LABORATORY IDENTIFICATION NEISSERIA SPECIES
SPECIMEN CHOICE AND COLLECTION
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The most important members of the genus
Neisseria are Neisseria gonorrhoeae and Neisseria
meningitidis
the proper collection and transport of clinical
specimens is critical for the isolation, identification
and characterization of agents that cause this
infection
the specimen choice and collection method for the
causative agent of gonorrhea (Neisseria
gonorrhoeae) depends on the testing technique
used in a laboratory, the age sex and sexual
orientation of the patient
cervical is the preferred site for specimens from
female for isolating Neisseria gonorrhoeae
urethral is the site for men or in women with no
cervix (for example those patients who undergoes
post-hysterectomy)
the material can be examined by direct microscopy
and by culture
for culture immediate plating is preferable – but in
most cases this is impossible
generally, a wooden applicator covered with
cotton wool are used to collect the material for
microscope and bacteriological examination for
Neisseria gonorrhoeae
however, some kinds of woods and cotton are
known to be toxic to gonococcus – and the use of
Dacron or rayon swab are recommended
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the method of transport of the infected material
plays a decisive role
especially when the transportation time exceeds a
few hours and the temperature is too high
when specimens are taken for both microscopy and
culture
the specimen taken first should be used for the
culture because it is likely to contain more material
than the second specimen – take note of that
GRAM STAIN
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a direct smear for gram staining may be performed
as soon as the swab specimen is collected from the
urethra, cervix, vagina or rectum to isolate Neisseria
gonorrhoeae
the swab should be rolled gently onto the slide to
preserve the cellular morphology
under oil immersion or 1000x magnification
smears from symptomatic patients usually contain
intracellular gram negative kidney shape diplococci
in polymorphonuclear leukocytes
the presence of which is required for the
presumptive diagnosis of gonorrhea
Neisseria meningitidis are gram-negative coffee
bean shape diplococci
that may occur intracellularly or extracellularly in
polymorphonuclear leukocytes
meaning it can be located inside the cell or outside
of the cell – specifically the neutrophils
CULTURE
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if bacterial meningitis is suspected – cerebrospinal
fluid is the best clinical specimen to use for
isolation, identification and characterization of
etiological agents
the collection of CSF is an invasive procedure – it
should only be performed by experienced personnel
under septic conditions
SPECIMEN TRANSPORT
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in vitro gonococcal growth is more difficult to
achieve than growth of many other pathogens
the genus in Neisseria nutritional growth
requirements are relatively demanding
atmospheric carbon dioxide is required for certain
isolates and others grow better with carbon dioxide
incubation temperatures are relatively exacting
with extremes at either and detrimental
and autolysis occurs after optimal growth
Neisseria species grows best aerobically in an
atmosphere containing 3-5% of carbon dioxide at a
temperature of 35 degrees centigrade
and it can be achieved by using a candle jar
the earliest culture media contained body fluids to
provide the gonococcal nutritional requirements
the chocolate agar was one of this early media
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Neisseria gonorrhoeae colonies on chocolate agar
are
small, gray to tan, translucent and raised
exhibits smooth consistency and defined margins as
you can see with
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in contrast the Neisseria meningitis
appear as large colorless to gray opaque colonies on
chocolate agar plate
prior to identification and characterization testing
procedures – k isolate should always
be inspected for purity of growth
a single colony should be restricted when necessary
to obtain a pure culture
Thayer-martin agar is used for the isolation of
pathogenic Neisseria from specimens containing
mixed flora of bacteria and fungi
it is used to isolate and cultivate Neisseria species
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OXIDASE TEST
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the genus Neisseria can be identified using covax
oxidase test and also carbohydrate utilization test
if the oxidase test is positive – j carbohydrate
utilization testing should be performed
if the carbohydrate utilization test indicates the
specific necessary species – serological tests to
identify the sero group should also be performed
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CARBOHYDRATE UTILIZATION TEST
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patterns of acid production from the carbohydrates:
glucose, maltose, lactose, sucrose and sometimes
fructose
are used to identify Neisseria and related species
in contrast to most bacteria which produce acid by a
fermentative pathway
Neisseria species produce acid by an oxidative
pathway
this is an important distinction because more acid is
produced by fermentation than by oxidation
the traditional test for determining acid production
by the Neisseria and related species has been the
cystine tryptic is agar test or CTA test
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in which a base medium was supplemented with a
1% final concentration filter sterilized
carbohydrate solution adjusted to a final pH of 7.3
the phenol red indicator is added to detect the acid
and this indicator changes from red alkaline to
yellow which indicates acid production
the CTA media contained in a screw cap tubes are
inoculated with heavy suspension of a 18 hour to 24
hour pure subculture from chocolate or equivalent
medium
the tubes are then incubated without
supplemental carbon dioxide at 35 degrees Celsius
for 24 hours
often tests must be incubated for at least 24 to 48
hours or longer - before the reaction patterns
can be determined
RAPID CARBOHYDRATE UTILIZATION TESTS (RCUT)
traditionally CTA supplemented with a 1%
carbohydrate solution was used to determine acid
production by Neisseria and related species
this test is no longer - so CTA is no longer
recommended now - and has been replaced by the
rapid carbohydrate utilization test or the RCUT
in this test patterns of acid production from
carbohydrates: glucose, maltose, lactose, sucrose
and fructose are used to identify Neisseria and
related species
so how is the RCUT test or the rapid carbohydrate
utilization test performed
a small amount 0.10 ml of phosphate buffered
saline solution containing phenol red as the pH
indicator are distributed in a series of tubes
these tubes are labeled as glucose, sucrose and
lactose
single drops of the individual carbohydrates are
added to individual tubes according to the labeling
example the glucose is added in the tube labeled as
glucose
then after that a heavy suspension of the test
isolate is prepared in a buffer without
carbohydrates
one drop of this suspension is added to each of the
labeled tubes
Neisseria species utilize a carbohydrate source
present in the tubes
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and produce acid by an oxidative pathway which is
in contrast to other organisms which produces acid
by fermentative pathway
the acid daft produced when exceeds the buffering
capacity of the solution
and the color of the medium changes from red
which is alkaline to yellow which is equivalent to
an acid production
these are the rapid acid detection test reactions for
Neisseria and related
species which is Moraxella catarrhalis