LAB S19 Flashcards
Specimens to be obtained for culture depend on
the nature of the
staphylococcal infection
G+ cocci, 0.5-1.5 micrometer, grape-like clusters
staphylococcus species
staphylococcal species’ specimens are obtained from
- Pus from wound and other pyogenic infections
- Blood from septicemia
- CSF from meningitis
- Sputum from respiratory infections
- Urine from UTI
- Nasal swab from suspected carriers
direct smears of staphylococcus species from pus or CSF usually reveals
G+ cocci arranged as
- single cells
- pairs
- tetrads
- short chains
direct smears of staphylococcus species from pus or CSF usually reveals
G+ cocci arranged as
- single cells
- pairs
- tetrads
- short chains
The direct smears of staphylococcus species from pus or CSF should be examined for the presence of
inflammatory cells
-these cells along with background tissue debris and proteinaceous
material generally stain pink to red in properly gram stained specimens
Staphylococcus species grow well on routine laboratory media such as
- Blood Agar Medium (BAM)
- Mannitol Salt Agar (MSA)
This an enriched medium that supports the growth of staphylococci and
permits observation of the pattern of hemolysis of blood
Blood Agar Medium (BAM)
A culture media that is both a selective and differential medium for
staphylococci
Mannitol Salt Agar (MSA)
Approximate formula / Liter of Mannitol Salt Agar (MSA)
- Pancreatic digest of Casein - 5g
- Peptic digest of animal tissue - 5g
- Beef extract - 1g
- Sodium chloride - 75g
- D-mannitol - 10g
- Phenol Red - 25mg
- Agar - 15g
Uninoculated MSA appears
light to medium rose red in color
The ______________ concentration of MSA inhibits the growth of
other organisms (except for enterococci), and selectively allows
the growth of halotolerant staphylococci
high salt (7.5% NaCl)
Mannitol fermentation results in acids is indicated by
a change in the phenol red indicator to yellow
this aids in the differentiation of staphylococcal species
Mannitol fermentation
Interpretation of colonial morphologies of staphylococcus species is usually performed after
24 to 48 hours of incubation at 35-37 degrees celsius
size of staphylococcal colonies on Blood Agar Medium (BAM)
medium-large (1-2 mm in diameter)
other characteristics of staphylococcal species on BAM
- color: off-white or gray
- surface: smooth
- margin: entire
- elevation: slightly raised, low convex
- opacity: opaque
- consistency: butyrous (butterlike)
size of S. aureus colonies on BAM
usually large (4.6 mm in diameter)
some strains in S. aureus colonies on BAM produce _________ pigments
golden yellow (lipochrome)
- hence they appear creamy yellow or yellow-orange
- aureus = meaning “golden”
other S. aureus strains may have _____ or ______ zone of B-hemolysis around the colonies
distinct; hazy
-this hemolytic property may become apparent only after prolonged incubation
characteristics of S. epidermidis on BAM
- size: small to medium
- color: gray-white colonies
- opacity: opaque
- most colonies are NONHEMOLYTIC
- Slime-producing strains are extremely sticky and adhere to the agar surface
characteristics of S. saprophyticus colonies on BAM
- size: large
- color: usually white but can be yellow or orange
- surface: smooth; very glossy
- margin: entire
- elevation: convex
- opacity: opaque
- consistency: butyrous (butterlike)
characteristics of Mannitol-fermenting staphylococci (S. aureus)
color: yellow colonies (accompanied by yellow discoloration of the medium) > typical for S. aureus
S. saprophyticus may also ferment mannitol and thus resemble S. epidermidis.
False. S. saprophyticus may also ferment mannitol and thus resemble S. aureus on MSA medium
S. epidermidis is also a mannitol fermenter.
False. S. epidermidis is non-mannitol fermenter.
-Hence, y S. epidermidis and several other species produce SMALL RED colonies with NO COLOR CHANGE to the medium.
Identification Tests are done in what order
-Catalase Test
-Coagulase Test
>Slide Coagulase Test
>Tube Coagulase Test
-Novobiocin Test
When performed with bacterial isolates that are
G+ cocci, this test distinguishes staphylococci
form streptococci
Catalase Test
because
-Staphylococci = strongly catalase (+)
-streptococci = catalase (-)
The principle of this identification test is based on the ability of the bacteria to produce the catalase enzyme that decomposes hydrogen peroxide into water and oxygen
Catalase Test
The evolution of oxygen in Catalase Test leads to
effervescence (rapid bubble formation)
what culture slant is unsuitable for Catalase Test
Blood Agar Slant
This test is done on catalase-positive, gram-positive cocci, placing them in the genus Staphylococcus
Coagulase Test
the single most reliable characteristic for identifying S.
aureus
Coagulase Test
The principle of this identification test relies on the coagulase enzyme produced by S. aureus that binds plasma fibrinogen and activates a cascade of reactions causing plasma to clot
Coagulase Test
Two types of Coagulase Test
- Slide coagulase test
- Tube coagulase test
The medium for both slide coagulase and tube coagulase
procedures
rabbit plasma with EDTA
Rabbit plasma has high amounts of
Coagulase-reacting Factor (CRF)
Preferred anticoagulant of rabbit plasma
EDTA
Why is human plasma not recommended as medium for slide coagulase and tube coagulase procedures?
because it contains variable
amounts of CRF and may contain anti-staphylococcal
antibodies
Why is citrated plasma not used in coagulase test
because some bacteria (enterococci) are able to utilize citrate which could yield to false positive results if they are mistaken for staphylococci
NOTE: ALWAYS PERFORM A CATALASE TEST FIRST
This is a screening test for S. aureus
Slide Coagulase Test
Strains that are negative with the
slide coagulase test must be
confirmed with a tube coagulase test
This identification test is based on the principle that the abundant presence of bound coagulase or “clumping factor” on the surface
of the cell wall of most strains of S. aureus.
Slide coagulase test
This factor reacts directly with fibrinogen in plasma and converts it into fibrin, causing rapid cell agglutination
bound coagulase or clumping factor
reaction between bound coagulase (clumping factor) and fibrinogen in plasma results in
formation of fibrin which leads to rapid cell agglutination
This indicates a positive result in Slide Coagulase Test
Formation of white precipitates or clumping
within 10-15 seconds of mixing the plasma with the bacterial suspension.
characteristic of the negative control in slide coagulase test
saline suspension remains smooth and milky
what are some human coagulase-negative species that produce clumping factor and may be slide coagulase-positive
S. lugdunensis and S. schleiferi subsp. schleiferi
slide coagulase test can be performed with growth from
blood agar or other nonselective medium
slide coagulase test should not be performed with growth from
media having high salt content (mannitol salts agar)
-because high salt causes some strains of S. aureus to AUTOAGGLUTINATE
This is a confirmatory test for S. aureus
Tube coagulase test
this identification test’s principle is based on the detection of free coagulase which is secreted extracellularly by S. aureus. The free coagulase reacts with a substance in the plasma called coagulase-reacting factor (CRF), which in turn reacts with fibrinogen in plasma to form fibrin (clot formation)
tube coagulase test
positive result in tube coagulase test
clotting of the plasma (because of the formation of fibrin clot)
what animal isolates may be tube-coagulase positive
- S. intermedius
- S. hyicus
- S. delphini
- S. schleiferi subsp. coagulans
- strains of S. schleiferi subsp. schleiferi
What to do when you are observing for any degree of clot formation (partial or complete) after incubation
-gently tilt the tube
-DO NOT AGITATE as small clot formed may be dislodged and will not
reform anymore
What to do to tube coagulase tests that are negative after 4 hours of incubation at 35 degrees celsius
- should be held at room temperature
- read again after 18 to 24 hours
why does negative tube coagulase tests need to be read again after 18 - 24 hours (at room temp)
because some strains will produce fibrinolysin (staphylokinase) on prolonged incubation at 35°C, causing dissolution of the clot during the incubation period
one of the methods to distinguish between coagulase-negative
staphylococci, S. epidermidis, S. saprophyticus
Novobiocin Test
this agent is involved in several
infectious processes
S. epidermidis
this agent has a recognized clinical significance in urinary tract infections
S. saprophyticus
in novobiocin test, a zone of inhibition of <12mm indicates that the agent is
novobiocin resistant
-presumptive for S. saprophyticus
in novobiocin test, a zone of inhibition of >16mm indicates that the agent is
novobiocin-susceptible
-coagulase-negative staphylococci (CoNS), S. aureus
Novobiocin test is performed as a
disk susceptibility test using a novobiocin disk (NB,5 µg)
other than S. saprophyticus, some human staphylococcal species resistant to novobiocin
- S. cohnii subspecies,
- S. hominis subsp. novobiosepticus
- S. xylosus
- some S. pseudolugdunensis
Additional culture media for isolation of Staphylococcus species
- Columbia CNA Agar
- Phenylethyl Alcohol (PEA) Agar
- Vogel-Johnson (VJ) Agar
- Chapman Stone Agar
- Baird-Parker Agar
This blood supports the growth of fastidious bacteria and allows
detection of hemolytic reactions in Columbia CNA Agar
Sheep blood
what causes Columbia CNA Agar to be selective for G+ bacteria by inhibiting G-bacteria
because it contains the antimicrobial agents: COLISTIN and NALIDIXIC ACID
sheep blood in phenylethyl alcohol supports the growth of fastidious bacteria particularly
G+ cocci.
The medium should not be used for determination of hemolytic
reactions since atypical reactions may be observed
phenylethyl alcohol (PEA) agar
Phenylethly Alcohol (PEA) Agar inhibits
G- bacteria
selectivity of Vogel-Johnson (VJ) Agar is achieved by
-potassium tellurite
-lithium
-chloride
-glycine
which inhibit non-staphylococcal organisms, both G+ and G- bacteria
Tellurite is reduced to metallic tellurium by S. aureus producing
colonies that are
black or gray-black in color
in vogel-johnson (VJ) Agar, mannitol is degraded
into acid which is indicated by _____________ which changes the color of medium to yellow
phenol red
the selectivity of Chapman Stone Agar is due to
relatively high salt content
this serves as a susbtrate for gelatinase activity in chapman stone agar
gelatin
this allows detection of gelatin hydrolysis in chapman agar which is characterized by clear zone around the colonies
ammonium sulfate
chapman stone agar is differential due to
mannitol fermentation
how to determine mannitol on chapman stone agar
add few drops of bromcresol purple to areas on the medium
-yellow color indicates mannitol fermentation
in chapman stone agar, what identifies S. aureus
yellow colonies surrounded by clear halo
in chapman stone agar, what identifies S. epidermidis
white nonpigmented colonies with or without clear zone
in baird-parker agar, these have inhibitory action for organisms other than staphylococci
- tellurite
- lithium chloride
- glycine
in baird-parker agar, this is incorporated to stimulate the growth of S. aureus without destroying the selectivity
Sodium pyruvate
reduction of tellurite to _______ produces a black discoloration
tellurium
Addition of egg yolk is used to demonstrate ____________ producing clear zones around the colonies.
- proteolysis by lecithinase
- lipolysis by lipase
in baird-parker agar, these are black, shiny, convex colonies 1-5 mm in diameter with a narrow white edge
S. aureus
in baird-parker agar, these are black, shiny, irregular shaped colonies with the development of opaque zone around the colonies after 24 hrs
S. epidermidis
S. aureus produces ________ and ___________ .
DNase; thermostable nuclease having endo- and exonucleolytic activities
what are the additional identification tests for staphylococcus aureus
- deoxyribonuclease (DNase) test
- Thermostable endonuclease test
this identification test is based the detection of bacterial deoxyribonuclease
deoxyribonuclease test
this is an extracellular enzyme that depolymerizes (breaks down) DNA into
subunits composed of nucleotides.
deoxyribonuclease
method of inoculation in deoxyribonuclease test could either be
- streak inoculation
- spot inoculation
a DNase test agar plate (containing deoxy-ribonucleic acid) must be incubated at _______ for ___________
35C for 24-48 hrs
after the incubation of a DNase agar plate flood the plate with _________ which will precipitate DNA
1N hydrochloric acid (HCl)
what is the indication of a positive result in DNase test
A clear area surrounding the
growth after addition of 1N
HCL
what is the indication of a negative result in DNase test
no clearing
and a cloudy precipitate
around the colonies
what is the alternative method for DNase test without the use of 1N HCl
usage of agar plates with dye
- methyl green
- toluidine blue
What causes the formation of color green medium when methyl green is used
Methyl green forms a complex with intact polymerized DNA to form green
color of the medium
what is the indication of a positive result in a DNase test using methyl green
DNAse activity depolymerizes the DNA, breaking
down the DNA-methyl green complex, which results in the formation of
colorless zones around the colonies .
what is the indication of a positive result in a DNase test using toluidine blue
DNAse activity depolymerizes the DNA, breaking
down the DNA-toluidine blue complex, and the toluidine blue takes
on its metachromatic color, forming pink to RED ZONES around the
bacterial growth.
what is the indication of a negative result in a DNase test using toluidine blue
A negative test is indicated when the medium remains
blue because the intact DNA-toluidine blue complex, the toluidine blue has a normal blue color
this test also use the same DNase test agar
thermostable endonuclease test
procedures involved in thermostable endonuclease test
-3-mm holes are cut into the agar with a sterile cork borer
-the wells are filled with a 24-hour broth culture
>from the test organism that has been boiled in a water bath (15 mins)
- The plate is incubated overnight at 35°C.
what staphylococcal specie is thermostable endonuclease-positive
S. aureus
what is the indication of a positive result in thermostable endonuclease test (S. aureus)
will show a pink zone surrounding the
well containing the boiled suspension
what is the indication of a negative result in thermostable endonuclease test
when the blue color in the
medium remains
