STUDY OF SOIL BACTERIAL DIVERSITY BASED ON DIFFERENTIAL AND STRUCTURAL STAINING

ABSTRACT

The study of soil bacterial diversity based on differential and structural staining were carried out on three different soil namely sandy soil, clay soil, and loamy soil, these samples were serial diluted and inoculated on three different agar plates: nutrient agar, macConkey agar and Eosin Methylene Blue (EMB) agar; after incubation diverse bacteria growth were isolated which include Bacillus subtilis, Escherichia coli, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa, Enterogene aerogenes, Staphylococcus aureus. Further identification techniques were used which involves differential and structural staining, under the endospore staining Bacillus subtilis was found to be a spore forming bacteria identified.

TABLE OF CONTENTS

Cover page   i

Title page ii

Certificate Page         iii                           

Dedication   iv

Acknowledgement                                         v

Contents                                                                             vi                        

List of Tables                                                              vii

Abstract                                       viii

CHAPTER ONE

1.0    INTRODUCTION                                                                                                        1

1.1     Justification of study                                                                                                      3

1.2     Specific objectives                                                                                                         3

1.3     General limitations in studying bacterial diversity                                                        4

CHAPTER TWO

2.0       LITERATURE REVIEW                                                                                          5

2.1       Introduction                                                                                                                5

2.1.1    Biogeochemical processes                                                                                          8                                                                                                                                                   

2.1.2   What we don’t know                                                                                                  10

2.1.3   Increasing cultivation efficiency                                                                                 11

2.2      Spatial heterogeneity                                                                                                   11

2.3      Inability to culture soil bacteria                                                                                   13

2.4      Limitations of molecular-based methods                                                                    14

2.5     Taxonomic ambiguity of microbes                                                                               15

2.5.1   Methods of studying bacterial diversity in soil                                                           17

2.6      Biochemical-based techniques to study bacterial diversity                                         18

2.7      Future perspective                                                                                                        19

CHAPTER THREE

3.0      MATERIALS AND METHODS                                                                                23            

3.1       Sample collection                                                                                                         24 

3.1.1    Determination of soil temperature                                                                               24                      

3.1.2   Determination of soil pH                                                                                              24

3.1.3   Sampling depth                                                                                                             25

3.2      Processing of sample and media preparation                                                                25

3.3      Isolation of bacteria from three different soil sample                                                   28

3.4      Determination of groups of isolates based on differential techniques                          29

3.5      Determination of groups of isolates based on structural staining                                 29

3.6      Biochemical tests                                                                                                          30

3.6.1   Indole test                                                                                                                     30

3.6.2   Catalase test                                                                                                                  30

3.6.3   Citrate utilization test                                                                                                   30

3.6.4   Coagulase test                                                                                                               30

3.6.5   Motility test                                                                                                                   31

3.6.6   Urease test                                                                                                                     31

3.6.7   Oxidase test                                                                                                                   31    

CHAPTER FOUR

4.0      RESULTS                                                                                                                    32 

CHAPTER FIVE

5.1       Discussion                                                                                                                     34

5.2       Conclusion     

 References

LIST OF TABLES

Table        Title                                                             Page

4.1    Bacterial isolates                    32

4.2   Colony count                                                        32

4.3   Morphology and Biochemical test                       33

                                              CHAPTER ONE

1.0       INTRODUCTION

The term soil refers to the outer loose material of the earth crust. It may be regarded as a three phase system composes of solid, liquid and gases, dispersed to form a heterogenous matrix. On the whole, the soil is composed of five major components, these includes mineral matter, water, organic matter, air and living organisms. The various components of the soil environment constantly changed and the quantity of these constituents are not the same in all soil but vary with locality. Living portion of the soil body includes small animals and microorganisms but it is generally considered that its microorganisms that plays the most important in the release of nutrients and carbon dioxide for plant growth. The bacteria and the most abundant group usually more numerous than the four combined. Soil bacteria can be rod (bacilli),cocci (spherical), spirilla(spirals) of these,bacillus are more numerous than the others. They are one of the major groups of soil bacteria population and are very widely distributed (Bhagabati et al.,2010). The number and type of bacteria present in a particular soil would be greatly influenced by geographical location such as soil temperature, soil type, soil pH, organic matter content, cultivation, aeration and moisture content (Davies et al., 2012).

Bacteria are some of the smallest and most abundant microbes in the soil. In a single gram of soil, there can be billions of bacteria. There are an estimated 60,000 different bacteria species, most which have yet to be even named and each has its own particular roles and capabilities. Most live in the top 10cm of soil where organic matter is present. Soil bacteria dominate or participate in a number of earth’s biogeochemical cycles,including the carbon, nitrogen, oxygen, phosphorus and sulfur cycles.(Giri et al.,2012). Some of the specific processes of soil bacteria include carbon fixation, decomposition of organic matter, respiration, nitrogen fixation, nitrogen mineralization, nitrification, denitrification, phosphorus mineralization and sulfur oxidation and reduction.   (Wu et al.,2013).

Such large scale effects by these tiny cells are possible because of their fantastic abundance. One gram of soil contains, on average a billion bacterial cells (Schloss et al.,2013),though not all of these may be viable. The majority of bacteria in soils are found in the top 1 meter of soil (Whitman et al.,2011) and according to the Food and Agriculture Organization (FAO) of the United Nations(2000),there are approximately 31,823000km² of non-constained agricultural soil on earth. Presuming one gram of soil is approximately equal to 15 cubic. Centimeters, then there are 2.12x10²⁴total bacterial cells in arable soil on earth.(Whitman et al.,2011),however determined there are approximately 2.6x10²⁹ total soil bacteria cells on earth by including the estimated bacterial abundance of desert and other  non-arable soils and adding a calculated abundance from depths of 2 to 8 meters.

1.1    JUSTIFICATION OF THE STUDY

The study points out that this is a biome with a vast reservoir of bacteria which degrades with increasing attitudes, and highlights the microbiological importance of the poorly studied soil range, justifying efforts to explore the prevalence of novel species in the biome.

1.2     SPECIFIC OBJECTIVES

1.   To isolate microorganisms from three different soil environment or three different soil types; sandy, clay and loamy soil.

2.   To determine the groups of the isolates based on the differentiation techniques.

3.   To determine the group of the isolates based on structural techniques.

1.3   GENERAL LIMITATIONS IN STUDYING  BACTERIAL DIVERSITY.

 There are problems associated with studying bacterial and fungal diversity in soil. These arise not only from methodological limitations, but also from a lack of taxonomic knowledge. It is difficult to study the diversity of a group of microorganisms when it is not understood how to categorize or identify the species present.

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