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. Identification Methods
of
Oral Microbes
Dr. Ali Yaldrum
Faculty of Dentistry
SEGi University, Kota Damansara, Malaysia
18-06-12

2. 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

3. 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

4. VS
1. prokaryotes
eukaryotes

5. prokaroyte
cell wall
peptidoglycan singular supercoiled
circular chromosome
flagellum
cytoplasm rich in plasmid
cellmembrane
ribosomes
(Fig.1)

6. eukaroyte
mitochondria
cell membrane
nuclear membrane
lysosome
cytoplasm
rough endoplasmic smooth endoplasmic
reticulum reticulum
Golgi apparatus
(Fig.2)

7. 2. Classification &
Identification

8. 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

9. taxonomic hierarchy (Fig.3)
KINGDOM
PHYLUM
DIVISION
CLASS
ORDER
FAMILY
GENUS
SPECIES
SUB SPECIES

10. identification
‘is the process of determining that a new isolate belongs to
particular taxon’
• Bacteria are identified using phenotype, immunological or
molecular characteristics

11. 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

12. Bacterial (Fig.4)
Classification
Shapes
Cell wall
Outer membrane
Teichoic acid protein
peptidoglycan
Thin Gram
peptidoglycan Reaction
layer
Gram +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 structures
DNA sequencing of key genes ; Ribosomal 16S gene DNA SEQUENCING

13. bacterial shapes
Coccus Vibrio
Bacillus Spirillum
Coccobacillus Spirochete
Fusiform bacillus
(Fig.5)

14. 3. sampling Oral
bacteria

15. • Oral Cavity contains a variety of different niches that harbour
distinctive communities of bacteria.
• Location and environment determines the diversity of eco
system.

16. Studies of various niches have shown distinctive microbial
profiles for different locations
• Tongue
• Tooth surface
• Gingival sulcus
• Buccal mucosa
• Gingival crevice

17. gingival sulcus
1
2
3
4
5
6
1= Enamel 4= Free gingivae
2= Dentine 5= Cementum (Fig.6)
3= Pulp 6= Alveolar bone

18. 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

19. sampling plaque
2 approaches can be used
1.Supragingival plaque
2.Subgingival plaque

20. Supragingival
• Curette is used to scrap the biofilm of the tooth surface (fig.
7)
• Can not be inserted more than 6mm

21. Periodontal Curette
(Fig.7)

22. 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

23. Endodontic Paper (paper point)
(Fig.8)

24. 4. Identifying Oral
bacteria

25. 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

26. 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

27. 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

28. 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
bacteria's
• Not used for fragile
bacteria

29. 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’

30. 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

31. 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

32. 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

33. variations of PCR
The basic PCR methodology is modified to provide
sophisticated analytical tools
•Nested PCR
•Multiplex PCR
•Real Time PCR

34. 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

35. Watch Video of
PCR & Gel Electrophoresis
https://www.youtube.com/watch?v=GLgt-EGkhZs&feature=related

36. 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

37. 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

38. S. mutans
all streptococci
Checkerboard hybridization
(Fig.9)

39. Watch video of
DNA Hybridization
http://www.phgfoundation.org/tutorials/dna/2.html

40. Watch video of
DNA Microarrays
http://www.phgfoundation.org/tutorials/dna/6.html

41. cultivation of bacteria
• Consist of diverse group of bacteria
• Requires a spectrum of physical & chemical for successful
growth
• Laboratory cultivation conditions must be adjusted

42. Bacterial identification process
GenusX
species 1
species 2
species 3
1 2 3 4 5
Sample & disperse, dilute & plate pick individual characterize by classify
transport Onto selective or non colonies & grow morphology & bio-
selective media pure cultures chemical tests
(Fig.10)

43. 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)

44. O2 requirements
• Facultative anaerobes
a) Streptococcus mutans
b) Lactobcillus
Both cause caries and can be grown in environment rich in O2

45. 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

46. Anaerobic Chamber
(Fig.11)

47. 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

48. 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

49. culture media
Special requirements
• Some bacteria have specific nutritional requirements
• Difficult to grow until those requirements are determined &
supplimented

50. Dispersion & Dilution DNA extraction
Non-selective & 16S rRNA gene amplification with
Selective agars universal primers
Incubate 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

51. 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