Culture Media
medium contains sodium isolation of Legionella.
- Chocolate agar and
chocolate agar based
selective media should be
used for recovery of
Neisseria gonorrhoeae from
urethral discharge. Chocolate
agar provides the nutrients
required by N. gonorrhoeae
and selective media contains
antimicrobial agents that
inhibits other organisms and
permits recovery of
pathogenic Neisseria
- Columbia CNA agar contains
colistin and nalidixic acid,
which inhibit most gram-
negative organisms. Eosin
methylene blue is selective
and inhibits gram-positive
organisms and modified
Thayer Martin is selective
and inhibits gram-positive
organisms, gram-negative
bacilli and yeast.
- Enriched media such as
chocolate agar has no
inhibitory effects on
bacterial growth and
contains additional
nutrients that support the
growth of fastidious
organisms such as H
influenzae and Neisseria
- Chocolate agar and
chocolate agar-based
selective media [Martin
Lewis] are routinely used
for the recovery of
Neisseria gonorrhoeae
from genital specimens.
Sputum and urine
specimens are routinely
processed using a general
purpose media [blood
agar] and a selective agar
[EMB or MacConkey]. In
addition chocolate agar is
routinely included to
enhance recovery of
fastidious organisms such
as H influenzae. CSF is
routinely processed using
blood and chocolate agars.
- CIN agar is a selective
and differential medium
for the isolation and
differentiation of Y
enterocolitica, This
desoxycholate, crystal
violet, cefsulodin, irgason
[triclosan], and
novobiocin as selective
agents, and mannitol as
the carbohydrate.
- Differential media contain
compounds, often
carbohydrates, that
provide
a presumptive
identification
based on colony color or a
precipitate around the
colony. Examples include
MacConkey, Hektoen and
xyloselysine
desoxycholate
agar.
- TCBS is a highly selective
and
differential medium for
the recovery of
most Vibrio species
including V
parahaemolyticus.
Hektoen and
Salmonella-Shigella agars
are selective
and differential for the
isolation and
differentiation of enteric
pathogens such
as Salmonella and
Shigella. EMB is a
selective and differential
medium for gram
negative enteric bacilli.
- Skirrow medium is an
enriched selective
blood agar medium used
for the isolation of
Campylobacter from
specimens with mixed
flora. CIN and bismuth
sulfite agars are
selective and differential
for Yersinia
enterocolitica and
Salmonella, respectively.
CNA agar is selective for
gram- positive
organisms.
- Buffered charcoal yeast
extract medium is
a specialized enrichment
medium for the
The nutritive base
includes yeast extract.
Charcoal is added to
the medium as a
detoxifying agent
- Regan-Lowe agar is an
enriched and
selective medium for the
isolation of B
pertussis. Cephalexin is
added to inhibit
nasopharyngeal flora. It
provides better
isolation of B pertussis
than Bordet-Gengou
medium.
- Francisella tularensis is
fastidious and
not readily recovered in
culture. Cysteine
blood glucose agar is an
enriched medium
with beef heart infusion,
peptones, glucose
and rabbit blood. It also
includes cystine,
which is required by F
tularensis for
growth.
- Sheep blood agar is
preferred because
clear-cut patterns of
hemolysis are
obtained.
- Detection of group B
Streptococcus
[GBS] in the genital and
gastrointestinal
tracts of pregnant women
can identify
infants at risk for GBS
infection. The
CDC currently
recommends the
collection of vaginal and
rectal swabs or
a single swab inserted first
into the
vagina and then the
rectum at 35 - 37
weeks gestation. The
swab[s] should be
inoculated into a selective
broth medium
such as LIM broth [Todd-
Hewitt broth
 with colistin and nalidixic
acid]. The use
of vaginal/rectal swabs
and selective
broth medium greatly
increases the
recovery of GBS.
- Nutritionally deficient
streptococci such as
Abiotrophia
do not grow on sheep
blood agar
without the addition of
cysteine or
proximity to S aureus
colonies.
Antibiotic
- Variations in the
- The MIC is a basic laboratory
measurement of the activity
of an antibiotic against an
organism. It is the
lowest concentration of
antibiotic that
inhibits visible growth of the
organism.
It does not represent the
concentration
of antibiotic that is lethal to
the organism.
- Antimicrobial resistance in
Neisseria gonorrhoeae is
widespread. The production
of beta lactamase
- Cefoxitin is used as a
[penicillinase] breaks
open the beta lactam ring of
penicillin, destroying its
activity.
Thus, N. gonorrhoeae that
produce
beta lactamase are resistant
to penicillin.
- The chromogenic
cephalosporin test using
nitrocefin is the most
sensitive and specific test for
detection of beta lactamase.
Acidimetric tests employing
penicillin are less expensive,
but not as sensitive, as the
nitrocefin assay.
- All Mueller-Hinton agar used
- Systemic enterococcal
infections, such
as endocarditis, are
commonly treated
with a cell-wall-active agent
and an
aminoglycoside. These
agents act
synergistically to kill the
organism. If the
organism is resistant to one
or both, there
is no synergy, and the
combination will
fail. It is important to detect
aminoglycoside and beta-
lactam
resistance in these cases.
Enterococci
have intrinsic moderate level
resistance to
aminoglycosides. Acquired
resistance
corresponds to very high
MICs [greater
than 500 mg/mL] for
gentamicin and is
termed high level resistance.
concentrations of
divalent cations primarily
calcium and
magnesium affect the results
of
aminoglycoside, tetracycline
and colistin
tests with P aeruginosa
isolates. A cation
concentration that is too
high results in
smaller zone sizes, and a
concentration
that is too low increases
zone sizes.
surrogate
for mecA-mediated oxacillin
resistance in S aureus. S
aureus
with cefoxitin MICs >4
mg/mL are
considered oxacillin
resistant. The
Clinical Laboratory Standards
Institute [CLSI] recommends
addition of 2% NaCl,
incubation at
35 +/- 2 C, and incubation for
24 hours when performing
susceptibility testing of S
aureus against oxacillin.
for disk diffusion
susceptibility testing
should be poured to a depth
of 4 mm. If the depth of the
media is <4 mm, this may be
associated with excessively
large zones and false positive
susceptibility results. Agar
that is > 4 mm in depth may
cause excessively small zone
sizes.
- The amount of antibiotic
used in disk
diffusion susceptibility
testing is
standardized and constant.
Once the disk
is placed on the inoculated
plate and
makes contact with the
agar, the antibiotic
in the disk begins to
diffuse out. As it
diffuses into the media,
the concentration
of antibiotic gets lower
the further it
diffuses from the disk.
- As many as 20%-40% of
H influenzae
produce beta-lactamases.
Detection of these
enzymes should be
performed on any isolate
considered to be a
pathogen using the
chromogenic
cephalosporin [nitrocefin]
test.
- For optimum detection of
oxacillinresistant S
aureus, a suspension with
a
turbidity equivalent to a
0.5 McFarland
standard should be
inoculated into a cation
adjusted Mueller-Hinton
broth with 2% NaCl.
Plates should be incubated
at 35 +/- 2 C for
24 hours. Temperatures
above 35 C may not
detect oxacillin resistance.
- Cyclohexamide, which
inhibits protein
synthesis, is the common
agent used in
Mycosel or mycobiotic
agar to inhibit
faster-growing
saprophytic fungi.
Penicillin and
streptomycin do not
inhibit
fungi. Amphotericin B is
 not routinely
used as an additive in
fungal media.
- Some bacteria such as - Cephalothin is a first-
Enterococcus
sp., H influenzae and
Neisseria
gonorrhoeae continually
produce betalactamase. S
aureus produces
betalactamase only after
exposure to an
inducing agent [such as
penicillin]. C - Penicillin inhibits
diphtheriae and S
pyogenes do not
produce beta-lactamase.
- Deterioration of the
antimicrobial agent
in the disk will cause the
zone sizes to be
too small [falsely
resistant].
Standardization of the
inoculum turbidity
to less than a 0.5
McFarland standard
would result in an
inoculum that is too - Susceptibility testing
light and resulting zone
sizes that are too
large. Incubation of the
plates at 35 C
and inoculating plates
within 10 minutes
of preparation would not
have an adverse
effect on zone sizes.
- The disk diffusion
procedure will
not differentiate S aureus
strains
with reduced
susceptibility to
vancomycin [MICs 4 -8 - Certain antimicrobials,
mg/mL]
from susceptible stains
even when
incubated for 24 hours.
- A delay of more than 15
minutes between placing
the disks
on an inoculated plate and
incubation permits excess
prediffusion of the
antimicrobial - Extended spectrum beta
agent from the disk. This
would
result in a larger than clavulanic acid.
expected Confirmatory tests for the
zone diameter presence of ESBL
are based on the enhanced
generation activity of a betalactam
cephalosporin, cefotetan antibiotic, usually
and cefotaxime or
cefoxitin are second- ceftazidime, when it is
generation tested with clavulanic
cephalosporins, and acid compared to the
ceftriaxone is activity of the betalactam
a third generation tested alone.
cephalosporin. -
Sterilization
penicillin
binding proteins that are - The traditional gravity
essential displacement
to peptidoglycan [cell steam sterilization cycle is
wall] 121 C for 15
synthesis. minutes at 15 pounds per
Chloramphenicol square inch.
inhibits protein synthesis, Ethylene oxide is an
colistin alternative
increases cell membrane sterilization method
permeability, and - Bacillus
sulfamethoxazole inhibits stearothermophilus is
folate commonly used as an
metabolism. indicator organism
for the appropriate
should be functioning of
performed when the autoclaves. Unlike most
susceptibility of the Bacillus
organism cannot reliably species, B
be predicted and stearothermophilus grows
resistance is known or at
suspected. 56 C.
Susceptibility testing of
Colony
penicillins for
treatment of S pyogenes - When 0.001 mL of urine is
does not need plated, the growth of one
to be performed routinely colony is equivalent to 1000
since CPU/mL. Thus 70 colonies
resistance has not been are 70,000 CPU/mL
documented. - Urine in the bladder is
normally sterile. Suprapubic
such as aspiration removes urine
nitrofurantoin and directly from the
bladder and yields a
norfloxacin, are used
specimen free
only or primarily to treat
of urethral contamination.
urinary tract
- The number of
infections. These agents
colonies isolated is
should not be
multiplied by 100
reported for pathogens
when a 0.01 mL
recovered from
loop is used for
other sites of infection
inoculation.
Grampositive, catalase
lactamases
negative cocci are
[ESBL] are inhibited by
 indicative of
streptococci.
Blood Culture
- Coagulase-negative
staphylococci are commonly
associated with
contaminated blood
cultures; however, they are
also increasing as a cause of
true bacteremia. Significant
bacteremia in a patient with
endocarditis is usually
continuous and low grade.
In most cases, all blood
cultures drawn will yield
positive results. The facts
that only 1 bottle of 1
set was positive, and that the
bottle did not become
positive until day 5 of
incubation, indicate that
this isolate is most likely a
contaminant
- The volume of blood
collected is the single
most important variable in
the recovery of
organisms in patients with
bloodstream
infections. Since many
cases of adult
bacteremia are of low
magnitude, there is a
direct relationship
between the yield of blood
culture [positivity] and
volume of blood
collected the collection of
multiple blood
culture sets from a single
venipuncture is an
unacceptable practice due
to the potential for
contamination. The
practice of terminal
subculture of blood
culture bottles at 5 days
is no longer recommended
The use of
chlorhexadine for skin
antisepsis does not
affect organism recovery,
but aids in
decreasing blood culture
contamination.
MOS air requirements
- Campylobacter coli/jejuni
require a microaerophilic
atmosphere for optimal
recovery. The use of
selective media is
recommended for recovery
from fecal specimens.
Selective media for
Campylobacter contains
antibiotics to inhibit the
growth of enteric gram-
negative flora. Unlike other
enteric pathogens, C
coli/jejuni grow well at
42 C.
- Facultative anaerobes are
organisms that can grow
under both aerobic and
anaerobic conditions
- The 2 most common
causes of failure of
the Gas Pak system is a
defective gasket
in the jar lid that allows
escape of gas from
inside the jar and
inactivated catalyst
pellets.
- Most Campylobacter
species grow best
under lower oxygen
tension in an
atmosphere of 5%
oxygen, 10% carbon
dioxide and 85% nitrogen.
E coli and
Proteus mirabilis are
facultative
anaerobes and
Pseudomonas aeruginosa
is an aerobe.
- Many anaerobic bacteria
are
commensal flora in the
oropharynx.
Anaerobic bacteria do not
cause
pharyngitis. The most
common cause of
pharyngitis is
Streptococcus pyogenes.
Other causes include
Arcanobacterium
haemolyticum,
Corynebacterium
diphtheriae, Neisseria
gonorrhoeae and
viruses.
Microscopy
- Due to the small size of
viruses, they
are not visible using light
microscopy.
Electron microscopy is
used to visualize
viruses and the internal
structure of
microorganisms.
Quality check
- Daily disk diffusion
quality control can
be converted to weekly
testing when 30
days of consecutive
testing
demonstrates no more
than 3
antibiotic/organism
combinations outside
of the acceptable limits
- Quality control zone sizes
that are
too small could indicate
that the
organism inoculum is too
high, plates
were poured too thick, or
that the
potency of the antibiotic
disks is too
low.
Specimen Requirements
- Urine is an appropriate
specimen for the
detection of renal
tuberculosis. Since feces
contain anaerobic
organisms as part of the
indigenous flora, it is an
unacceptable specimen
for anaerobic culture.
Foley catheter tips are
also not acceptable for
culture, because they
are contaminated with
colonizing organisms.
Gram stain smears of
rectal swabs for N
gonorrhoeae should also
not be performed,
since the presence of
organisms with similar
 morphologies may lead to - Problems with analysis
over interpretation of of Gram staining
smears. generally
- Materials collected from result from errors
sites not harboring including
indigenous flora [sterile interpretation of the slide
body fluids, abscess [smear prepared too
exudate and tissue] should thick],
be cultured for excessive heat fixing, and
anaerobic bacteria. improper decolorization.
However, since anaerobes Inadequate decolorization
normally inhabit the skin with acetone/alcohol
and mucus membranes results in a smear in
as part of the indigenous which host cells
flora, specimens such [neutrophils and
as urine, sputum, and squamous cells], as well
vaginal, eye and ear as bacteria, all appear
swabs are not acceptable blue.
for culture. - Moraxella osloensis is a
- The use of swabs for gram-negative
collection of coccobacillus that is often
specimens for anaerobic plump and
culture is occurs in pairs and
discouraged. Aspiration demonstrates a
with a needle and morphology similar to
syringe is recommended. Neisseria. The
Whenever possible presence of this organism
cultures should be in endocervical
obtained before the specimens contaminated
administration of with vaginal
antibiotics to optimize secretions can lead to over
organism recovery. interpretation
of smears for N
Fecal Specimens gonorrhoeae.
- Campylobacter continues
Biochemical Test
to be
the most common enteric - The indole test is used
pathogen isolated from for determining an
patients with diarrhea. organism's ability to
Routinely fecal produce indole from
specimens should be deamination of tryptophan
cultured for by tryptophanase.
Salmonella, Shigella and - Through the action of the
Campylobacter. Fecal enzyme beta
specimens galactosidase, ONPG
are not routinely cultured cleaves into galactose and
for o-nitrophenol [a yellow
enterotoxigenic E coli or compound]
C - the
botulinum. E hartmanni is use of E coli and Proteus
a mirabilis
nonpathogenic parasite will produce a positive
and does and
not cause diarrhea. negative result for indole,
respectively. The
Gram Stain remainder of the
organisms are all positive
for the
test described
- The organism in this urine
culture is a
Staphylococcus
species. Coagulase will
differentiate S aureus
from
coagulase-negative
staphylococci
[CNS] and novobiocin
susceptibility will
differentiate S
saprophyticus from other
CNS.
Saprophyticus is a
common cause
of urinary tract infections
in young
females
- Bile solubility testing of
alpha-hemolytic
streptococci differentiates
S pneumoniae [soluble]
from other alpha-
hemolytic
streptococci, such as
viridans streptococci
[insoluble].
- Coagulase is the
biochemical test used to
distinguish S aureus
[positive] from coagulase
negative staphylococci
[negative].
- Optochin susceptibility
is used to differentiate S
pneumoniae, which are
susceptible, from other
alpha-hemolytic
streptococci, which are
resistant.
- Optochin susceptibility
is used to differentiate S
pneumoniae, which are
susceptible from other
alpha-hemolytic
streptococci, which are
resistant.
- Group D streptococci and
Enterococcus produce a
positive bile esculin test;
however, of these two,
only
Enterococcus grows in the
presence of 6.5% NaCl.
- Bile esculin, PYR,
bacitracin
 and hippurate are growth describe a
biochemicals/tests used in Staphylococcus species.
the Catalase production
presumptive or definitive confirms
identification of beta- that the organism
hemolytic belonged to
streptococci such as S the genus Staphylococcus
pyogenes, S agalactiae and
and coagulase is used to
Enterococcus. differentiate S aureus
- Streptococcus from
agalactiae is coagulase-negative
catalase and bile staphylococci.
esculin hydrolysis - Staphylococcus aureus
negative and produces an enterotoxin
bacitracin and that is associated with
optochin resistant. short- incubation food
Streptococcus poisoning.
agalactiae - Organisms that used to be
hydrolyzes categorized as
hippurate. nutritionally
- Group A variant or deficient
streptococci streptococci
[Streptococcus have been reclassified into
pyogenes] are the
susceptible to genera Abiotrophia and
bacitracin and Granulicatella
CAMP test
negative.
- Group B streptococci
[Streptococcus agalactiae]
are
resistant to bacitracin and
CAMP test
positive.

Diseases

- Noninfectious sequelae
associated with infection
with Streptococcus
pyogenes are
glomerulonephritis and
rheumatic fever.
- Streptococcus
pyogenes is the cause
of exudative pharyngitis,
commonly called strep
throat.
- Staphylococcus
epidermidis is the most
common cause of
prosthetic valve
endocarditis.

Staphylococcus and Streptococcus

- The Gram stain and

culture