Staphylococcus species Flashcards
Specimens for LABORATORY DIAGNOSIS of Staphylococcus species
- 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
G+ cocci, 0.5-1.5 µm in diameter, and [?]
grape-like clusters
G+ cocci arranged as [?]
single cells, pairs, tetrads, and short chains
The direct smears should also be examined for the presence of
inflammatory cells
These cells, background tissue debris and proteinaceous material generally stain
pink to red
Enriched medium that supports the growth of staphylococci
Blood agar medium (BAM)
permits observation of the pattern of hemolysis of blood
Blood agar medium (BAM)
both a selective and differential medium for staphylococci
Mannitol salt agar (MSA)
Uninoculated MSA appears
light to medium rose red in color.
inhibits the growth of other organisms (except for enterococci)
high salt (7.5% NaCl) concentration
selectively allows the growth of halotolerant styaphylococci
high salt (7.5% NaCl) concentration
Mannitol fermentation results in acids indicated by a change in the
phenol red indicator to yellow
aids in the differentiation of staphylococcal species
Mannitol fermentation
Interpretation of colonial morphologies is usually performed after
24 to 48 hours of incubation at 35-37 oC
medium to large (1-2 mm in diameter)
off-white or gray, smooth, entire, slightly raised, low convex, opaque, and butyrous in consistency
Staphylococcal colonies On BAM
usually large (4-6 mm in diameter)
produce golden yellow pigments “lipochrome”
may have a distinct or hazy zone of β-hemolysis around the colonies (apparent after prolonged incubation)
Staphylococcal colonies On BAM
produce small to medium, opaque, gray-white colonies;
most colonies nonhemolytic;
slime-producing strains are extremely sticky and adhere to the agar surface
S. epidermidis On BAM
colonies are large, entire, very glossy, smooth, opaque, butyrous, convex; usually white but colonies can be yellow or orange.
S. saprophyticus On BAM
produce growth of yellow colonies with yellow discoloration of the medium
Mannitol-fermenting staphylococci
- S. aureus
- S. saprophyticus
produce small red colonies with no color change to the medium
Non-mannitol fermentation
- S. epidermidis
distinguishes staphylococci which are strongly catalase-positive from streptococci (catalase-negative)
Catalase test
Catalase test
Based on the ability of the bacteria to produce catalase enzyme decomposes hydrogen peroxide into [?]
The evolution of oxygen results in [?]
water and oxygen
effervescence
single most reliable characteristic for identifying S. aureus; slide or
tube procedures
Coagulase test
Coagulase test is done on
catalase-(+)
gram-(+) cocci
Coagulase test
The coagulase enzyme produced by S. aureus binds [?]and activates a cascade of reactions causing plasma to clot.
plasma fibrinogen
The medium for both slide coagulase and tube coagulase procedures is [?], which is commercially available in lyophilized form.
rabbit plasma with EDTA
Rabbit plasma has high amounts of [?]
coagulase-reacting factor (CRF)
is the preferred anticoagulant.
EDTA
must not be used because it contains variable amounts of CRF and may contain anti-staphylococcal antibodies.
Human plasma
should not be used as some bacteria are able to utilize citrate (such as enterococci) will yield positive results if they are mistaken for staphylococci; always perform a catalase test first.
Citrated plasma
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
Slide coagulase test
Based on the presence of bound coagulase or [?] on the surface of the cell wall of most strains of S. aureus. This factor reacts directly with and converts fibrinogen in plasma into fibrin, causing rapid cell agglutination
“clumping factor”
Some human [?] produce clumping factor and may be slide coagulase-positive.
coagulase-negative species
- S. lugdunensis
- S. schleiferi subsp schleiferi
Slide coagulase test
The test can be performed with growth from [?]
But should not be performed from media having a [?] (MSA) since the high salt causes some strains of S. aureus to autoagglutinate
blood agar (or nonselective medium)
high salt content
This is a confirmatory test for S. aureus since free coagulase is secreted by virtually all strains of S. aureus.
Tube coagulase test
Tube coagulase test
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).
Some animal isolates [?] may be tube coagulase- positive.
Other phenotypic tests distinguish them form S. aureus.
S. intermedius S. hyicus S. delphini S. schleiferi subsp. coagulans strain of S. schleiferi subsp. schleiferi
When observing for any degree of clot formation (partial or complete) after incubation, [?]; do NOT agitate as a small clot formed may be dislodged and will not reform anymore.
gently tilt the tube
Tube coagulase tests that are negative after [?] should be held at room temperature and read again after 18 to 24 hours, because some strains will produce [?] on prolonged incubation at 35°C, causing dissolution of the clot during the incubation period.
4 hours incubation at 35°C
fibrinolysin (staphylokinase)
one of the methods to distinguish between coagulase-negative staph, s. epidermidis and s. aureus
Novobiocin test
Novobiocin test is performed as a disk susceptibility test using a
novobiocin disk (NB,5 µg)
Other than S. saprophyticus, some human staphylococcal species [?] are resistant to novobiocin.
S. cohnii subspecies
S. hominis subsp. novobiosepticus
S. xylosus
S. pseudolugdunensis
Sheep blood supports the growth of fastidious bacteria and allows detection of hemolytic reactions.
Columbia CNA Agar
Antimicrobial agents, colistin and nalidixic acid, render the medium selective for G+ bacteria by inhibiting G- bacteria.
Columbia CNA Agar
Sheep blood supports the growth of fastidious bacteria particularly G+ cocci.
Phenylethyl Alcohol (PEA) Agar
The medium should not be used for determination of hemolytic reactions since atypical reactions may be observed.
Phenylethyl Alcohol (PEA) Agar
PEA inhibits
G- bacteria
Selectivity of the medium is achieved by potassium tellurite, lithium chloride and glycine which inhibit nonstaphylococcal organisms, both G+ and G- bacteria
Vogel-Johnson (VJ) Agar
Vogel-Johnson (VJ) Agar
is reduced to metallic tellurium by S. aureus producing colonies that are black or gray-black in color
Tellurite
Vogel-Johnson (VJ) Agar
is degraded into acid which is indicated by phenol red, which changes the color of the medium to yellow
mannitol
It is selective due to relatively high salt content, although it is reduced to 5.5%
Chapman Stone Agar
serves as a substrate for gelatinase activity
Gelatin
allows detection of gelatin hydrolysis which is characterized by clear zone around the colonies
ammonium sulfate
Chapman Stone Agar is differential due to
mannitol fermentation
Yellow colonies surrounded by clear halo presumptively identifies
S.
aureus.
White nonpigmented colonies with or without clear zone probably is
S.
epidermidis.
Baird-Parker Agar
have inhibitory action for organisms other than staphylococci.
Tellurite, lithium chloride and glycine
Baird-Parker Agar
is incorporated in order to stimulate the growth of S. aureus without destroying the selectivity.
Sodium pyruvate
Reduction of tellurite to tellurium produces a
black coloration
Addition of egg yolk is used to demonstrate proteolysis and lipolysis by [?] and [?] respectively producing clear zones around the colonies.
lecithinase and lipase
Black, shiny, convex colonies 1-5 mm in diameter with a narrow, white edge.
S. aureus
Black, shiny, irregular shape. Opaque zone develop around the colonies after 24 hours.
S. epidermidis
Other tests for S. aureus correlate highly with [?]
coagulase production
S. aureus produces both [?] and a [?] having endo- and exonucleolytic activities.
deoxyribonuclease (DNase) and a thermostable nuclease
DNase test detects bacterial deoxyribonuclease which is an [?] that depolymerizes (breaks down) DNA into subunits composed of nucleotides.
extracellular enzyme
Inoculate, either by [?] or [?] , a DNase test agar plate (containing deoxy-ribonucleic acid), and incubate at [?]; after incubation, flood with [?] which will precipitate DNA.
streak inoculation or spot inoculation
35 oC for 24-48 h
N hydrochloric acid
A [?] surrounding the growth after addition of 1N HCL indicates a positive reaction for DNase activity.
A negative reaction is indicated by no [?] around the colonies
clear area
clearing and a cloudy precipitate
Alternative method:
The test may also be carried out in agar plates with dye, such as [?] and [?]. This eliminates the need to add 1N HCl.
methyl green toluidine blue
The same DNase test agar is used;
[?] holes are cut into the agar with a sterile cork borer
wells are filled with a [?]
boiled in a water bath for [?].
plate is incubated overnight at [?]
3 mm
24-hour broth culture
15 minutes
35°C
S. aureus which is thermostable endonucleasepositive will show a [?] surrounding the well containing the boiled suspension.
Negative test is when the [?] in the medium remains
pink zone
blue color
determines whether bacteria can utilize glucose in the presence or absence of oxygen
Oxidation-Fermentation (O-F) test
Oxidation-Fermentation (O-F) test
sole carbohydrate
pH indicator is used
glucose
bromthymol blue
Oxidation-Fermentation (O-F) test
Two tubes are inoculated by stabbing; one tube
(“[?] tube”) sterile mineral oil which shields it from oxygen in air
(“[?] tube”) is exposed to oxygen
closed
open
Micrococci are [?] because they are aerobic bacteria;
produce acids from [?] only in the presence of oxygen
Medium in the [?] turns yellow
oxidative
glucose
open tube
Staphylococi are [?]
glucose = produce acids in the presence and absence oxygen
This is evidenced C. bound coagulase by yellow color change of the medium in both open and closed tubes.
fermentative; facultative anaerobes
performed as an agar disk susceptibility procedure using commercially available disks
Susceptibility to Bacitracin, 0.04 U and Furazolidone, 100 µg
Micrococci are
?; zones of inhibition: 9mm
bacitracin - susceptible
furazolidone - resistant
endopeptidase enzyme that can lyze the cell wall of staphylococci but not micrococci.
Lysostaphin test
Lysostaphin solution is added to a bacterial suspension and incubated at
35-37 oC for 2 hour
Micrococci are lysostaphin-resistant — bacterial suspension remains
turbid; it does not clear
Staphylococci are lysostaphin-susceptible — there is
clearing of the bacterial suspension
is also known as the microdase test; it is recommended for gram-positive, catalase-positive cocci only
Modified oxidase test
Modified oxidase test uses filter paper disks impregnated with [?] in dimethyl sulfoxide (DMSO).
A colony from the growth medium is removed with an applicator stick and rubbed onto the disk
tetramethyl-pphenylenediamine dihydrochloride
Micrococcus species are modified oxidase-[?]; the test is indicated by development of a [?].
modified oxidase-positive
blue-purple color within 30 seconds
Staphylococcus species are generally modified oxidase-[?]
modified oxidase-negative
no change in color
Production of acids from glycerol in the presence of erythromycin uses a medium containing
glycerol (1%) and erythromycin (0.4 µg/mL)
It is prepared with an [?] containing [?] indicator and poured into Petri plates.
enriched agar base
bromcresol purple
Micrococci will not produce acids from glycerol, so the medium will remain [?] in color.
purple
Staphylococci will produce acids from glycerol causing a [?] color
change in the medium.
yellow
