Identification of N. meningitidis Serogroup and Performing the
SASG Test
Twelve serogroups, based on the biochemical composition of capsular
polysaccharides, are currently recognized: A, B, C, H, I, K, L, W135, X, Y,
Z, and 29E (Z’). Serogroup D is no longer recognized as a serogroup. Serogroups A, B, C, W135 and Y are the 5
most common causes of bacterial meningitis. Serogroup A has been the most common cause
of epidemics in Africa and Asia. Serogroups C, W135, and X have also been reported as
causes of epidemics in several parts of Africa as well. Serogroup-specific antisera for these
major serogroups are available commercially.
It is not always practical to test for all serogroups for which antisera
are available in a laboratory. Testing algorithms may be set up in laboratories with previous knowledge of
the predominance or lack of serogroups within that particular geographic region in order to
test for the most common serogroups first. Modifications may be made to the testing algorithm
for any laboratory based on information about current strains that are circulating in the
region. For example, in Africa, testing with antisera for serogroups A and W135 (and X in some
regions) as well as with a saline control to detect nonspecific autoagglutination should be adequate
to characterize most case isolates.
Strains reacting negatively with A, W135, and X antisera should then be
tested with other available antisera, particularly C, Y, and B. Nearly all
case isolates are serogroupable, if they are tested against a comprehensive
panel of antisera and proper controls are used. In rare instances, an
isolate that does not react with any of the serogroup-specific antisera or
reacts with more than one serogroup-specific antisera is considered
nongroupable (NG). Polyvalent antisera containing combinations of
serogroups (e.g., A, B, and C or X, Y, W135, and Z) may also be used.
It is essential that reference laboratories have the capacity to isolate,
identify, and characterize the serogroup of isolates of N. meningitidis. This valuable data
provides laboratories and public health authorities with the tools to identify outbreaks controllable by
vaccination campaigns, recognize serogroups causing sporadic disease, and detect emergence of new
outbreak strains.
A. Slide agglutination serogrouping (SASG) test for serogrouping N. meningitidis isolates
Formalin-killed meningococcal suspensions should be used for SASG testing
rather than saline suspensions of living organisms to maintain a safe working environment. A
solution of 5% formalinized physiological saline is sufficient to kill the bacteria.
However, formalin is a carcinogen and must be stored and handled with great care.
Alternatively, work should be performed under a biosafety hood if formalin
is not used. Antisera should be stored in the refrigerator at 4°C and
warmed to room temperature (25°C) before use. It must be put back in the
refrigerator as soon as testing is finished to prevent the loss of binding activity of the antibody.
Read Also : Carbohydrate Utilization by N. meningitidis: Cystine Trypticase Agar (CTA) Method
B. Performing the SASG test
1. Grow the isolate(s) to be tested for 18-24 hours on a BAP at 35-37°C
with ~5% CO2 (or in a candle-jar).
2. Clean a glass slide with alcohol (optional if slides are pre-cleaned).
3. Divide the slide into equal sections (e.g., twelve 11 X 22 mm sections
on a standard 50 X 75 mm slide) with a liquid impermeable pen or a wax pencil. Each isolate will
require as many sections on the slide as the individual serogroup-specific
antisera that will be tested as well as a saline negative control.
4. In the lower portion of each of the sections of the glass slide
described in step (2), add 10 μl of the 5% formalinized saline with a micropipettor. The instructions
specify using a micropipettor with sterilized filtered tips to measure the
10 l of the 5% formalinized saline to suspend the bacteria. The
micropipettor will transfer precise and equal measurements for a proper
SASG reaction.
-If a micropipettor and tips are not available, sterile, disposable 10 l
inoculation loops can be used to transfer 10 l of the 5% formalinized saline, but often do not
deliver accurate amounts (between 5-10 l).
5. Use a sterile, disposable 10 l inoculating loop to collect a few
colonies from the surface of the overnight culture incubated on the BAP.
6. Suspend the bacteria in the 5% formalinized saline solution in the lower
portion of each of the sections of the slide. The suspension should be moderately opaque. Do not allow the cell suspension to dry before adding the
antisera.
-If the bacteria are difficult to suspend directly on the slide, make a
moderately milky suspension (comparable to McFarland 6 standard) of the
test culture in a small vial with 250 l of 5% formalinized saline and
briefly vortex the suspension to mix and break up any pellets. Add 10 l of
this suspension to the lower portion of the slide.
7. In the upper portion of each of the sections of the glass slide
described in step (2), add 10 μl of the serogroup-specific antisera to be tested as well as unformalinized
saline or phosphate buffered saline (PBS) for a negative control with a micropipettor.
-DO NOT use the dropper provided with the antisera because it usually
delivers larger amounts than is necessary and can easily be contaminated.
-If a micropipettor and tips are not available, sterile, disposable 10 l
inoculation loops can be used to transfer 10 l of the antisera, but often do not deliver
accurate amounts (between 5-10 l).
-Dispose of the tip or loop used to transfer the antisera to the slide in a
waste container after each use to avoid contamination of the antisera. If the source of antisera
is contaminated, a new vial must be used.
8. Gently tilt the slide to mix the cell suspensions with the antisera in
each section. Continue to gently rock the slide for 1 to 2 minutes to allow the lower and upper
portions to completely blend. Do not use a circular motion while rocking, as it can cause the
sections with different serogroup-specific antisera to run together and contaminate each other.
9. After 2 minutes, examine the SASG reactions under a bright light and
over a black background. Use the rating system in Figure 6 to determine the
intensity of the agglutination reaction in each section of the slide. Disregard any agglutination that
occurs after the 2 minute time period.
10. Record the SASG results in the laboratory log book.
C. Reading the SASG results
1. Rating the intensity of the agglutination reaction
Agglutination occurs when the antisera bind to the bacterial cells causing
the cells to agglutinate or clump together, thus making the cell suspension
appear clearer. The intensity of the agglutination reaction may vary
according to the density of the cell suspension or the antisera used. A description on the intensity ratings shown in Figure 6 are listed
below.
4+ All of the cells agglutinate and the cell suspension appears clear
3+ 75% of the cells agglutinate and the cell suspension remains slightly
cloudy
2+ 50% of the cells agglutinate and the cell suspension remains slightly
cloudy
1+ 25% of the cells agglutinate and the cell suspension remains slightly
cloudy
+/- Less than 25% of the cells agglutinate and a fine granular matter
occurs
0 No visible agglutination; the suspension remains cloudy and smooth
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