The Resident Oral Microbiota Flashcards
What is plaque?
The community of microorganisms found on the tooth surface as biofilm embedded in a matrix of polymer of salivary and bacterial origin
What do you call plaque found on the fissures of teeth?
Fissure plaque
How much bacteria can be in fissure plaque?
1-33 x10^6
1,000,000-33,000,000
What do you call plaque found on the side of the tooth?
Approximal plaque
What do you call plaque found on the gingival of the teeth?
Sub ginigival plaque
How much bacteria can be found in sub gingival plaque?
10^3 - 10^6
Why do plaque found in different sites of the mouth have different characteristics
Because the mouth offers varying conditions at different sites in the mouth so only certain bacterial species can survive in certain environments
How much bacteria can be found in the saliva
Up to 10^9 bacteria per mL
1,000,000,000
How much bacterial can be found on the tongue?
100 bacteria
What was the first thing microbiologist do to identify oral bacteria?
They tried to isolate the microbes and grow them in a liquid or solid medium
Give an example of a solid medium microbiologist may have used to grow microorganisms
Agar (soft solid) where you can subculture bacteria on the surface and they use the nutrients in the agar to grow
How many types of growth media and what are they?
2 types:
- Selective
- Non selective
What is a non selective medium
It contains as many nutrients as possible for microbes to grow
What is a selective medium
Creating an environment for only a specific micro organism to grow
Requires you to do further research on specific microorganisms to see what conditions are favourable to them
Give some examples of non selective medium
Blood agar has horse and/ or sheep blood
Give some examples of deceive mediums
Mitis salivarius bacitracin agar allows only mutants streptococcus to grow
Sabouraud agar only allows yeast to grow
What determines the gram reaction of bacteria
Its cell wall
What is the cell wall of bacterial made of?
peptidoglycan
What is the main difference between prokaryotes and eukaryotes
Prokaryotes don’t have a nucleus so their generic material just floats around as plasmids in the cell
What is the DNA coiled into in bacteria
A circular double stranded nucleoid
What is the cytoplasm
A viscous substance that fills in the entirety of the cell and it is packed with ribosomes
What is a flagellum
A tail like extension on some bacteria
How can we identify different bacteria?
Using differential characteristics like: Gram stains Morphology Haemolysis Pigment Metabolic activity Antigens Cellular composition Nucleic acid
Why are not all bacteria cultivable in a lab
Because we are unable to reproduce the unique requirements that each bacterial species may have to grow and multiply
Approximately how many oral bacteria species are we capable of growing in the lab?
70%
What do all bacteria have
A plasma membrane surrounded by a cell wall made up of peptidoglycan
What does it mean if a bacteria is described as being gram-positive
The peptidoglycan on the outside of the cell wall is very thick
There are lipoteichoic acids that transverse and anchor into the membrane
What does it mean if a bacteria is described as being gram-negative
They have a thinner peptidoglycan layer on the outside of their cell wall
It has a second phospholipid bilayer on the outside of the peptidoglycan later
This second phospholipid bilayer has lipopolysaccharides (O antigens) and lipid A which are very unique to the gram negative
What do lipopolysaccharides (O antigens) and lipid A do?
They are recognised by host cells and may be involved in triggering the defence mechanisms and triggering inflammation
Describe how you would carry out gram staining
- You’d take a sample of a colony using a serile loop and you make a smear on a glass slide
- You let that dry using a Bunsen layer
- The heat fixates the cells into the glass slide 4. Use a crystal violet dye to flood that glass slide for a minute
5 Rinse - Then use iodine which will foxate the crystal violet within the peptidoglycan layer
- Decolorisation using acetone (or any type of alcohol)
- Add a counter stain (usually safranin) that will should stain everything pink
Gram positive cells: will be purple
Gram negative cells: will be pink
What result will a gram positive bacteria give during gram staining and at what stage?
A gram positive cell will go purple when alcohol (like acetone) is added
This is because the iodine will have fixated the crystal violet into the peptidoglycan and so the acetone will not decolorise the sample
What result will a gram negative bacteria give during gram staining and at what stage?
When acetone is added a gram negative bacteria will go white
This is because it has a second philolipid membrane that covers the thin peptidoglycan
So the crystal violet will not be fixated into the peptidoglycan layer. The crystal violet will be completely washed off and so the cells will be completely decolourised
If lipoteicholic acids are found in a bacteria cell what type of gram staining will it give
Positive
Where are lipoteicholic found in the cell
They are transverse and anchor into the membrane
How do staphtococci divide? And what gram stain do they give?
On multiple planes in clusters
They are gram positive
How do streptococci divide?
On one plane in chains
If you carry out a gram staining test and at the end the sample is a pink colour what type of bacteria do you have
Gram negative
How can cell morphology be used as a differential characteristic to differentiate between bacterial cells
Bacteria have many different shapes such as Rods Cocci Coccobacilli Curved rods (vibrio) Spirilla
How can colony morphology be used as a differential characteristic to differentiate between bacterial cells
As bacterial colonies can have different:
Shapes
Pigments
Haemolysis
What are the different haemolysis?
And what is haemolysis?
There are three types of haemolysis:
Alpha (oxidation of the haem in iron)
Beta
Gamma
Haemolysis helps you see if the bacteria produces the haemolytic enzyme that helps them to destroy specific substrates
What would you see if a bacterial colony was alpha haemolytic?
A greenish tint would appear on top of the colony
What would you see if a bacterial colony was beta haemolytic?
You would end up with zones of clearing as the haemolytic enzymes completely destroy the haem groups
What would you see if a bacterial colony was gamma haemolytic?
There’s no hydrolysis
How can metabolic activism be used as a differential characteristic to differentiate between bacterial cells
Certain types of bacteria may produce unique enzymes to help them in fermentation
Some enzymes may produce unique acids or gases
Or some bacteria may utilise certain carbohydrates or proteins
How can antigens be used as a differential characteristic to differentiate between bacterial cells
Some bacteria may present specific antigens that we can use to detect them
How can cellular composition be used as a differential characteristic to differentiate between bacterial cells
Like gram staining shows us the type of peptidoglycan
You could test for unique lipids or amino acids that bacteria may have
How can DNA be used as a differential characteristic to differentiate between bacterial cells
Where dna is extracted from the cell
It is the most accurate way of identifying bacteria
What are the main groups of gram positive bacteria found in the mouth
- Cocci :Streptococcus
2. Rods: Actinomyces, Eubacterium, Lactobacillus
What are the main groups of gram negative bacteria found in the mouth
Cocci: Neisseria, Veillonella
Rods: Aggregatibacter, Fusibacterium, Prevotella, Treponema
What is the plural of genus?
Genera
Name the main gram positive cocci found in the mouth
Streptococcus Oralis (found in health) Streptococcus mitis (found in health) Streptococcus sanguinis (found in health) Streptococcus intermedius (found in abscesses) Streptococcus mutans (found in dental caries) Streptococcus sobrinus (found in dental caries)
How can Streptococcus sanguinis be found in the heart and what is the effect of this?
They can travel through the blood stream to the heart and colonise there
They make biofilm in the heart which can lead to heart disease and heart attack
Which bacteria are the early colonisers of the mouth
Streptococci salivarius
Which bacteria is catalase negative specific to
Streptococci oralis
What does the catalase negative enzyme do?
Degrade hydrogen peroxide into water and hydrogen
This is v beneficial as hydrogen peroxide is very dangerous to bacteria
How can hydrogen peroxide be used to differentiate between streptococci bacteria?
Add hydrogen peroxide to the glass slide and if bubble of oxygen appear you have a catalase positive result
If no bubbles appear then you have Streptococcus oralis
Name some gram positive rod bacteria
Actinomyces
Lactobacillus
Eubacterium
Where is Actinomyces commonly found?
Dental plaque
Implicated in root surface caries
They are opportunistic pathogens
They are gram positive branches rods
Describe Eubacterium
They are gram positive rods and filaments
They are difficult to grow in the lab
They are found in plaque
Describe lactobacillus
They are gram positive rods
Implicated in advanced dental caries
They are acidoogenic
Name some gram negative bacteria
Neisseria
Veillonella
Treponema
Fusobacterium
Describe Neisseria
They are gram negative coccus
Early colonisers of the tooth
Describe Veillonella
They are gram negative coccus
Prefer anaerobic conditions
Utilise lactic acidosis is plaque
Describe treponema
They are gram negative
Strictly anaerobic
Found in sub gingival plaque
Implicated in periodontal disease
Describe fusobacterium
They are gram negative filaments
Anaerobic
Common in plaque
They are key bridging organisms between early and late colonisers
How can we identify bacteria that we can’t culture
We use DNA analysis
What is the method of choice of microbiologist for dna analysis of bacteria
The 16S ribosomal RNA gene (16srRNA)
Why can we use the 16S ribosomal RNA gene?
As eukaryotes have different RNA To prokaryotes (they are different sizes and so have a different gene sequence )
Prokaryotes ribosomes have two sub units large and small
We are interested in the small 16S supplement that codes the 16S ribosomal RNA gene
Scientists found that this gene has really interesting properties
It is about 1500base pairs long and it has different regions
What interesting properties does the 16S ribosomal RNA gene have?
It is about 1500base pairs long and it has different regions and the conserved regions are the same for every 16s rna ribosomal Gene
What are the different regions of the 16S ribosomal RNA gene?
The conserved region (the same for every 16S rna ribosomal gene in every prokaryote)
Variable region
How can we use the 16S ribosomal RNA gene?
Extract the dna
Amplify the 16s gene using PCR and then sequence it
Then compare it to the online database
The closer related the organisms are the more similar their variable region
How many phylotypes have been detected in the mouth using the 16S rRNA gene sequencing?
Around 700 phylotypes (species
Around 400 of these are in the sub gingival domain
Around 35 have not yet been cultured
What’s another word for species
Phylotypes
Name some molecular typing methods
16A rRNA gene amplification
FISH
Denaturing gradient gel electrophoresis (DGGE)
DNA-DNA Checkerboard
Human oral microbe identification microarray (HOMIM)
Most of these are obsolete and the one preferred by scientists now a days is:
Next generation sequencing
What can you say if a green tint is seen on a bacterial colony
It displays alpha haemolysis
If there’s no hydrolysis what type of haemolysis does the bacterial colony display
Gamma haemolysis
If there are clear zones in the bacterial colony what can you say about its haemolysis
It displays beta haemolysis
Streptococci salivarius are described as what type of colonisers?
Early colonisers
Streptococci oralis are the only bacteria that have which enzyme
Catalase negative
