resident oral microbiota Flashcards
what 3 types of plaque are present in the mouth
fissure (1.33 x 10^6 bacteria/fissure)
approximal
sub-gingival (10^3-10^6 bacteria/crevice)
each has a varying bacterial load
as anatomy + biogeography at these sites varies greatly
define plaque
community of micro-organisms found on the tooth surface as a biofilm embedded in a matrix of polymers of salivary and bacterial origin
how many bacterial cells are in 1ml of saliva
up to a billion
100 bacteria/cell on tongue dorsum
what does saliva under a darkfield microscope show and why
shows saliva is v lively / teeming w life + not static
darkfield is
- different to gram staining
- uses light with dark background
- light reflected onto LIVING organism so can view them when alive - for most staining procedures you have to kill the organism you’re trying to view
how do we study microbes
- grow / culture in lab
- use broth + agar plates
what are the types of agar
NORMAL = contains all nutrients required for bacterial growth (sugars, minerals, protein in form of yeast / meat extracts)
BLOOD AGAR = contains either horse or sheep blood (found to be rlly nutritious + allow growth of many microorganisms)
what is a columbia blood agar
- non-selective medium
- allows growth of most bacteria
give some examples of selective media
MITIS SALIVARIUS BACITRACIN AGAR (selective for S. mutans)
SABOURAUD AGAR (selective for yeasts - yeasts are NOT bacteria but eukaryotic microorganisms + members of fungus kingdom)
how do we incubate selective media
- anaerobically or in an environment of high CO2 (~5-10%)
- overnight at 37 degreesC
how do we incubate columbia blood agar plates (non-selective media)
in CO2
what is seen on the agar after inocultating overnight / after 10 hours
exponential growth of bacteria
colonies visible by naked eye
what are some features of bacterium
- cytoplasmic membrane (phospholipid bilayer) surrounded by a cell wall made of peptidoglycan
- genetic material floats freely in the cytosplasm + is associated w proteins + is coiled into a nucleoid
- cytoplasm = viscous, packed with ribosomes
- on the outside of the cell there may be
1) polysaccharide capsule
2) flagella (for motility)
what does the cell wall structure of bacteria divide them into
2 main groups
- gram +ves
- gram -ves
why arent all species culturable in the lab
- not always able to reproduce the conditions / nutritional requirement that certain species need to grow
how many microbes of the world have been cultures in labs
less than 1% (huge diversity ie environmental microbes from soil to oceans)
how many microbes of the oral microbiota have been cultures in labs
70%+
well studied
which tools / aspects of the bacterium can microbiologists use to characterise bacteria from a clinical sample
- identification using differential characteristics
3) Gram-stain (bacterial cell wall determines outcome)
4) Morphology
5) Haemolysis
6) Pigment
7) Metabolic activities
8) Antigens
9) Cellular composition
10) Nucleic acid
what is the difference between gram +ves and gram -ves
gram +ve = thick cell wall traversed with lipoteichoic acid molecules
gram -ve = thin peptidoglycan layer surrounded by an outer phospholipid membrane attached to cell wall via lipoproteins
surface of outer membrane displays lipopolysaccharides (O antigens) and lipid A molecules which are v specific to gram -ve bacteria and are recognised by host cells (trigger host defence mechanisms ie inflammation)
what are the 5 stages of gram staining (after smearing sample of the grown colony on a glass slide)
1) fixation
2) crystal violet
3) iodine
4) decolourisation using acetone
5) counter stain with safaranin
explain the fixation stage of gram staining
use heat from a Bunsen burner
just enough to stick / seal the cells on the glass slide
this process kills the bacterial cells
explain the crystal violet stage of gram staining
flood surface of the glass slide w crystal violet (a purple dye) for 1 min
then rinse the slide (tap water)
explain the iodine stage of gram staining
flood slide with iodine
at this stage = crystal violet dye has been retained on the bacterial cells esp on the gram +ve cells
why do gram +ve cells especially retain crystal violet dye
peptidoglycan v thick + exposed on the outside as the cell wall absorbs it
iodine seals it in (cannot be washed off)
explain the decolourisation using acetone (or methanol or ethanol) stage of gram staining
washes crystal violet dye away in case of gram -ve
so we can differentiate…
gram +ves = purple
gram -ves = cannot see
explain the counter stain with safaranin stage of gram staining and why is it used
safarin is redy-pink
for gram +ve = remains purple
for gram -ve = turns pink
we can visibly see mixture of the 2 and differentiate them
used because if its all been decolourised + bacterial cells we were studying were gram - (would be all completely decolourised + if viewed under microscope you wouldn’t be able to see anything) but if gram + (would be seen in purple)
why cant gram -ve cells retain crystal violet dye
thin peptidoglycan cell wall surrounded by a phospholipid membrane
how can we further look at cell morphology after gram staining
- microscope to see if rods or cocci (round shape cells)
- really helps their identification
how do bacteria reproduce
binary fission
1 cell splits equally into 2 identical daughter cells
how do streptococci and staphylococci divide/grow differently (both gram +ves)
streptococci = divide on one plane into chains staphylococci = divide on multiple planes into clusters
which shapes and forms can bacteria come in
rods / bacilli cocci coccobacilli vibrio / curved rods spirilla filamentous (filament like cells are typical of fusobacteria)
what factors make up colony morphology and when is this used
shape
pigment
haemolysis
- 1st characteristic used to differentiate bacteria when grown on an agar plate
what does the size of a bacterium range by
1-10um
invisible to eye
what are some bacterial species classified base on when cultured on blood agar plates
what 3 categories is this divided into
haemolytic properties
1) alpha haemolytic
2) beta haemolytic
3) gamma haemolytic
what do alpha haemolytic species do when cultured on blood agar plates
- cause oxidation of iron in haemoglobin contained in the agar (contain/posses genes to do this)
- leads to greenish colour / shine on blood agar plates around the bacterial colonies
what do beta haemolytic species do when cultured on blood agar plates
- cause complete rupture of red blood cells contained in the agar medium
- appears as clear areas surrounding the colonies
what do gamma haemolytic species do when cultured on blood agar plates
dont cause any haemolysis
which 4 differential characteristics can be used to further differentiate bacteria
1) metabolic activities
2) antigens
3) cellular composition
4) DNA
how can metabolic activities be used to further differentiate bacteria
certain species have specific metabolic activities
- fermentation +/- acid or gas production
- preformed enzyme production
- use specific carbs or proteins (use in selective media for their growth)
how can antigens be used to further differentiate bacteria
find specific antibodies that bind to antigens that certain bacteria may produce
use these as a way to identify these bacteria
how can cellular composition be used to further differentiate bacteria
peptidoglycan = used in gram staining method to differentiate between gram +ve and -ve
similarly other specific components (lipids / amino acids etc) may be used / targeted in differentiationo between specific species