Published on March 21, 2013
Identiﬁcation Methods of Oral Microbes Dr. Ali Yaldrum Faculty of DentistrySEGi University, Kota Damansara, Malaysia 18-06-12
Learning ObjectivesAt 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
Infection? Virus? culture 2 1 diagnosis Clinician + 3 request treatment 8 What the!!!*A-A-ah Diagnostic interpretation Cycle collection data ﬂow 4 transportation 7 labortary analysis 5 6
prokaroyte cell wall peptidoglycan singular supercoiled circular chromosomeﬂagellum cytoplasm rich in plasmid cellmembrane ribosomes (Fig.1)
eukaroyte mitochondria cell membrane nuclear membranelysosome cytoplasm rough endoplasmic smooth endoplasmic reticulum 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
taxonomic hierarchy (Fig.3) KINGDOM PHYLUM DIVISION CLASS ORDER FAMILY GENUS SPECIESSUB SPECIES
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 (Fig.4) Classiﬁcation Shapes Cell wall Outer membrane Teichoic acid proteinpeptidoglycan Thin Gram peptidoglycan Reaction layerGram +ve Gram -ve Obligate aerobes requires O2 Plasma membrane Microaerophiles requires reduced O2 Atmosphere Obligate anaerobes requires no O2 Facultative anaerobes anaerobic or aerobic Capnophiles requires increases CO2 Spores Key Enzymes Bacteria lacking certain enzymes Interaction of antibodies with certain Serological Reaction surface structuresDNA sequencing of key genes ; Ribosomal 16S gene DNA SEQUENCING
bacterial shapes Coccus Vibrio Bacillus SpirillumCoccobacillus SpirocheteFusiform bacillus (Fig.5)
3. sampling Oralbacteria
• 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 sulcus 1 2 3 4 5 61= Enamel 4= Free gingivae2= Dentine 5= Cementum (Fig.6)3= Pulp 6= Alveolar bone
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 plaque2 approaches can be used1.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 Oralbacteria
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 primers 2. Quantitative PCR 3. DNA hybridization assays 4. Ribosomal 16S cloning & sequence analysis 5. 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’ Detergents Bead Beating & Proteinase K• Target one type of bacteria • Lyse a wide spectrum of • Bacteria are mixed with• Variable intensity bacteria small slurry of tiny glass• High speciﬁcity • Unable to lyse Gram-ve beads bacteria • Vile is placed in a vibrating apparatus • Will lyse the most sturdy bacterias • 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 PrimerThe 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 speciﬁcstrand of DNA from a speciﬁc specie
what is a Primer‘A primer is a strand of nucleic acid that serves as a startingpoint for DNA synthesis. They are required for DNA replicationbecause the enzymes that catalyze this process’*Primers are usually short, chemically synthesized oligonucleotides, with a lengthof 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 PCRThe basic PCR methodology is modiﬁed to providesophisticated 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 of PCR & Gel Electrophoresishttps://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
S. mutans all streptococciCheckerboard hybridization (Fig.9)
Watch video of DNA Hybridization http://www.phgfoundation.org/tutorials/dna/2.html
Watch video of DNA Microarrayshttp://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
Bacterial identiﬁcation process GenusX species 1 species 2 species 3 1 2 3 4 5Sample & disperse, dilute & plate pick individual characterize by classifytransport Onto selective or non colonies & grow morphology & bio- selective media pure cultures chemical tests (Fig.10)
O2 requirements• Amount of O2 in the atmosphere is critical for bacterial growth• Most Oral Bacteria are 1. Facultative anaerobes or 2. Anaerobes 3. Capanophilic anaerobes (A. actinomysetemcomitans)
O2 requirements• Facultative anaerobes a) Streptococcus mutans b) LactobcillusBoth 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 gas iii. Commercially available pre reduced media
Anaerobic Chamber (Fig.11)
culture mediaNon-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 mediaSelective 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 mediaSpecial requirements• Some bacteria have speciﬁc nutritional requirements• Difﬁcult to grow until those requirements are determined & supplimented
Dispersion & Dilution DNA extraction Non-selective & 16S rRNA gene ampliﬁcation with Selective agars universal primersIncubate under appropriate Cloning & partial atmospheric conditions sequencing for various times Search for homology Colony count in database Construction of speciﬁc Identiﬁcation scheme 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
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