Professional Documents
Culture Documents
Methods To Find Which Types of Bacteria Are Present in The Mouth
Methods To Find Which Types of Bacteria Are Present in The Mouth
Methods To Find Which Types of Bacteria Are Present in The Mouth
mouth of a patient
Dr.Sonalee SHAH
Bacterial identification is the first step in establishing the bacterial etiology of a
particular disease. It includes the procedures and techniques used to correctly
identify the bacterial pathogens responsible for disease. Bacteriologist employs a
wide variety of techniques, based upon known characteristics of specific bacteria,
to arrive at the identity of a given specimen. The traditional way of detecting and
identifying bacteria from given specimen is based on :
culturing,
enumeration and
Principles of identification:
Conventional methods for further subtyping of bacteria include - the study of the
phenotypic characteristics of the microorganisms.
Serotyping and Phage typing : serological and phage typing concentrate more on
the surface structure differences of bacteria.
Most of the bacterial species found in the mouth belong to microbial communities,
called "biofilms", a feature of which has inter-bacterial communication, mediated
by two distinct phenomena.
The first is through direct cell-cell contact, which is mediated by specific protein
"adhesins" and often, as in the case of inter-species aggregation, also by
complementary polysaccharide receptors.Intra (auto-) and inter-species (co-)
aggregation both promote ordered successional integration of species into the
biofilm.
The rapid development of molecular techniques during the past decade has
revolutionized the field of microbiology.
Of considerable benefit also, has been the fact that the same nucleic acid-based
molecular approaches can be applied in all microbial environments, ranging from
the oral cavity, to the surfaces of historical monuments, to the depths of open
oceans.
By comparative analysis of fully- sequenced oral bacteria and between them and
non-oral relatives, it is clear that horizontal gene transfer (HGT) is extremely
frequent in oral pathogens and that this process probably also takes place also
between bacteria and oral fungi. It is likely that HGT is facilitated, in oral biofilms
such as dental plaque, by the close physical contact between phylogenetically
distant bacteria. .
In some cases, certain sets of genes are shared between more than two species co-
inhabiting the mouth ecosystem, supporting the idea that there is an environment-
associated gene pool formed by sequences of special adaptive importance. This
habitat gene reservoir is also present in other natural ecosystems.
To extract the bacterial DNA, the bacterial cell wall must be lysed with out damage
to the DNA. Several methods are available.
Methods that yield high recovery of DNA in one organism might not yield same
amount in another.
Fig-4: METHODS OF DNA RECOVERY
1a. Primer
Almost every DNA based method uses PCR as it allows detection of DNA from as
low as one cell which is possible to do due to extensive, detailed analysis .Thus it
allows : Specific amplification of DNA from a target species even in the presence
of hundred of species.
Nested PCR
Multiplex PCR
Disadvantage of qPCR
It measures-
It is the gold standard to distinguish bacterial species, with a similarity value
greater than 70% indicating that the compared strains belong to distinct species.
Thus, it is used to identify the bacterial species by the criteria that, every bacterial
species may have at least one or many unique sequences of DNA & so is the
superior method for establishing bacterial species.
The gene target most commonly used for bacterial identification & classification
is the 16S rRNA gene, which is an approximately 1500-bp gene encoding a portion
of the 30S ribosomal subunit .
16S rRNA is a type of RNA that plays a major role in synthesis of protein. rRNA
contains very well-conserved regions among biological species, which makes the
comparison of 16S rRNA sequences possible in studies of molecular evolution.
16S rRNA sequences also enable the identification of microorganisms because the
16S rRNA contains variable sequences that change according to different species.
More than one 16S rRNA sequences may exist in a single bacterium.
It is considered to be fast and better alternative to other methods of bacterial
identification. Along with its use identifying the bacteria, 16S sequencing can also
be used to re-categorize the bacteria into new species.
The microflora within the oral biofilm is quiet complex & determination of the
microbial populations responsible for oral infections is even more difficult. Also,
Bacteria present in the oral cavity are both, gram positive and gram negative.
Therefore, knowledge of the ecological relationships among bacterial species in a
habitat of oral cavity can direct investigations on the critical bacterial interactions.
Hence,16S rRNA genes have been used to investigate the composition samples
from the human oral cavity. The 16S rRNA gene sequences are compared with
other known 16S rRNA sequences to identify the bacteria species. So far, around
700 orally derived 16S sequences are deposited in GenBank .
Advantages of 16SrRNA:
16S rRNA gene sequence analysis is not only widely used as a taxonomic
tool but also recognized as an effective reference method for bacterial
identification .
Samples for 16S rRNA sequence analysis have been expanded from the
bacteria in environments such as the oceans and soils to clinical settings.
It allows to obtain accurate information regarding contaminated
environments within health care facilities, & comprehensive reevaluation of
microorganisms using culture-independent methods .
16S rRNA gene sequence analysis shows higher sensitivity to detect
specific bacteria than the usual culture method
16S rRNA gene sequence analysis has been used to identify novel and
emerging pathogens and to define complex microbial communities . This
analysis is especially valuable for detecting bacteria that are slow growing,
biochemically inert or variable, and fastidious.
In addition, 16S rRNA gene sequence analysis has enhanced our
understanding of previously unrecognized, often opportunistic pathogens.
According to recent studies, this molecular biology analytical method has
been used in the search of pathogens of infectious diseases, reporting a
higher rate of detection than the usual culture method .
Indeed, 16S rRNA gene sequence analysis provides a comprehensive
assessment of microbial diversity compared with culturing alone, and is an
excellent complement to the culturing approaches.
Limitations of 16SrRNA method:
Although 16S rRNA sequencing is being used widely for bacterial
identification, there are no specific guidelines for using the technique and
interpreting the sequence data. As , no threshold values are available, one
of the major drawback in the interpretation of sequence data is concerned
with the assigning the bacterial species in response to the likeness of search
results.
This technique may not be useful when two different bacterial species share
almost the same 16S rRNA sequence.
5. FISH & Microscopy :
The detection of whole-bacterial cells via the labeling of specific nucleic
acids with fluorescently labeled oligonucleotide probes is called
Fluorescence In Situ Hybridization (FISH).
The rRNA molecule, which exists in multiple copies in the cell (up to 104-
105), is an excellent target for fluorescently labeled oligonucleotide probes
which are directed against regions on the rRNA molecule specific for a
bacterial group, genus or species.
The small probes (16-20 nucleotides) cross the bacterial cell wall and
hybridize with their complementary target sequence.
Evaluation of the test result is done by epifluorescence microscopy.
1. Cultivation of bacteria:
Culture involves placing some of the specimen in conditions where the organism
or organisms of interest can grow and multiply. Once grown in culture, most
bacterial populations are easily observed without microscopy and are present in
sufficient quantities to allow laboratory testing to be performed. The successful
transition from the in vivo to the in vitro environment requires that the nutritional
and environmental growth requirements of bacterial pathogens be met.
■ To determine which of the bacteria that grow are most likely causing infection
and which are likely contaminants or colonizers.
Growth media are used in either of the two phases: liquid (broth) or solid (agar)
In some instances, a biphasic medium that contains both a liquid and a solid phase
is used.
Liquid (broth) media : These are liquid media in which bacteria grow uniformly
producing general turbidity. Certain aerobic bacteria and those containing fimbriae
(Vibrio & Bacillus) are known to grow as a thin film called ‘surface pellicle’ on
the surface of undisturbed broth. Liquid media tend to be used when a large
number of bacteria have to be grown from an inoculum suspected to be low in
bacterial numbers.
Solid (agar) media: These are are made by adding a solidifying agent to the
nutrients and water. Agar agar (simply called agar) is the most commonly used
solidifying agent. Agar doesn’t contribute any nutritive property, it is not
hydrolyzed by most bacteria and is usually free from growth promoting or growth
retarding substances.
Enrichment media
Supportive media
Selective media
Differential media
The four most critical environmental factors affecting microbial growth in culture
They include :
2. temperature,
3. pH, and
These tests usually provide rapid results because they can be performed on
organisms already grown in culture & are commonly used to determine the
presence of a single enzyme.
These tests are easy to perform and interpret and often play a key role in the
identification scheme.
Although most single enzyme tests do not yield sufficient information to provide
species identification, they are used extensively to determine which subsequent
identification steps should be followed.
For example - the catalase test can provide pivotal information and is commonly
used in schemes for gram-positive identifications. The oxidise test is of
comparable importance in identification schemes for gram-negative bacteria.
Various EB tests are:
To summarise :