A REVIEW ON THE HUMAN ORAL

MICROFLORA AND THE EFFECTS OF

SEVERAL COMMERCIAL AVAILABLE
PRODUCTS ON THE MICROFLORA.

MADE BY:- Ankita Dogra

Bsc biotechnology (hon’s) VI SEMESTER
PRESENTED TO:- Ms Shruti Gupta
ABSTRACT
The presence of nutrients, epithelial debris and secretions make the mouth a
favoral habitat for a great variety of microorganisms.

Oral bacteria include:- Streptococci, lactobacilli, staphylococci,

corynebacteria, with a great number of anaerobes, especially bacteroides.

An investigation was made of the effects on the human micro-flora resulting

from daily consumption of four commercial available products:- tea,
toothpaste, chocolate, and carbonated drinks. The aim of the study is to
determine the effects of these four products on the human microflora of
different students.

The finding of this study have illustrated that:-

 Results from the effect of toothpaste shows that these are active
toothpaste because of their inhibition zones, these toothpaste have
accetptable to antibiotic potency and they also contain fluoride which
helps to prevent tooth decay.

 Extracts of chocolate, tea was found to have no inhibitory effect on

human mouth flora

 Extracts of sugar-flavored acid beverages was found to have inhibitory

effect on the human mouth flora.
INTRODUCTION
In an healthy animal, the internal tissues are free of microflora, but the
external tissues such as oral cavity may effect with different types of
microorganisms.

Oral cavity is the initial part of the gastrointestinal tract, due to the regular
supply of food it makes an environment for the growth of microorganisms.

Oral Microflora has numerous organisms which include Bacteria, Fungi,

Protozoa mostly and rarely virus. Among these organisms, bacteria play a
vital role in causing diseases.

The ecological condition changes from primary to permanent dentition. The

microorganisms vary depending on the food intake, saliva and antibiotics
consumed.

The gingival crevice area (which supports the structures of teeth) provides a
habitat for broad group of anaerobic species. Oral microorganisms cause
major dental diseases namely periodontal disease and dental caries.
CLASSIFICATION
1. Oral Bacteria Classification has been

classified based on Gram’s Staining

➢ Gram Positive

➢ Gram negative

2. Based on the effect of oxygen bacteria

are classified as:

➢ Obligate aerobe

➢ Micro aerophilic

➢ Facultative anaerobes

➢ Obligate anaerobe

Based on the Gram’s Staining the bacteria can be classified into the following (Table 1):
ORAL MICROFLORA
The oral cavity of infant is free from bacteria but rapidly normal flora will be
colonized such as Streptococcus salivarius. As these microorganisms colonise
the dental surface and gingiva, Colonization of Streptococcus
sanguinis and Streptococcus mutans are formed on the teeth. Other strains
of streptococci adhere strongly to the cheeks and gums but not to the teeth.
Oral flora of an Adult is Due to the
Characteristics
Dental plaque formation
The dental plaque formation involves huge colonies of bacteria. Dental
Plaque is a biofilm on the surface of the teeth . Due to the large number of
colonies of microorganisms, they produce more amount of metabolites
which results in dental disease on the teeth and gingival tissues. If the teeth
is not hygienated through brushing or flossing, then the plaque forms into
tartar (its hardened form) and leads to periodontal disease or gingivitis.

Biofilm formation
Some of the indigenous bacteria construct biofilm on a surface tissue or

they colonize a biofilm by taking the help of another bacterial species.

Development of the Resident Oral Microflora

Generally, the foetus in the mother’s womb will be sterile. In children below
3 years of age, their mouth comprises of microbes, which are passed by
passive transfer from mother i.e; through milk, microbes which are present in
water, milk and the general environment will be transferred to children
including saliva, it is the main vehicle for transmission .
Functions of the resident Oral Microflora
in Health
The resident microflora plays an important in health of human. Microflora
acts against the pathogens and protects the body from entering of several
microbes. Resident Microflora helps in the metabolism of the body.

Some strains such as Streptococcus salivarius produces bacteriocin called as

salivaricin. It shows activity against Lancefield Group A streptococci.
Production of bacteriocin by such strains in the pharynx will reduce bacterial
colonies in the mouth. Similarly, many oral bacteria produce other inhibitors
such as hydrogen peroxide, volatile fatty acids, they change local
environmental conditions (e.g. redox potential or pH), which may exclude
exogenous species and suppress opportunistic pathogens. For example, the
production of hydrogen peroxide by the members of the Streptococcus
mitis which can suppress the growth in the dental plaque of periodontal
pathogens, such as A. actinomycetemcomitans.
MATERIALS AND METHODS
 Saliva (1ml)

 Inoculating loop

 petriplates

 Nutrient agar and broth

 Gram staining kit( crystal

violet,iodine,decoloeizer-
ethanol or
acetone,counterstain-
safranin)

 L-shaped spreader

 Incubator

 Clean beaker, test tubes

 Sterile pipettes and tips

 Glass rod

NUTRIENT AGAR,BROTH
GRAM STAINING
PROCEDURE
CULTURING AND ISOLATION

 Collect and filter the saliva in a test tube (1ml),

 Pipetted the 1ml saliva onto the nutrient agar plate and spread with the
help of L-shaped spreader,

 Incubate the plate at 37*C for 24 hours,

 Count the different colonies, pick and streak them on the agar plate.

STREAKING

 Different colonies were picked up and streaked on the nutrient agar

plate,

 Incubate the plates at 37*C for 24 hours,

 Observe the different colonies (masterplate).

IDENTIFICATION (GRAM STAINING)
Gram stain or Gram staining, also called Gram's method, is a method
of staining used to distinguish and classify bacterial species into two large
groups (gram-positive and gram-negative). The name comes from the
Danish bacteriologist Hans Christian Gram, who developed the technique. The
Gram stain is almost always the first step in the preliminary identification of a
bacterial organism.

Staining mechanism

Gram-positive bacteria have a thick mesh-like cell wall made

of peptidoglycan (50–90% of cell envelope), and as a result are stained purple
by crystal violet, whereas gram-negative bacteria have a thinner layer (10% of
cell envelope), so do not retain the purple stain and are counter-stained pink
by safranin. There are four basic steps of the Gram stain:

Applying a primary stain (crystal violet) to a heat-fixed smear of a bacterial

culture. Heat fixation kills some bacteria but is mostly used to affix the bacteria
to the slide so that they don't rinse out during the staining procedure.

The addition of iodide, which binds to crystal violet and traps it in the cell

Rapid decolorization with ethanol or acetone

Counterstaining with safranin.[10] Carbol fuchsin is sometimes substituted for

safranin since it more intensely stains anaerobic bacteria, but it is less
commonly used as a counterstain.
Purple-stained gram-positive (left) and pink-
stained gram-negative (right)
PREPARATION OF BACTERIAL LAWN

 Nutrient broth medium was prepared and 10ml was distributed in the
test tubes, plugged and autoclaved,

 Loopful of culture was transferred aseptically in the nutrient broth, the

tube was incubated at 37*C for 24 hours

 Pipetted 1ml of the broth and spread onto the nutrient agar plate and
incubate at 37*C for 24-48 hours,

 Observe the bacterial lawn (individual colonies merge to form a field or

mat of bacteria).

PREPARATION OF AGAR WELLS

 Using a sterile cork borer to punch holes on the centre of the agar plate
or to cut wells use a pipette tips and gently press the tip on the gel,
going all the way to the bottom of the gel layer, release the
compression, this should pull out the gel plug out of the well and into
the tip.

EXTRACT PREPARATION

Tea :-

-Take a tea and boil into the water, let it cool.

-Only 1-2 drops of solutions are needed to fill the well with the help of the
pipette.

-The plates were incubated at 37*C for 24 hours.

Toothpaste (Colgate):-

-Take a Colgate and dilute into the water and mix by swirling.
-only 1-2 drops of solutions are needed to fill the well with the help of the
pipette.

-The plates were incubated at 37*C for 24 hours.

Chocolate :-

-Take a chocolate and dilute into the water.

-Dissolve the Chocolate in the water by swirling.

-Only 1-2 drops of solutions are needed to fill the well with the help of the
pipette.

-The plates were incubated at 37*C for 24 hours.

Cold drink (Mirinda):-

-Take Mirinda in a beaker.

- Only 1-2 drops of solutions are needed to fill the well with the help of the
pipette.

-The plates were incubated at 37*C for 24 hours.

RESULTS AND DISCUSSION
IDENTIFICATION OF BACTERIA (GRAM STAINING)

Type of bacteria shape

Gram –ve bacteria bacillus

EFFECTS OF EXTRACTS

CARBONATED DRINKS zone was formed Inhibiting effect on

microflora

TEA No zone was formed No effect

CHOCOLATE No zone was formed No effect

TOOTHPASTE Zone was formed Inhibiting effect on

microflora
EFFECT OF TOOTHPASTE
EFFECT OF TEA
NO ZONE FORMATION
NO ZONE FORMATION

EFFECT OF CARBONATED
DRINKS

ZONE IS FORMED

EFFECT OF CHOCOLATE

NO ZONE FORMATION

EFFECT OF TEA

NO ZONE FORMATION
BIBLIOGRAPHY
 A review on the human oral microflora

Author:- Sowmya Y M.sc Mirobiology, Andhra University

 The antibacterial effect of toothpastes on the salivary flora

J. Moran  M. Addy R. Newcombe

 The Effects of selected toothpaste on the microbial flora

C.Nwakanma1 , C.J.Ejim2 and M.N.Unachukwu2

 https://www.slideshare.net/wali303/oral-flora-25031102

 Wikipedia.