THE ISOLATION, IDENTIFICATION, AND ENUMERATION OF BACTERIA IN THE ORAL CAVITY

ABSTRACT

This study aimed to isolate, identify, and enumerate bacteria present in the oral cavity of students from Michael Okpara University of Agriculture, Umudike. The oral microbiota, comprising a complex community of microorganisms, plays a crucial role in maintaining oral health and influencing disease outcomes. Samples were collected using sterile swabs from students before and after brushing their teeth, providing insight into bacterial load variations. The research sought to isolate and identify bacterial species, quantify their presence, and examine their roles in oral health and disease.

Bacterial isolation was performed using the pour plate method, followed by enumeration through colony counts on Nutrient and MacConkey agar. Serial dilutions were prepared, and bacterial colonies were cultured and identified using biochemical tests and Gram-staining techniques. A variety of bacteria were isolated, including Streptococcus spp., Staphylococcus spp., Veillonella spp., Lactobacillus spp., and Fusospirochetes spp. These results align with previous studies, indicating the oral cavity's diverse microbial population.

The study revealed that Streptococcus spp. was the most prevalent bacterium, playing a critical role in the formation of biofilms and contributing to dental caries. Lactobacillus spp. was identified as part of the normal flora, known for creating an acidic environment that inhibits the growth of other bacteria. Staphylococcus spp., typically transient in the oral cavity, was associated with periodontal disease. Other bacteria, such as Veillonella spp., were found to thrive in acidic environments and may slow the progression of dental caries.

The findings highlight the dynamic balance of oral microbiota, where some bacteria protect against disease, while others can promote infection under certain conditions, such as poor oral hygiene. Bacterial counts were significantly reduced after brushing, demonstrating the importance of oral hygiene in controlling bacterial growth. The study emphasizes the need for maintaining proper oral health practices to prevent infections and periodontal diseases.

TABLE OF CONTENTS

CHAPTER ONE

1.0       Introduction

1.1     Biofilm and Oral Microbiota  

1.2     Justification of Study

1.3     General Aims

1.4       Specific Aims                                                                                                      

CHAPTER TWO

LITERATURE REVIEW

2.0       The Mouth As A Microbial Habitat

2.1     Heterogeneity of Oral Microbiota

2.2     Development of Resident Microflora

2.3     Characteristics Interactions between Oral Microorganisms.

2.4.   Cell-Cell Communication

2.5    Oral Bacterial

2.5.1  Streptococci

2.5.2  Lactobacillus

2.5.3  Actinobacillus Actinomycetemcomitans

2.5.4 Veillonella

2.5.5 Fusospirochetes

2.6     The Oral Microbiota and Disease

2.7     What Can We Do To Minimize Disease Caused By The Oral Microbiota?

2.8     Roles of Resident Microbiota in the Oral Cavity

CHAPTER THREE

3.0     MATERIALS AND METHOD

3.1     Study Area

3.2     Collection of Sample

3.3    Media and Media Preparation

3.4     Isolation and Enumeration of Bacterial from Oral Cavity

3.5     Identification of Bacteria Isolates

3.5.1   Gram-Staining

3.5.2   Biochemical Reaction Tests

3.5.2.1   Coagulase test

3.5.2.2   Citrate test

3.5.2.3   Oxidase test

3.5.2.4 Motility test

CHAPTER FOUR

4.0  RESULTS

CHAPTER FIVE

5.0     Discussion and Conclusion

5.1     Discussion

5.2     Conclusion

References

LIST OF TABLES

Table 4.1 :       Microbial (bacterial) count of oral cavity.

Table 4.2:        Bacteria isolated from oral cavity and their percentage occurrence

Table 4.3:        Biochemical test reaction

CHAPTER ONE

1.0      INTRODUCTION

The human oral cavity is a diverse environment with hard and soft tissues comprising a total area of 215cm² bathed in saliva (Eleni Kanasi, 2008). The oral environment is thus optimal for their architecture and characteristics to biofilm in nature (Auschill et al., 2001). Specific to oral biofilms is the ability of sessile oral bacterial cells to tolerate short-term abundance of external nutrient supply and to withstand nutrient restraint. Nutrient deprived biofilm cells after reactivation have shown to exhibit low reactivity invitro, suggesting that slower reactivation of these cells might be a survival strategy (Chavez de paz et al., 2008).

The oral cavity of healthy individuals contains hundreds of different bacterial, viral and fungal species. Many of these can associate to form biofilm which are resistant to mechanical stress and antibiotic treatment. It has been a long journey from the beginning to the study of oral microbiota consisting of an unknown number of bacteria. But not all species residing in the oral microbiota have been identified yet and an estimated 750 different species are anticipated (Jenkinson and Lamont, 2005;Paster et al.,2006).

Most of these micro organisms exist in our oral cavity in a symbiotic capacity maintaining relationships with the hosts that are based on mutual benefits (los Alamos National Library, 2009). Not only do they not cause harm, but also the commensal populations may keep pathogenic species in check by not allowing them to adhere to mucosal surfaces. Numer4oyus factors impede the isolation of this vast number of species. First and foremost, many of the species are not cultural with today’s laboratory technologies, and genomic similarities do not allow for organismal determination based on short read lengths. Because of the limitations, researchers have begun by identifying with the communities of healthy mouth: Streptococcus, Actinomyces, Staphylococcus, Veillonella, Neisseria, Eubacteria, Leptotrichia, Fusobacterium etc.(Jenkinson and Lamot 2005; Wilson, 2005).

1.1   BIOFILM AND ORAL MICROBIOTA  

Some bacterial invaders overcome the immune response by farming mixed biofilms consisting of commensals and potential pathogens to cover fly hide within the host (costerten, 2007). The formation of biofilm may occur on a wide variety of surfaces in the oral cavity. Thus epithelial cells, saliva-coated enamels, dental surfaces, primary colonizing bacteria and orthodontics all create suitable environments for the establishment of mixed species biofilms (Jekinson and Lamont, 2005; Marsh 2006). Bacteria cells in oral biofilms interact by various recognized ways including co-aggregation ( Kolenbrander et al.,2006), metabolic exchange, cell-cell communication ( Li et al., 2006) and exchange of genetic material ( Robert et al., 2001). These mechanisms benefits bacterial survival and can make dental biofilms difficult therapeutic targets in dental diseases. At the beginning of biofilm formation, initial surface attachment occurs by the primary colonizers, resulting in disposition of a microbial monolayer.

The primary colonizers in the oral microbiota for both mucosal and tooth surfaces are usually streptococci which make up approximately 80% of early biofilms (Maria Avila et al., 2009).

The oral microbiota also faces challenges that are not experienced by other microbiotas. The host has the option to maintain good personal hygiene, the cleansing mechanism especially deliberate oral hygiene can alter the natural succession that would otherwise define the climax community. This contributes to the quantity and composition of mixed species biofilm in the oral cavity. In response to eating, salivating, tooth brushing, tongue movement, flossing, and other agitation, the oral microbial communities have evolved the skills to survive these inhibitory practices.

Although biofilms are required for health of the oral cavity, biofilms are also known to contain pathogens (Ruby and Barbeau, 2002). Periodontal diseases such as chronic gingivitis and periodontitis can result from an increase in the crevice. The biofilms are typically comprised mainly of Gram-positive facultative anaerobes, but in the absence of proper hygiene, the percentage of Gram-negative species in the biofilms increases, contributing to periodontal inflammation (Ruby and Barbeau, 2002).

1.2   JUSTIFICATION OF STUDY

With our population aging and living longer lives, health management and disease prevention become increasingly important. Improved knowledge of the oral microbiota and their impact/role on our health will contribute greatly to health of the society as a whole. Also a better understanding of their dynamic complexity of oral microbiota will contribute to the next-level in medical diagnostic tool. Ideally, this study should also lead to providing the potential to manipulate the microbiota to optimize personal health.

1.3   GENERAL AIMS

The objective of this project research is to isolate, identify, enumerate and investigate the role of the oral microbiota using microbiological and clinical methodology.

1.5      SPECIFIC AIMS                                                                                                             

v  To isolate and identify bacteria from the oral cavity.

v  To enumerate oral microbiota.

v  To identify the roles of these organisms.

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