Lab 1 Flashcards
GAS
Group A streptococci or streptococcus pyogenes
GBS
Group B streptococci or Streptococcus agalactiae
viridans streptococci
streptococcus species that are a- haemolytic or gama- haemolytic and cannot be categorized using lancefield antigen grouping
streptococcal arrangements
genera Enterococcus and Lactcoccus
gonococcus
Neisseria gonorrhoeae
meningococcus
Neisseria meningitidis
pneumococcus
Streptococcus pneumoniae
Diagnosis
is a conclusion or decision based on a careful examination of relevant information
The evaluation process of hypothesis
A valid hypothesis has three characteristics.
1) it is testable
2) it is unbiased
3) it is repeatable
What are the assumptions of CFUs
that each colony arises from a single living cell
(note however, two ajacent cells may clump their colonies together to apper as one.)
that all living cells will produce colonies
(note VBNC cells may exist)
thatthat all cells within a colony are genetically identical to each other and to the original single cells
Selective Media
inhibits the growth of some groups of organims from growing while allowing others groups to grow
Differential Media
can distinguich between different types of bacteria based on biochemical changes in the media after inoculationa nd incubation
differential and selective properties of Manitol Salt Agar
selects for halophiles and inhibits non-halophiles.
Of the salt tolerant organisms that grow, the medium differentiates those that caan ferment the carbohydrate mannitol from those that cannot ferment mannitol
Purpose of a four quadrent streak plate
isolate CFUs by streaking different cell densities (decreasing) across each quatrent of an agar plate
Often selective media is used to help isolate a specifc microbe
some also can tell the difference between microbes are are thus selective and differential
Blood agar
differential based on haemolytic traits
TSA with %5 sheeps blood
haemolysins= haemolytic enzymes dyscolour the surrounding media from the break down of the RBCs
Haemoglobin -> metemoglobin (green and opaque)
= alpha haemolysis
complete distruction of haemoglobin (translucent yellow)
= beta haemolysis
no breakdown (agar remains the same) =gamma haemolysis
Note: Blood agar is an enriched medium and can help to accelerate the growth of fastidious organisms
The Gram stain
primary stain = crystal violet with iodine which acts as a mordant to enhance the colour (makes a complex with CV and is harder to wash away)
decolourization with ethanol removes CV-iodine from Gram- cells (this strips away the lipids of the outer membrane which allows the CV to leak out)
secondary stain = safranin to stain G- pink
false negative gram stain
G+ is pink
because the result appers to be G- when the organism is G+ (ie a false result)
False positive gram stain
G- is purple
because the organism falsely appersa G+
Problems with Bacillus and Staphylococus Gram stains
give false negatives after 24 hrs because they lose their resistance to the decolourization step
Catalase test
During aerodic respiration, aerobic, facultative anaerobic and microaerophilic bacteria use O2 as a TEA
Some e- leak from the ETC and form superoxide and hydrogen peroxide
to combat this these organisms use superoxide dismutase and catalase hydrolaze to turn these products to oxygen and water
superoxide dismutase catalyzes the reaction of superoxide to hydrogen peroxide
The inability of anaerobes to dismantle superoxide shows us why oxygen is toxic to these organisms
Catalase + organisms will result in vigourus bubbling immediately after the addition of hydrogen peroxide
What is the purpose of blood agar
good indication of G+ cocci like Streptococcus species
and to promote the growth of fastidious organisms
What is the purpose of the catalase test
differentiate catalase positive organisms like Staphylococcus species from catalase negative Streptococcus species
What is the purpose of the Coagulase test
differentiation of Staphylococcus species.
S. aureus produces the coagulase enzyme which converts fibrinogen to fibrin
the ability for SA to trigger coagulation allows a clot to form around and protect the colony from host defenses
bound-coagulase
clumping factor
coagulase as a extracellular // exoenzyme
free coagulase // coagulase-reacting factor
Coagulase slide test
only detects bound coagulase, since the one to two minutes it takes for a positive test is not sufficient for significant levels of exoenxyme to be released
DNase Test Agar
tests for exoenzymes = DNase that can hydrolyze DNA into oligonucleotides
contains peptides as an energy and carbon source, DNA and toluidine blue with forms complexes with DNA but not nuclotides
+ test = plate turns from blue to clear around the colony
What is the purpose of the DNase Test agar
differential and selective media
Toluidine blue will inhibit the growth of some G+ cocci. and with Staphylococcus, heavy innoculation is needed to overcome this.
Staphylococcus aureus is a strong positive for this test (clear agar)
Staphylococcus epidermidids is a weak positive
This agar can also differentiate between Serratia species (+) from Enterobacter species (-) and Moraxella (+) from Neisseria (-)
Mannitol salt agar
has pheol red which is yellow below pH 6.6, orange at pH 6.6 and red above 7.2 and fushia above 8.
the carbohydrate mannitol when fermented produces organic acids which lower the plates pH
Novobiocin Susceptibility Test
Novobiocin is an aminocoumarin antibiotic produced by the actinomycete Streptomyces niveus
Aminocoumarins inhibit bacterial gyrase from using ATP to manipulate circular chromosomes, preventing DNA replication
done via a Kirby-Bauer antibiotic suseptibility assay (paper disk), and done on enriched Mueller-Hinton agar
Novobiocin is effective against Staphylococcus epidermidis but not Staphylococcus saprophyticus
(this is usually done after the catalase test to differentiate the two since these guys are both -ve)
Lancefield antigen serotyping
The classification of Streptococci based on serotyping via antigens
Group A Strep
Streptococcus pyogenes
Streptococcus with no Lancefield groups
Streptococcus pneumoniae
Are enterococcus and Lactococcus part of the Streptococcus genus?
No, they were reclassified in 1984
Group B Strep
Streptococcus agalactiae
CAMP test
detects the haemolytic substance (CAMP factor) that is produced by Streptococcus afalactiae; A group B streptococcus.
Detects beta haemolytic streptococcus producing CAMP factor, can be transferred via HGT but is rare enough that this is a good indicator of Group B Strep.
CAMP test Synergy
CAMP factor reacts synergistically with the B-haemolysin that is produced by Staphylococcus aureus
when drawn in a T shape with group B strep, an area of synergy will be present.
Bacitracin Suseptibility test
Bactracin is a narrow spectrum antibiotic that inhibits the growth of Staphylococcus aureus, Staphylococcus, epidermidis, and Streptococcus pyogenes.
S. pyogens unlike most other b-haemolytic streptococcus species is not resistant and thus this antiobiotic can be used as an identification tag of Group A Strep.
Bile esculin Test
esculin is a glycoside that can be hydrolyzed by Enterococcus spcies and streptococcus bovis group amound the streptococci.
This hydrolysis is usually done in the presence of bile salts
Cow bile is added and inhibits the growth of most streptococci.
Upon esculin hydrolysis, the ferric citrate indicator is oxidized to form a brown ppt indicating a positive result
Streptococcus bovis group
S. equinus
S. galloolyticus
S. infantarius
S. alactolyticus
What tests differentiate between Streptococci and Staphylococci
Gram stains
What test can be used to help to identify human staphylococcal pathogens
Catalase
Coagulase
DNase
Mannitol Salt Agar
Novobiocin Susceptibility
What tests can be used for the identificaiton of human Streptococcal Pathofens
Lancefield Antigen serotyping
CAMP
Bacitracin suceptibility
Bile Esculin
What test can be used for the identificaiton of Streptococcus pneumoniae
Viridan Streptococci
Optochin susceptibility
Bile solubility
Negative stains
Viridan Streptococci
need to determine if A-haemolytic strep is Strp pneumoniae because it is the most pathogenic
These will produce a green tinge on blood agar
Includes a and g haemolytic streptococcus that includes the mutans group, the anginosum group, the bovis group, the mitis group, and the salivarius group;
organisms that are commonly found in the moutn, upper respiratory tract and urogenital tract
Optochin susceptibility test
optochin can be used to treat Streptococcus pneumonia infections
S pneumonia is susceptible to optochin, but most other a haemolytic streptococcus are resistant (ie the virdans streptococci)
any clearing around the disk = susceptibility = Taxo P = S. pneumonia
Bile solubility test
differentiates between pathogenic streptococcus pneumoniae and other a haemolytic streptococcal species
the activate of an autolytic enzyme that induces cell death is increased in the presence of bile salts (sodium desoxycholate= bile salt lowers the surface tensi9on of the plasma membrane and causes S. pneumoniae to digest its own cell wall.)
CELLULAR DEBRIS FROM LYSED CELLS lead to a decrease in turbidity and thus a potitive result
Negative stains
utalizes an acidic stain that does not penetrate the negatively charged cell wall of bacteria
cell remains clear
useful for observation of spirochetes
heat fixed, as opposed to methanol to maintain structural integrity
also is useful for the dection of capsules
Tests for the examination of normal Flora
Chocolate agar
Tellurite agar
Sabouraud agar
SIngle step Staphylococcus (4S) agar
TSY+G agar
Mitis Salivarius-bacitracin agar
Chocolate Agar
non-selective enriched media
casein, peptone, haemoglobin and corn starch
RBC is already lysed from heat treatment
used for the growth of fastidious organisms like pathogenic Neisseria species or Haemophilus species that require X and V blood factors
Tellurtie Agar
Potassium tellurite inhibits most Gram+ and Gram- organisms, some Staphylococcus species, some Enterococcus species and most Cornebacterium species are able to reduce tellurite to tellurium to produce a grey/ black colony
most other species cannot grow on this
peptone as a carbon source
Sabouraud Agar
for the growth requirements of yeast and fungi
incubated at room temp to encourage the fungi to grow (heaat = bad)
no selective or differential properties
4S Agar
SIngle step Staphylococcus (4S) agar
= Baird Parker agar
based ont he ability of Staphylococcus aureus to grow and produce lecithinase in the presence of a high salt conc. and potassium tellurite
the agar has an egg yolk emulsion that will break down in the presence of lecithinase
the reduction of tellurite to telluride will give the colonies a grey colour
SA will produce characteristic grey colonies with white halos
Staphylococcus saptrophyticus may also produce this colony morphology but can be differentiated but the longer incubation time
TSY+G Agar
enriched medium
Tryptic soy broth with Yeast and glucose
used to culture Propionibacterium acnes, an aerotolerant anaerobic Gram+ non-motile bacillus that is frequently found on the skin
Propionibacterium acnes
an aerotolerant anaerobic Gram+ non-motile bacillus that is frequently found on the skin
grows well in anerobic enviroments like blocked pores and can cause acne vulgaris and folliculitis
co-infection and inflammation with staphylococcus species lead to disease, and the by product propionic acid decreases skin pH to 3/5 and supresses the growth of other commensal microbes
Mitis Salivarius-bacitracin Agar
MSB agar
selective and differential medium
selevtive against G+ b/c of Crystal violet
selective agaisnt G- b/c of tellurite
contains trypan blue with will cause most Streptococcus and Enterococcus species to develop a blue colony morphology
S mutans MSB agar
Indicative of Streptococcus mutans whihc will produce a raised, conves, undulate, opaque, pale-blue colonie that are granular “frosted-glass” sinc ethe colonies produce EPS from the sucrose in the medium
S salivarius MSB agar
Streptococcus salivarius forms large, pale-blue, mucoid colonies that are glistening “gum-drop” in apperance
S mitis MSB agar
Streptococcus mitis forms small, flat, hard colonies, blue in colour with a domed centre
Enterococci MSB agar
Enterococci produce blue-black shiny and slightly raised colonies
