RDT for Pneumococcal Meningitis and QC of Primary Culture Media
RDT commercial kits are also available for S. pneumoniae detection
by immunochromatography.
The principle is the same as the one described above for N.meningitidis. Follow the manufacturer's instructions on the
package insert.
II. Primary Culture
A. Selection of primary culture media
The best medium for growth of S. pneumoniae is a blood agar plate
(BAP), which is a trypticase soy agar (TSA) plate containing 5% sheep
blood. Human blood is NOT an acceptable substitute for the blood in the agar because the antibodies contained in human blood
may inhibit bacterial growth. S. pneumoniae will also grow on a chocolate agar plate
(CAP).
For H. influenzae, a CAP made with heat lysed blood or
supplemented with hemin (X factor) and nicotinamide-adenine-dinucleotide (NAD; V factor) should be used. Growth of H. influenzae on a BAP may be achieved by adding a source of NAD, traditionally done by
cross-streaking the inoculated medium with a Staphylococcus aureus or Enterococcus species strain. H. influenzae forms satellite colonies along the length of the staphylococcal or
enterococcal growth.
Additionally, applying filter paper (or disks) saturated with hemin and NAD
to the surface of the BAP after the medium has been inoculated will produce a halo of growth
around the strip or disk.
N. meningitidis
grows on both BAP and CAP. Because N. meningitidis grows well in a
humid atmosphere, if an infection with N. meningitidis is
suspected, laboratorians may choose to add a shallow pan of water to the bottom of the incubator or add a dampened paper
towel to the candle
jar. The moisture source should be changed regularly to prevent
contamination with molds.
Ordinarily, both BAP and CAP are used for subculture. If only one type of
plate is available, a CAP should be used because it contains the hemin and NAD needed for H. influenzae, whereas a BAP does not.
If primary cultures appear to be contaminated, selective media may improve
the isolation of these bacteria from specimens containing a mixed flora of bacteria and/or
fungi. For primary isolation of N. meningitidis, a chocolate agar base containing
vancomycin, colistin, nystatin, and trimethoprim can be used. For isolation of S. pneumoniae, tryptic
soy agar with 5% sheep blood and either gentamicin, neomycin or sulfamethoxazole-trimethoprim (SXT) may
be useful. For isolation of H. influenzae, chocolate agar with bacitracin can be
used. Other antibiotic formulations are also available.
B. QC of primary culture media
All primary culture media should be tested for QC to ensure that the media
will support the proper growth of N. meningitidis, S. pneumoniae, and H. influenzae. One plate from each new lot of media received should be tested using an appropriate,
well-characterized reference strain of N. meningitidis, S. pneumoniae
, and/or H. influenzae. In addition, all media should be tested periodically (every 3 months) to ensure that it can support the growth of
appropriate bacteria.
One uninoculated plate from each new lot should also be tested in order to
check for contamination of mold or other organisms in the laboratory and/or
incubator. QC should be repeated on plates from a lot if they have been exposed to temperatures
above 4oC or if there is reason to suspect that the plates have been contaminated since the initial
QC was performed.
Procedure for QC of primary culture media
1. Inspect the media for any visible microbial contamination,
discoloration, drying, deterioration, or other physical defects that may
interfere with use.
2. Inoculate the media with pure colonies from 18-24 hour growth of a
well-characterized reference strain. Streak for isolation and incubate for
18-24 hours at 35-37°C with ~5% CO2 (or in a candle-jar).
3. Incubate an uninoculated plate for 18-24 hours at 35-37°C with ~5% CO2
(or in a candlejar).
4. Examine the inoculated and uninoculated plates after 18-24 hours.
Reading QC testing results
· Passing result: proper growth of the reference strain on appropriate
media and no growth on uninoculated media.
· Failing result: no growth or poor growth of the reference strain on
appropriate media and growth of organisms on the uninoculated media.
III. Inoculation of primary culture media from CSF specimens
A. Primary culture directly from CSF
1. If the CSF can be transported to a microbiology laboratory immediately
(within 1 hour from the time of collection) for culture and analysis,
inoculate 1-5 drops of CSF (depending on volume received in laboratory)
directly onto both a BAP and CAP within 1 hour after collection.
· If the CSF was centrifuged, use 1 drop of the well-mixed sediment for
primary culture.
2. Using a sterile bacteriological loop, cross-streak the inoculum to
obtain single, isolated colonies.
· Disposable loops are preferred, but if using a wire loop, it must be
sterilized prior to each step of the plate-streaking process.
· BAP and CAP that have been properly streaked are shown in Figures 6, 7,
and 8.
3. A back-up broth (e.g., brain-heart infusion broth with proper
supplements) should be inoculated with some of the sediment pellet.
4. Agar plates and broth inoculated with the CSF sediment should be
incubated for 18-24 hours at 35-37°C with ~5% CO2 (or in a candle-jar).
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