Neisseria

neisseria
is a large genus of bacteria that colonize the mucosal surfaces of many animals. of the 11 species that colonize humans, only two are pathogens, n. meningitidis and n. gonorrhoeae.
microscopy
n. meningitidis are gram-negative, coffee-bean shaped diplococci that may occur intracellularly or extracellularly in pmn leukocytes. n. meningitidis is a fastidious organism, non motile, non spore forming, nonencapsulated, which grows best at 35-37°c with ~5% co (or in a candle-jar). it can grow on both a blood agar plate (bap) and a chocolate agar plate (cap). colonies of n. meningitidis are grey and unpigmented on a bap and appear round, smooth, moist, glistening, and convex, with a clearly defined edge. n. meningitidis appear as large, colorless-to-grey, opaque colonies on a cap. prior to identification and characterization testing procedures, isolates should always be inspected for purity of growth and a single colony should be re-streaked, when necessary, to obtain a pure culture. for the following identification and characterization procedures, testing should be performed on 18-24 hour growth from a bap (figure 1) or a cap (figure 2) at 35-37°c with ~5% co (or in a candle-jar).
clinical samples:
chapter 7
identification and characterization of neisseria meningitidis
blood and/or spinal fluid for (n. meningitidis ), vaginal or penile discharge (for n. n. meningitidis are gram-negative, coffee-bean shaped diplococci that may occur intracellularly
goroexntraocerllrulharloy einapmen) leukocytes. n. meningitidis is a fastidious organism, which grows best
at 35-37°c with ~5% co2 (or in a candle-jar). it can grow on both a blood agar plate (bap) and
a chocolate agar plate (cap). colonies of n. meningitidis are grey and unpigmented on a bap
and appear round, smooth, moist, glistening, and convex, with a clearly defined edge. n.
meningitidis appear as large, colorless-to-grey, opaque colonies on a cap. prior to identification identification
and characterization testing procedures, isolates should always be inspected for purity of growth
and a single colony should be re-streaked, when necessary, to obtain a pure culture. for the following identification and characterization procedures, testing should be performed on 18-24
the following tests are recommended to confirm the identity of cultures that morphologically
hour growth from a bap (figure 1) or a cap (figure 2) at 35-37°c with ~5% co2 (or in a
candle-jar).
appear to be n. meningitidis (figure 3). n. meningitidis can be identified using kovac’s oxidase the following tests are recommended to confirm the identity of cultures that morphologically
appear to be n. meningitidis (figure 3). n. meningitidis can be identified using kovac’s oxidase
testsatndacanrbdohycdratre butiolizhatyiodn. riaf tthe oxuidtaisleitezstaistiposniti.ve,icfarbtohyedraote xutildizatisonetestiengst is positive, carbohydrate utilization testing
should be performed. if the carbohydrate utilization test indicates that the isolate may be n.
smhenoinguitlidis, sbereologpicealrtefsots rtomidendtif.y thiefsertohgreoupcshaourlbd boehpeyrfdormraedt.ethuis tseiqluiezncaetoifon test indicates that the isolate may be n.
testing is an efficient way to save costly antisera and time. additional methods for identification
mandecnhairanctgeriztaitidonisof,n.smeernoingliotidgisiucsianglmtoelescutlsar ttools airde deenscrtiibfedyin tchaeptesr 1e0r: opcgrroup should be performed. this sequence of
methods and chapter 12: molecular methods.
testing is an efficient way to save costly antisera and time. additional methods for identification
biosafety level 2 (bsl-2) practices are required for work involving isolates of n. meningitidis,
and characterization of n. meningitidis using molecular tools are pcr methods and other
as this organism presents a potential hazard to laboratory personnel and the surrounding working
environment. please refer to chapter 4: biosafety in order to follow the guidelines that have
molecular methods.
been established for laboratorians working in bsl-2 facilities as many of the tests described in
this chapter require opening plates with live cultures and are often performed outside of a
biosafety cabinet (bsc).
figure 1. n. meningitidis colonies on a bap
figure 2. n. meningitidis colonies on a cap
figure 2. n. meningitidis colonies on a cap
figure 1. n. meningitidis colonies on a bap
1
inoculate bap or cap
examination of growth on bap or cap shows round, moist, glistening, and convex colonies
kovac’s oxidase test
oxidase oxidase positive negative
sterile site specimen
(e.g., csf or blood) from suspect casepatient
perform gram stain on csf for clinical decision-making
not n. meningitidis
gram-negative other morphology or diplococci= stainingcharacteristics n. meningitidis = not n. meningitidis
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kovac’s oxidase test
kovac’s oxidase test determines the presence of cytochrome oxidase. kovac’s oxidase reagent, tetramethyl-p-phenylenediamine dihydrochloride, is turned into a purple compound by organisms containing cytochrome c as part of their respiratory chain. this test aids in the recognition of n. meningitidis, but other members of the genus neisseria, as well as unrelated bacterial species, may also give a positive reaction. positive and negative quality control (qc) strains should be tested along with the unknown isolates to ensure that the oxidase reagent is working properly.
performing kovac’s oxidase test filter paper method
1. grow the isolate(s) to be tested for 18-24 hours on a bap at 35-37°c with ~5% co (or in a candle-jar).
2. on a nonporous surface (i.e., petri dish or glass plate), wet a strip of filter paper with a few dropings of kovac’s oxidase reagent.
3. let the filter paper strip air dry before use.
4. use a disposable plastic loop, a platinum inoculating loop, or a wooden applicator stick to
pick a portion of a colony from overnight growth on the bap and rub it onto the treated
filter paper (figure 4).
5. observe the filter paper for color change to purple.
6. perform steps 3 and 4 with a positive and negative qc strain to ensure that the oxidase
6. perform srtepas 3gaennd 4t wisithwa posritkivienangd npergaotipveeqrclyst.rain to ensure that the oxidase reagent is working properly.
figure 4. kovac’s oxidase test: a negative and positive reaction on filter paper
figure 4. kovac’s oxidase test: a negative and positive reaction on filter paper
plate method
1c. .grrowetahedisionlatge(st)htoebeotexsteidfaors1e8-2t4ehsotursroensaubaltpsat 35-37°c with ~5% co2 (or in a candle-jar).
2. dispense a few dropings of kovac’s oxidase reagent directly on top of a few suspicious colonies
• positive reactions will develop within 10 seconds in the form of a purple color where the
growing on the 18-24 hour bap.
bacteria were applied to the treated filter paper. delayed reactions are unlikely with n. • do not flood the entire plate as the bacteria exposed to the reagent are usually not viable for
subculture.
3. tilt the plate and observe colonies for a color change to purple.
4. perform steps 1 and 2 with a positive and negative qc strain to ensure that the oxidase 2 reagent is working properly.
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and negative results should not be interpreted before 72 hours of incubation. a color change to
&
yellow without turbidity is usually not a positive reaction. carbohydrate utilization results for differentiating n. meningitidis from other neisseria spp. and bacteria are listed in table 1.
meningitidis.
development of visible turbidity and a yellow color in the upper portion of the medium indicates
growth of bacteria and production of acid and is interpreted as a positive test (figure 5).
although reactions may occur as early as 24 hours after inoculation, some reactions are delayed
• negative reactions will not produce a color change on the treated filter paper. yellow without turbidity is usually not a positive reaction. carbohydrate utilization results for
and negative results should not be interpreted before 72 hours of incubation. a color change to
differentiating n. meningitidis from other neisseria spp. and bacteria are listed in table 1.
figure 5. cta sugar reactions for n. m(aeni)ngitidis with utilization of glucose (dextrose) and (b)
maltose, indicated by acid production (color change to yellow), and no utilization of lactose or
sfucirgosue re 5. cta sugar reactions for n. meningitidis with utilization of glucose (dextrose) and
figure 5. sugar reactions for n. meningitides(a) with utilization of glucose (dextrose) and maltose, indicated by acid production (color change to yellow), and no utilization of lactose or table 1. carbohydrate utilization by some neisseria and moraxella spp.
maltose, indicated by acid production (color change to yellow), and no utilization of lactose or
sucrose
sucrose, (b) n. gonorrhoeae
tnea organism glucose1 maltose lactose sucrose
.agirc neisseria lactamica
ahrybdoraht+eyudtrilaiztaet+iuontibliyzsao-
isbs b
l
e1m.e
elrei
1
a
ncin
tibdios
neisseria gonorrhoeae
neisseria sicca
+ +2
+ -
+ - + -
+ - -
- 1 glucose
-
-
moraxella catarrhalis
aci+d production from: -
sucrose
organism
maltose lactose
acid production from:
1
2 some strains of n. gonorrhoeae are weak acid producers and may appear to be glucose
neisseria meningitidis
+ - -
glucose may also be referred to as dextrose.
neisseria lactamica iiin.ideeinstisfiecartiaongofonn. moernrinhgoitiedias eserogroup
https://www.google.com/search?q=neisseria
negative in the cta medium.
neisseria gonorrhoeae
ysesrioamaned nmoeriasxsellraiasppa.nd moraxella spp. tmi oe nn ebi s-
+
+ + + -
+2 - - - neisseria sicca 6 + + - + moraxella catarrhalis - - - -
1 glucose may also be referred to as dextrose.
2 some strains of n. gonorrhoeae are weak acid producers and may appear to be glucose neisseria gonorrhoeae, also known as gonococci (plural), or gonococcus (singular), is a species
laboratory cultures. to be specific, they grow on chocolate agar with carbon dioxide. these cocci
are facultatively intracellular and typically appear in pairs (diplococci), in the shape of coffee beans. of the eleven species of neisseria that colonize humans, only two are pathogens. n. gonorrhoeae is the causative agent of gonorrhea and is transmitted via sexual contact.
culture and isolation
neisseria is usually isolated on thayer-martin agar (or vpn agar)—an agar plate containing antibiotics (vancomycin, colistin, nystatin, and trimethoprim) and nutrients that facilitate the
negative in the cta medium.
of gram-negative coffee bean-shaped diplococci bacteria responsible for the sexually transmitted infection gonorrhea.
iii.identification of n. meningitidis serogroup
neisseria are fastidious gram-negative cocci that require nutrient supplementation to grow in
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growth of neisseria species while inhibiting the growth of contaminating bacteria and fungi. further testing to differentiate the species includes testing for oxidase (all clinically relevant neisseria show a positive reaction) and the carbohydrates maltose, sucrose, and glucose test in which n. gonorrhoeae will oxidize (that is, utilize) only the glucose.
meningitidi sicca gram stain
meningitidis bacteria
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references
ryan kj ray cg (editors) (2004). sherris medical microbiology (4th ed.). mcgraw hill. isbn 0-8385-8529-9.
a.prof. dr. zainab al-mahdi
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meningitidis
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