I.

Introduction
a. Background
The family Neisseriaceae currently contains the genus Neisseria are
aerobic, nonmotile, non-spore forming, cytochrome exidase and
catalase-positive, gram-negative diplococcic. Their adjacent sides are
flattened, commonly described as coffee bean or kidney shaped.
Neisseria gonorrhoea, Neisseria meningitides, Neisseria catarrhalis,
Neisseria sicca and Neisseria flavescence are some of the species
which belong to this group. The pathogenic spp. are Neisseria
gonorrhoea and Neisseria meningitidis. Furthermore, they require
complex substances in order for them to grow with the presence of
atmosphere of 10% carbon dioxide on primary isolation. Whereas, non-
pathogenic spp may be found in the throat of man and can be grown in
simple media in an ambient environment.

b. Purpose
To be able to identify the different genera of the Family
Neisseria and the most commonly isolated genus
To identify different laboratory techniques used in identifying the
non-pathogenic Neisseria
To be able to identify the Neisseria specie from a performed
laboratory test

II. Materials and Methods
a. Materials
1
st
laboratory period
o Alcohol lamp
o Inoculating loops
o Sterile cotton swab
o Nutrient agar plates (2pcs)
o Clinical specimens (throat swabs)
o Broth or plated pure culture of Genus Neisseria
2
nd
laboratory period
o Gram staining solutions (crystal violet, iodine, ethyl
alcohol, safranin)
o Cedar wood oil
o Oxidase reagent
o Nutrient agar slants (2pcs)
o Staining racks
o Microscope
o Glass slides (2pcs)
3
rd
laboratory period
o Carbohydrate fermentation tests
Glucose
Fructose
Maltose
Sucrose
Lactose
b. Methods
1
st
laboratory period
1. Ask your patient to seat comfortably, tilt the head back with the
mouth wide open. Collect a throat swab by touching the tip of a
sterile cotton swab at the back portion of the throat near the
tonsils. Use a tongue depressor to hold the patients down
during the process.
2. Using the cotton swab with the clinical specimens, touch the
surface of the NAP and do multiple interrupted streaking at the
top of area A. Make as many streak lines as you can without
overlapping. Rotate the swab between thumb and forefinger as
you streak to expose all the swab surface to the plate.
3. Inoculate bacteria into region B by moving a sterile loop once
or twice through region A and then making more streak lines
that neither overlap nor enter region A again. Then stab the
agar two or three times.
4. Sterilize the loop and inoculate region C using the same
technique. If done carefully, this technique is (almost)
guaranteed to produce isolated colonies in some area of the
plate.
5. Do the same with the pure culture of Genus Neisseria.
6. Label the plates and incubate at 37 degree Celsius for 18-24
hours.
2
nd
laboratory period
1. Examine the cultures for bacterial growth. Study the colonial
morphology of the bacteria particularly the edge, size and
elevation of the colony. Describe their consistency after a
subculture is done. Refer to your Microbiology references for
guidance in the colonial characteristics of non-pathogenic
Neisseria species.
2. Choose a colony most likely similar to the colonial morphology
of the members of the non-pathogenic specie of the Genus
Neisseria and perform gram staining. The group should be able
to get gram(-) diplococci, which is the microscopic morphology
of genus Neisseria.
3. Choose a colony most likely similar to the colonial morphology
of genus Neisseria or a look-a-like colony which shows gram (-)
diplococci and streak on nutrient agar slants. The purpose of
this sub-culturing is for the preparation of a pure culture.
Incubate at 37 degree C for 18-24 hours.
4. Get a filter paper and place one drop of oxidase reagent. With a
sterile inoculating loop, pick one colony as described above (#3)
and streak to the saturated paper.
5. Report result of the oxidase test:
a. (+) result = formation of violet color immediately or within
10-30 seconds
b. (-) result = remain colorless
3
rd
laboratory period
1. Carbohydrate Fermentation Tests
a. Inoculate 1-2 colonies of the suspected Neisseria
species into each of the 5 CHO fermentation tubes
b. Incubate at 37 deg C for 24 hours.
4
th
laboratory period
1. Report results of the CHO fermentation.
(+) result = yellow color on broth which indicates that the CHO
is oxidized to an organic acid.
(-) result = no change in color (red)
2. Submit your isolates to your instructors with the corresponding
identity of your organisms.

III. Experiment Results
COLONIAL MORPHOLOGY
Throat culture Pure culture

Size: small
Edge/margin: smooth
Elevation: raised
Color: yellow
Consistency:creamy
Size: pinpoint
Edge/margin: smooth
Elevation: raised
Color: light yellow
Consistency: creamy

MICROSCOPIC MORPHOLOGY
Throat culture Pure culture

Gram reaction: (-)
Microscopic morphology: diplococci
(with presence of chains)
Gram reaction:
Microscopic morphology: diplococci
(with presence of chains)

CHO fermentation tests Throat culture Pure culture
Glucose
Maltose
Lactose
Sucrose
Fructose
Final ID of isolates

IV. Discussion
a. Interpretation of results
b. Expectations
V. Answers to Questions for Research
1. Identify clinical specimens that may be used to isolate the different
species of Neisseria.
Neisseria species Clinical specimen
Neisseria gonorrhoea Rectum, pharynx,
joint fluid
Neisseria meningitidis Nasopharyngeal
swabs
Neisseria sicca Nasopharyngeal
swabs, saliva, sputum
Neisseria catarrhalis Upper respiratory
tract
Neisseria flavescence Nasopharyngeal swab

2. What other testss can be performed (other than those performed) to
differentiate the pathogenic species of Neisseria from the non-
pathogenic species.
Tests Principle
Identify expected (+)
results
QuadFERM +
Uses an acidmetric method
for detection of
carbohydrate utilization and
DNAse and penicillase
activity. Organisms that
utilize carbohydrates will
produce an acid
environment that exceeds
the buffering capacity of the
substances resulting in color
change of phenol indicator
Yellow to orange (DNase
organisms & penicillase
producing organisms)
Chromogenic substrate tests
Detect enzymes that
hydrolyze colorless
substrates and produce
colored end products
Yellow or blue
Coagglutination test
Uses monoclonal antibodies
to detect Neisseria spp
(Neisseria gonorrhoea)
Agglutination within one
minute


3. Why is it that the reagent is not directly placed on the colonies on BAP
in the performance of oxidase test?
4. Differentiate the artificial growth requirements of pathogenic and non-
pathogenic species of Neisseria.
Pathogenic species Non-pathogenic species
Pathogenic Neisseria are very
fastidious; they grow only in an
enriched media such as BAP or
CAP, which provides nutrients,
including iron, hemin (X-factor),
Non pathogenic Neisseria are not
fastidious and can grow on simple
media such as Nutrient agar plate
at 35-37 deg C.
and co-enzyme I or nicotinamide
adenine dinucleotide (NAD, V
factor). Furthermore, these
organisms are sensitive to
temperature changes and should
be protected from cold.

5. Describe through a flowchart the different procedure used in the IS and
ID of Neisseria species starting with the inoculation of clinical specimen
on NAP. Not necessarily those which were only performed, include all
those (even those not performed) which could be used to differentiate
the genus and species.



VI. Updates