5.Its grow on agar based culture medium (like NAM or BP) and incubated for 24 hours then colonies appears.The colonies appear to be 2-4mm in diameter.The colonies appear is circular, smooth or shiny.

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SAMPLE COLLECTION

Gole ka mandir – Rasmalai

Hajira – Barfi

Murar – Rabaddi

Thatipur – khoe ka laddu

Kilagate – Rasmalai

Morena – rabaddi

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METHODS AND RESULT

1. Isolation Of Bacteria

It is isolated on Nutrient Agar Plate or Baired Parker Medium.

NAM plates show whitish colonies are grow.

BP plates shows cremish colonies are grow.

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Bacteria grow on specific media is Mannitol Salt Agar Plates.

MSA plates show yellowish colonies are grow.

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2. Morphological Identification

• Gram’s Staining – Its smear showing blue colour coccus are present in bunches like grapes means staphylococcus are conform and show the colony in 40x.

Shows gram staining are positive.

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3. Biochemical Characterization

INDOLE TESTAbsence of red colour was given by bacterial culture. So Staphylococcus aureus are negative.

MR TESTRed colour was shown by bacterial culture. So Staphylococcus aureus are positive.

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VP TEST

VP-1 TESTNo change in colour was shown by bacterial culture. So Staphylococcus aureus are negative.

VP-2 TESTPinkish colour was shown by bacterial culture. So Staphylococcus aureus are positive.

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SIMMON CITRATE UTILIZATION TEST

No change in colour of the medium was shown by bacterial culture. So Staphylococcus aureus are positive.

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GELATIN HYDROLYSIS TESTGelatinase produce was shown by bacterial culture. So Staphylococcus aureus are positive.

Gelatin Hydrolysis test before adding Mercuric chloride.

Gelatin Hydrolysis test after adding Mercuric chloride.

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STARCH AGAR TESTzone of bacterial colonies are diffuse after adding Gram’s Iodine. So Staphylococcus aureus was negative.

Starch hydrolysis test before adding Gram’s Iodine.

Starch hydrolysis test after adding Gram’s Iodine.

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TRIBUTYRIN AGAR TESTThe presence of clear area surrounding the bacterial growth was shown. So Staphylococcus aureus was positive.

After 24 hours of bacterial colonies grow.

After 48 hours of bacterial colonies are hydrolysis.

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CATALASE TEST

Bubbles were produced by bacterial culture. So Staphylococcus aureus are positive.

OXIDASE TESTNo colour change was given by bacterial culture. So Staphylococcus aureus are negative.

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ANTIBIOTIC TEST

Resistant antibiotics Resistant zoneAmphicillin 28 mmCefazolin 20 mmCefotaxime 22 mmOxacillin 25 mmPenicillin 27 mm

Measure of zone of inhibition of antibiotics on NAM media plate.

Measure of zone of inhibition of antibiotics on NAM media plate.

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Result for sensitive antibiotics-

• Moxiflox• Sepraflox• Azymicin• Lemizal penicillin• Norfloxa• Tobra• Gentamycine

Result for resistance antibiotics-

• Amphicilline• Cefazolin• Cefotaxime• Oxacillin• Penicillin

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CONCLUSION

We have isolated food posioning Staphylococcus aureus from different sweets samples of differents areas.This study indicates that the prevalence of S. aureus among food handlers was high. Contamination of food was also high indicating lack of hygienic practices among the food handlers.All the isolates were resistance to oxacillin and penicillin.

24Cleanliness Generates Healthiness