NEISSERIA

GONORRHOEAE

 SPECIMEN COLLECTION:
 Genital sources
 Specimen of Choice
 MEN: Urethra
 Collected directly onto a swab for
culture
 NO discharge: swab is inserted up to
2cm into anterior urethra and rotated
slowly
 WOMEN: Endocervix
SWABBING
 NEISSERIA
GONORRHOEAE

• SPECIMEN
COLLECTION:
• Other sites of
collection:
• Rectum
• Inserted
4-5cm
into the
anal
canal
• Pharynx
• Joint Fluid
 • SPECIMEN COLLECTION
CONSIDERATIONS:
• CALCIUM ALGINATE and
COTTON SWABS are
inhibitory to N. gonorrhoeae
• PREFERRED SWAB: Dacron
NEISSERIA and rayon
GONORRHOEAE
• N. gonorrhoeae: Extremely
susceptible to drying and
temperature changes 
DIRECT PLATING of the
specimen to gonococcal
selective media gives optimal
results
 NEISSERIA
GONORRHOEAE

• MICROSCOPY:
• Gram negative kidney/ coffee
bean shaped diplococci
 NEISSERIA
GONORRHOEAE

• GRAM
STAINING:
• Prepared from
Urogenital
specimens
 • GRAM STAINING
CONSIDERATIONS:
• For asymptomatic cases
(particularly Women) culture is
needed for confirmation
• A gram stain of >5
NEISSERIA
GONORRHOEAE POLYMORPHONUCLEAR
NEUTROPHILS/FIELD BUT
NO BACTERIA suggests:
• Non gonococcal urethritis
• Presence of: C. trachomatis
or Ureaplasma urealyticum
 NEISSERIA
GONORRHOEAE

 CULTURE:
 Transport systems:
 Transgrow medium
 JEMBEC Medium
 Gonopak
 NEISSERIA
GONORRHOEAE

 CULTURE:
 Transport systems:
 Transgrow medium
 JEMBEC Medium
 Gonopak
 NEISSERIA
GONORRHOEAE

 CULTURE:
 Transport systems:
 Transgrow medium
 JEMBEC Medium
 Gonopak
 NEISSERIA
GONORRHOEAE

• CULTURE:
• Medium of choice:
• CHOCOLATE
AGAR MEDIUM
• Supports the
growth of
many other
organisms
found as
commensals
in specimens
collected for
the recovery
of gonococci
 NEISSERIA
GONORRHOEAE
 COLONIES:
 Small (1-2mm), translucent, raised, moist,
grayish-white-tan with entire, lobate margins
 Colonial types:
 T1, T2 : small, glistening, raised, piliated,
isolated from primary culture
 T3, T5 : larger, non glistening, flattened,
nonpiliated, isolated form subculture
 AHU
 NEISSERIA
GONORRHOEAE
 NEISSERIA
GONNORHOEAE
CULTURE
MEDIUM

• THAYER MARTIN:
• Chocolate agar with enrichment
supplement (ISOVITALEX)
• Colistin
• Nystatin
• Vancomycin
 MODIFIED
THAYER
MARTIN

• Isovitalex
• Colistin
• Nystatin
• Vancomycin
• Trimethoprim
 MARTIN LEWIS

• Isovitalex
• Colistin
• Anisomycin
• Vancomycin : increased
concentration
• Trimethoprim
 NEW YORK
CITY

• Transparent medium with:

Lysed horse blood
Horse plasma
Yeast dialysate
Colistin
Amphotericin B
Vancomycin
Trimethoprim
 NEISSERIA
MENINGITIDI
S

• SPECIMEN
COLLECTION
• CSF
• Blood
• Nasopharyngeal
swabs and aspirates
• Joint fluids
• Sputum
• Urogenital sites
 • INCUBATION:
• SBA and CHOC AGAR
NEISSERIA
MENINGITIDIS • Same conditions as N.
gonorrhoeae and
examined daily for 72
hours
 NEISSERIA
MENINGITIDI
S

• MICROSCOPY
• GRAM STAIN
• Meningococci
appears as
gram negative
diplococci
 FA M I LY
MORAXELLACEAE

• Genera:
• Moraxella
• Acinetobacter
• Psychrobacter

• M. catarrhalis:
commensal of the URT
 NEISSERIA
MENINGITIDI
S

• CULTURE:
• Colonies:
• Round, convex
blue gray with a
smooth
glistening
surface
• Mucoid and
typically larger
than
gonococcus
colonies
 • SPECIMEN COLLECTION:
• Middle ear effusion
• Nasopharynx
MORAXELLA
C ATA R R H A L I S • Sinus aspirates
• Sputum aspirates
• Bronchial aspirates
 MORAXELLA
CATARRHALIS

• CULTURE AND
INCUBATION:
• SBA and CHOC
AGAR
• Colonies:
• Smooth, opaque,
gray-to-white
colonies
• “Hockey puck”
colonies
• Older colonies:
“wagon-wheel”
appearance
• 28C
 • KEY POINTS:
• Inhibited on gonococcal
selective agars by COLISTIN
• Oxidase and Catalase Positive
MORAXELLA
C ATA R R H A L I S • Assacharolytic
• DNAse Positive
• Butyrate esterase positive
reactions
 • HABITAT: upper respiratory tract
• 3rd most common cause of OTITIS
MEDIA and SINUSITIS in children

MORAXELLA • May cause infection of lower

C ATA R R H A L I S respiratory : adults
• Predisposing factors: age,
immunodeficiency,
neutropenia and chronic
debilitating disease
CYTOCHROME OXIDASE
TEST
CATALASE TEST
 OTHER TESTS

• Penicillin Disk test

• Nitrate reduction test
• DNA hydrolysis test
• Superoxol test
 Principle: Based on morphologic reaction of
microbes to penicillin

 Procedure
1. Trypticase Soy Blood Agar
2. Dispense 10μ of penicillin disk
PENICILLIN 3. Incubate at 35°C; Increased CO2 for 24
DISK TEST hours
4. Gram stain colonies around the zone of
inhibition

Neisseria and Moraxella catarrhalis:

Diplococci
Cocobacillary Moraxella: cocobacilli
  Procedure
1. Inoculate Nitrate broth
 1-2 drops(broth) of young colonies
2. Incubate at 48 hours at 35°C
3. Wait for obvious growth
 Maximum of 7 days
4. Test microorganisms
 24 hrs. after obvious growth
5. Apply 5 drops of Reagent A (Sulfanilic
NITRATE Acid)
REDUCTION 6. Apply 5 drops of Reagent B (α-
TEST naphthylamine)
7. Observe for color change

Neisseria spp NEGATIVE

Moraxella catarrhalis: POSITIVE
 Neisseria spp NEGATIVE
Moraxella catarrhalis: POSITIVE

DNA
HYDROLYSIS
TEST
SUPEROXOL TEST
 BIOCHEMICAL
TESTS

• CARBOHYDRATE
UTILIZATION TEST
• ELISA
• RIA
• MOLECULAR ASSAYS
• PARTICLE
AGGLUTINATION
METHODS: PHADEBACT,
GONOGEN II TEST
 Principle: Most Neisseriae oxidize
carbohydrates, producing acid but
not gas, and their carbohydrate
patterns are a means of
distinguishing them

CARBOHYDRAT Medium: Cystine-Trypticase Agar

E UTILIZATION (CTA) medium containing
TEST 1%Carbohydrate

Phenol red pH indicator

CTA
CTA
2. CARBOFERM TEST 3. PHADEBACT
4. GONOCHECK II
 CHROMOGENIC ENZYME TEST

• N. gonorrhaeae
• -hydroxylprolyl aminopeptidase

N. Meningitiddis
- Gamma glutamyl aminopeptidase
OTHER NEISSERIA SPP