1 - Diagnostic and typing methods Flashcards
What bacteria are associated periodontal disease?
- porphyromonas gingivalis
- actinobacillus actinomycetemcomitans (A. a.)
- prevotella intermedia
- bacteroides forsythus
What bacteria are associated with caries?
- streptococcus mutans
- lactobacilli
What bacteria are associated with root canal infections?
- porphyromonas endodontalis
- fusobacterium nucleatum
What is microbiological culture?
- bacteria are cultured on agar medium
- bacteria isolated
- identified by characteristics of enzyme activity and sugar fermentation tests
What is molecular biology?
DNA probes and PCR used to identify bacteria
What is vancomycin?
Selective agent for gram positive bacteria
How are cultures prepared?
- vortex mix sample for 30s
- serial dilution (10^-6)
- placed on fastidious anaerobe agar (FAA) medium with defibrinated horse blood
- incubated anaerobically for 10 days
- bacteria counted
What is a metronidazole disc used for?
Anaerobic bacteria are sensitive to metronidazole so die within the disc
How are bacteria identified from culture?
- metronidazole disc
- gram stain
- rapid API 32 A test (enzymes and sugar fermentation)
What colour do gram positive bacteria stain?
Pink
What colour do gram negative bacteria stain?
Do not retain stain so appear lighter
What are DNA probes?
Segments of DNA that have been labelled with chemoluminescent, luminescent or radioactive agents
What are the different types of DNA probe?
- whole genomic
- cloned gene
- oligonucleotide (20-50 bases)
What is the problem with whole genomic DNA probes?
Can cross-react with other bacteria
How are oligonucleotide probes made?
- 16S ribosomal RNA extracted and sequenced
- unique base sequence selected for desired bacteria
- complimentary DNA synthesised
- DNA tagged
- oligonucleotide probe complete
What is the 16S ribosomal RNA gene?
- found in all bacteria
- gene sequenced for all known bacteria
- highly variable regions means that every bacteria is unique
- probes are therefore very accurate
What is the conserved region?
- broad range of bacteria can be detected etc with same probe due to similarity in areas
- consensus probes used to detect general bacterial presence
What is PCR-RFLP?
- polymerase chain reaction restriction fragment length polymorphism
- used to identify bacteria isolates
- creates patterns (fingerprints) for individual species
Why is it important to subtype bacteria?
- track routes of transmission during disease outbreak
- pathogenicity of specific strains
What is subtyping?
Identifying different strains of the same bacteria
What are the different methods of subtyping bacteria?
- serotyping
- biotyping
- restriction enzyme analysis (REA)
- gene probe typing
- ribotyping
- 16S-23S intergenic spacer region (IGS)
- DNA sequencing
What is REA?
- restriction enzyme analysis
- digest whole genomic DNA with restriction enzymes
What ribotyping?
- ecoli rRNA operon used as DNA probe
- operon is common in many bacteria genomes
- migrate across plate to separate DNA to create “fingerprint”
What is 16S-23S IGS?
- 16S-23S intergenic spacer region
- very variable sequence used
- amplified by PCR
- digested with restriction enzymes to obtain strain-specific “fingerprints”
What is DNA sequencing?
Can detect single base differences between strains
What percentage of clone sequences determines if a bacteria is known?
98%
