ABSTRACT

Bacillus subtilis capable of producing bacitracin was isolated from soil and screened for the production of bacitracin on nutrient media against test pathogenic organisms viz: Micrococcus luteus, and Staphylococcus aureus. Bacitracin was produced by submerged fermentation from the isolated bacteria and checked for their antimicrobial activity by agar well diffusion method. By optimizing the media composition and culture conditions, the effect of different pH (5, 7, 8 and 9), time (24, 48,72 and 96 hours), temperature (25, 27, 37 and 40 °C) and salinity (0% w/v (no salt), 1% w/v, 2% w/v and 5% w/v ) on the production of bacitracin were investigated by agar diffusion assay as detected by the size of zones of inhibition. Peak bacitracin production was observed judging by their zones of inhibition after incubating at 40 °C of pH 7.0 for 96 hours at 0% w/v salinity.

TABLE OF CONTENTS

Certification ii

Dedication iii

Acknowledgements iv

Table of Contents v

List of Tables viii

List of Figures ix

Abstract x

CHAPTER ONE

1.0 Introduction 1

1.1 Aim 3

1.2 Objectives 3

CHAPTER TWO

2.0 Literature review 4

2.1 Bacitracin 4

2.1.1 History 4

2.1.2 Synthesis 4

2.1.3 Composition 4

2.1.4 Spectrum of activity and susceptibility data 5

2.1.5 Mechanism of action 5

2.1.6 Clinical use 5

2.2 Bacillus subtilis 6

2.2.1 Habitat 7

2.2.2 Reproduction 7

2.2.3 Chromosomal replication 8

2.2.4 Genome 9

2.2.5 Transformation 9

2.2.6 Uses 10

2.2.7 Safety 12

2.2.7.1 In humans 12

2.2.7.1 In animals 14

CHAPTER THREE

3.0 Materials and methods 15

3.1 Source of material and collection 15

3.2 Preparation of culture media.  15

3.3 Sterilization of materials 15

3.3 Isolation of bacillus subtilis 15

3.3.1 Morphological Appearance 16

3.3.2 Gram reaction 16

3.3.3 Endospore stain 16

3.3.4 Biochemical characteristics 17

3.3.5 Growth at 55Oc 19

3.4 Screening of bacitracin production 19

3.5 Variation of culture conditions 19

3.5.1 Variation of incubation time 20

3.5.2 Variation of temperature 20

3.5.3 Variation of ph 20

3.5.4 Variation of salinity 21

3.6 Effect of varying culture conditions on bacitracin activity 22

CHAPTER FOUR

4.0 Results 23

CHAPTER FIVE

5.0 Discussion and Conclusion 30

5.1 Discussion 30

5.2 Conclusion 31

REFERENCES 32

APPENDIX 35

LIST OF TABLES

Table Title Page

1a Characterization and identification of bacillus subtilis from the samples collected. 24

1b Confirmation of the test organisms Staphylococcus aureus and 

Micrococcus lateus. 25

LIST OF FIGURES

Figure Title Page

1 The effect of temperature on bacitracin production 26

2 The effect of incubation time on bacitracin production 27

3 The effect of pH on bacitracin production 28

4 The effect of salinity on bacitracin production 29