1 - Diagnostic and typing methods

Question 1

What bacteria are associated periodontal disease?

Answer 1

• porphyromonas gingivalis
• actinobacillus actinomycetemcomitans (A. a.)
• prevotella intermedia
• bacteroides forsythus

Question 2

What bacteria are associated with caries?

Answer 2

• streptococcus mutans
• lactobacilli

Question 3

What bacteria are associated with root canal infections?

Answer 3

• porphyromonas endodontalis
• fusobacterium nucleatum

Question 4

What is microbiological culture?

Answer 4

• bacteria are cultured on agar medium
• bacteria isolated
• identified by characteristics of enzyme activity and sugar fermentation tests

Question 5

What is molecular biology?

Answer 5

DNA probes and PCR used to identify bacteria

Question 6

What is vancomycin?

Answer 6

Selective agent for gram positive bacteria

Question 7

How are cultures prepared?

Answer 7

• 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

Question 8

What is a metronidazole disc used for?

Answer 8

Anaerobic bacteria are sensitive to metronidazole so die within the disc

Question 9

How are bacteria identified from culture?

Answer 9

• metronidazole disc
• gram stain
• rapid API 32 A test (enzymes and sugar fermentation)

Question 10

What colour do gram positive bacteria stain?

Answer 10

Pink

Question 11

What colour do gram negative bacteria stain?

Answer 11

Do not retain stain so appear lighter

Question 12

What are DNA probes?

Answer 12

Segments of DNA that have been labelled with chemoluminescent, luminescent or radioactive agents

Question 13

What are the different types of DNA probe?

Answer 13

• whole genomic
• cloned gene
• oligonucleotide (20-50 bases)

Question 14

What is the problem with whole genomic DNA probes?

Answer 14

Can cross-react with other bacteria

Question 15

How are oligonucleotide probes made?

Answer 15

• 16S ribosomal RNA extracted and sequenced
• unique base sequence selected for desired bacteria
• complimentary DNA synthesised
• DNA tagged
• oligonucleotide probe complete

Question 16

What is the 16S ribosomal RNA gene?

Answer 16

• found in all bacteria
• gene sequenced for all known bacteria
• highly variable regions means that every bacteria is unique
• probes are therefore very accurate

Question 17

What is the conserved region?

Answer 17

• broad range of bacteria can be detected etc with same probe due to similarity in areas
• consensus probes used to detect general bacterial presence

Question 18

What is PCR-RFLP?

Answer 18

• polymerase chain reaction restriction fragment length polymorphism
• used to identify bacteria isolates
• creates patterns (fingerprints) for individual species

Question 19

Why is it important to subtype bacteria?

Answer 19

• track routes of transmission during disease outbreak
• pathogenicity of specific strains

Question 20

What is subtyping?

Answer 20

Identifying different strains of the same bacteria

Question 21

What are the different methods of subtyping bacteria?

Answer 21

• serotyping
• biotyping
• restriction enzyme analysis (REA)
• gene probe typing
• ribotyping
• 16S-23S intergenic spacer region (IGS)
• DNA sequencing

Question 22

What is REA?

Answer 22

• restriction enzyme analysis
• digest whole genomic DNA with restriction enzymes

Question 23

What ribotyping?

Answer 23

• ecoli rRNA operon used as DNA probe
• operon is common in many bacteria genomes
• migrate across plate to separate DNA to create “fingerprint”

Question 24

What is 16S-23S IGS?

Answer 24

• 16S-23S intergenic spacer region
• very variable sequence used
• amplified by PCR
• digested with restriction enzymes to obtain strain-specific “fingerprints”

Question 25

What is DNA sequencing?

Answer 25

Can detect single base differences between strains

Question 26

What percentage of clone sequences determines if a bacteria is known?

Answer 26

98%