identification methods for oral microbes
DESCRIPTION
Develop an understanding of Taxonomy (classification) of Oral Microorganisms Describe how to obtain samples from Oral Cavity Describe Molecular techniques of identification Describe techniques that requires culture for identificationTRANSCRIPT
Identification Methods of
Oral MicrobesDr. Ali Yaldrum
Faculty of DentistrySEGi University, Kota Damansara, Malaysia
18-06-12
Learning Objectives
At the end of this session, the student should be able to:
• Develop an understanding of Taxonomy (classification) of Oral Microorganisms
• Describe how to obtain samples from Oral Cavity• Describe Molecular techniques of identification• Describe techniques that requires culture for identification
1
DiagnosticCycle
Infection?Virus? culture
2
Clinician request
*A-A-ah
3
collection
4 transportation
5
labortary analysis
6
data flow
What the!!!
7
interpretation
8diagnosis +treatment
1. prokaryotes VS eukaryotes
prokaroyte
cell wallpeptidoglycan singular supercoiled
circular chromosome
cytoplasm rich in ribosomes
plasmid cellmembrane
flagellum
(Fig.1)
eukaroyte
nuclear membrane
mitochondriacell membrane
cytoplasm
smooth endoplasmic reticulum
lysosome
rough endoplasmic reticulum
Golgi apparatus
(Fig.2)
2. Classification & Identification
classification
‘Classification is the arrangement of Organisms into groups (taxa) on the basis of their similarities and differences.’
The science of classification is called taxonomy
KINGDOM
PHYLUM
CLASS
ORDER
FAMILY
GENUS
SPECIES
SUB SPECIES
DIVISION
taxonomic hierarchy (Fig.3)
identification
‘is the process of determining that a new isolate belongs to particular taxon’
• Bacteria are identified using phenotype, immunological or molecular characteristics
why this is important
• Revealing their identity• Behavior and likely response to treatment• Also to predict their pathogenicity• Isolate microorganisms that spread in community & cause
serious disease
Bacterial Classification
Shapes
Atmosphere
Obligate aerobesMicroaerophiles
Obligate anaerobesFacultative anaerobes
Capnophiles
requires O2
requires reduced O2
requires no O2
anaerobic or aerobic
requires increases CO2Spores
Key EnzymesBacteria lacking certain enzymes
Serological ReactionInteraction of antibodies with certain surface structures
DNA SEQUENCINGDNA sequencing of key genes ; Ribosomal 16S gene
peptidoglycan Teichoic acid
Plasma membrane
Outer membraneprotein
Thin peptidoglycan
layer
Cell wall
Gram +ve Gram -ve
GramReaction
(Fig.4)
Coccus
Bacillus
Coccobacillus
Fusiform bacillus
Vibrio
Spirochete
Spirillum
bacterial shapes
(Fig.5)
3. sampling Oral bacteria
• Oral Cavity contains a variety of different niches that harbour distinctive communities of bacteria.
• Location and environment determines the diversity of eco system.
Studies of various niches have shown distinctive microbial profiles for different locations
• Tongue • Tooth surface• Gingival sulcus• Buccal mucosa• Gingival crevice
gingival sulcus1
2
3
4
5
6
1= Enamel2= Dentine3= Pulp
4= Free gingivae5= Cementum6= Alveolar bone
(Fig.6)
sampling saliva
• Easily sampled• Contains a mix of bacteria (planktonic)• Patient is asked to chew paraffin prior to collecting saliva• Results in enriched tooth derived saliva• Used for collection of large population samples
sampling plaque
2 approaches can be used
1.Supragingival plaque2.Subgingival plaque
Supragingival
• Curette is used to scrap the biofilm of the tooth surface (fig. 7)
• Can not be inserted more than 6mm
Periodontal Curette(Fig.7)
Subgingival
• Endodontic paper (paper point) can be used (fig. 8)• For pockets deeper than 6mm• Wicks up fluid containing bacteria• Large number of bacteria can be obtained
Endodontic Paper (paper point)(Fig.8)
4. Identifying Oral bacteria
Approaches to identifying bacteria can be grouped into 2major categories
• Techniques that do not require culture (molecular identification techniques)
• Techniques that require culture
*At present combination of both techniques is used to characterize the full compliment of organisms
molecular identification
• are most often based on sequence analysis of the ribosomal 16S genes
• Common techniques for molecular detection of bacteria:1. PCR with specific primers2. Quantitative PCR3. DNA hybridization assays4. Ribosomal 16S cloning & sequence analysis5. FISH and microscopy
bacterial DNA recovery
• 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
dna recovery
COMMERCIAL “KITS’
• Target one type of bacteria• Variable intensity• High specificity
Detergents& Proteinase K
• Lyse a wide spectrum of bacteria
• Unable to lyse Gram-ve bacteria
Bead Beating
• Bacteria are mixed with small slurry of tiny glass beads
• Vile is placed in a vibrating apparatus
• Will lyse the most sturdy bacteria's
• Not used for fragile bacteria
what is PCR
• Or “Polymerase Chain Reaction”
‘It is the process which results in cyclic amplification of target DNA using specific primers, theoretically from one single cell’
what is a Primer
The simplest explanation of a primer is to consider it as a “key”, as every key is specific to a particular lock.
So every primer is a strand of nucleic acid specific to a specific strand of DNA from a specific specie
what is a Primer
‘A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. They are required for DNA replication because the enzymes that catalyze this process’
*Primers are usually short, chemically synthesized oligonucleotides, with a length of about twenty bases
PCR
• Almost every DNA based method uses PCR• Allows detection of DNA from as low as one cell• Possible to do extensive, detailed analysis• Specific amplification of DNA from a target species even in
the presence of hundred of species
variations of PCR
The basic PCR methodology is modified to provide sophisticated analytical tools•Nested PCR•Multiplex PCR•Real Time PCR
Real-time PCR
• Conventional PCR requires Gel-electrophoresis for amplification analysis
• Labelled probes are used
• Multiple amplifications can be analysed at specific time period during reaction period
Watch Video ofPCR & Gel Electrophoresis
https://www.youtube.com/watch?v=GLgt-EGkhZs&feature=related
why is PCR widely used
• Even a minuscule quantity of DNA can be studied, as a single DNA molecule is adequate for amplification
• Rapid Clinical diagnostic procedures. Sensitivity of PCR enables rapid diagnosis
• Enables identification of different species. PCR allowed researcher to identify uncultivable bacteria
DNA Hybridization
• Measures the degree of genetic similarity between pools of DNA sequences (fig. 9)
• Possible to determine the genetic distance between two sequences
• Because of the complexity of bacterial ecology, necessary to identify many species of bacteria from single sample
Checkerboard hybridization
S. mutans
all streptococci
(Fig.9)
Watch video ofDNA Hybridization
http://www.phgfoundation.org/tutorials/dna/2.html
Watch video of DNA Microarrays
http://www.phgfoundation.org/tutorials/dna/6.html
cultivation of bacteria
• Consist of diverse group of bacteria• Requires a spectrum of physical & chemical for successful
growth• Laboratory cultivation conditions must be adjusted
Sample & transport
1disperse, dilute & plateOnto selective or nonselective media
2pick individualcolonies & grow pure cultures
3characterize by morphology & bio-chemical tests
4
GenusXspecies 1species 2species 3
5classify
Bacterial identification process
(Fig.10)
O2 requirements
• Amount of O2 in the atmosphere is critical for bacterial growth
• Most Oral Bacteria are1. Facultative anaerobes or2. Anaerobes3. Capanophilic anaerobes (A. actinomysetemcomitans)
O2 requirements
• Facultative anaerobesa) Streptococcus mutans b) Lactobcillus
Both cause caries and can be grown in environment rich in O2
CO2 requirements
• Subgingival species are exclusively anaerobic• Must be grown in special chambers containing low levels of
CO2 (fig.11)
O2 can be removed from the transport medium i. Boiling the media ii. Flushing with O2 free gasiii. Commercially available pre reduced media
(Fig.11)
Anaerobic Chamber
culture media
Non-selective media
• Blood agar supports growth of many oral species• Oral sample will produce diverse array of colony
morphologies• Difficult to sort out individual species• Species comprising of small percentage might not be seen
culture media
Selective media
• Contains ingredients that inhibit growth of all but a few species• Useful in isolating individual species• Enables detection of bacteria that are present in low levels
culture media
Special requirements
• Some bacteria have specific nutritional requirements• Difficult to grow until those requirements are determined &
supplimented
Dispersion & Dilution
Non-selective & Selective agars
Incubate under appropriate atmospheric conditions
for various times
Colony count
Identification scheme
DNA extraction
16S rRNA gene amplification with universal primers
Cloning & partialsequencing
Search for homologyin database
Construction of specific probes for subsequent
analysis
references• Philip D. Marsh, Michael V Martin, “The Resident Oral Microflora” in Oral Microbiology, 5th Edition, Churchil Livingstone,
2009, pp 24-29
• Philip D. Marsh, Michael V Martin, “Methods of Determining Composition of the resident oral Microflora” in Oral Microbiology, 5th Edition, Churchil Livingstone, 2009, pp 50-54
• Eugene J. Leys, Ann L. Griffen, Purnima S. Kumar and Mark F. Maiden, “Isolation, classification and identification of Oral
Microorganisms” in oral Microbiology and Immunology, ASM Press pp 73-88.
• PCR - DNA Fingerprinting https://www.youtube.com/watch?v=GLgt-EGkhZs&feature=related
• DNA Microaarays http://www.phgfoundation.org/tutorials/dna/6.html
• Hybridization http://www.phgfoundation.org/tutorials/dna/2.html
• FISH http://www.phgfoundation.org/tutorials/dna/3.html
http://www.dnalc.org/view/15924-Making-many-copies-of-DNA.html