Primary Culture and Presumptive Identification ofNeisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenza
Microbiology laboratories commonly receive cerebrospinal fluid (CSF) or
blood specimens from patients with meningitis, pneumonia, or unexplained febrile illness.
Laboratories may also receive joint fluid, pleural fluid, or other sterile
site specimens from these patients. Presumptive identification of N. meningitidis, S. pneumoniae, and H. influenzae can be made on the basis of a cytological examination of the CSF, specific colony morphology on blood
and/or chocolate agar, staining properties on a Gram stain, or by detection of specific antigens
in the CSF by a latex agglutination test or using a rapid diagnostic test
(RDT). Methods for confirmatory identification of N.meningitidis, S. pneumoniae, and H. influenzae are
presented in the next chapters of this laboratory manual article.
Personnel who are at risk of routine exposure to aerosolized N. meningitidis should strongly consider vaccination. Additional
health and safety information can be found in the next article Biosafety.
While laboratory-acquired infections with S. pneumoniae or H. influenzae are not as extensively reported, fatal infections with these bacteria can occur, and
vaccination against these organisms may be recommended in some laboratories.
Because the primary purpose of this manual is to aid in the identification
of N. meningitidis, S. pneumoniae
, and H. influenzae from clinical specimens collected from
suspected cases of bacterial meningitis, the methods described here will not allow for
identification of other isolates that may be of clinical importance but are less likely encountered.
Microbiologists should refer to clinical microbiology manuals, such as the American Society for
Microbiology’s Manual of Clinical Microbiology
, for procedures to identify other bacteria.
I. Processing CSF Specimens
A note about centrifugation: g (1 x gravity) represents relative
centrifugal force (RCF), but the recommended centrifugation speed is often listed in protocols as
revolutions per minute (RPM).
RCF is dependent on the length of the radius of the rotor, thus the same
RPM may not generate the same g force in another centrifuge. Therefore, RCF should be used to
describe the centrifuge speed. If only RPM is given, RCF can be calculated using this formula:
RCF = 0.00001118 x r x RPM2
r = radius of rotor in centimeters
Once the CSF arrives in the microbiology laboratory, the volume of CSF
available for analysis should be noted. If < 1 ml of CSF is available, it should not be
centrifuged; instead, the CSF should be plated directly onto a blood agar plate (BAP) and onto a
chocolate agar plate (CAP) and also used for the Gram stain. If > 1 ml of CSF is available (i.e.,
if the specimen volume is sufficient for centrifugation), it must be centrifuged at a force
sufficient to sediment the bacteria.
Typically, centrifugation at 1000 x g for 10-15 minutes is
sufficient to sediment bacteria. After the specimen has been centrifuged, the supernatant should be drawn
off with a Pasteur pipette and reserved if antigen detection by latex agglutination is
planned. The sediment should be vigorously mixed (e.g., in a closed tube using a vortex machine). Once
it has been wellmixed, one or two drops of sediment should be used to prepare the Gram stain and
one drop should be used to streak the primary culture media.
A. Cytological Examination of the CSF
Laboratory examination of the CSF is usually the first step to confirm the
presence of bacterial meningitis. Note that cytological examination should precede centrifugation
and heating of the CSF. Typical CSF abnormalities associated with bacterial meningitis include
the following:
-Turbidity
-Increased opening pressure (>180 mm water)
-Pleocytosis (usually of polymorphonuclear (PMN) leukocytes); WBC counts
>10 cells/mm3
-Decreased glucose concentration (<45 mg/dl)
-Increased protein concentration (>45 mg/dl)
Note: normal cytology of the CSF of an infant is 10-30 WBC/mm3 (50% PMNs).
B. Presumptive identification by Gram stain, latex agglutination, or
rapid diagnostic test (RDT)
In combination with a clinical picture and CSF examination consistent with
bacterial meningitis, a presumptive diagnosis of bacterial meningitis caused by N. meningitidis, S. pneumoniae, or H. influenzae
can be made after performing a Gram stain of the CSF sediment or by
detection of specific antigens in the CSF by a latex agglutination test or using RDTs.
Positive results for any of these tests can rapidly provide evidence of infection, even if cultures
fail to grow.
1. Performing a Gram stain
The Gram stain is an empirical method for differentiating bacterial species
into two large groups based on the chemical and physical properties of their cell walls.
Gram-positive bacteria retain the primary stain while gram-negative bacteria take the color of the
counterstain. A Gram stain can also serve to assess the quality of a clinical specimen. The CSF should
be properly centrifuged in order to obtain the sediment for the procedure.
Proper smear preparation using the CSF sediment should produce a monolayer of organisms sufficiently dense for
easy visualization but thin enough to reveal morphological characteristics. Clean, new glass
slides should be used.
Positive and negative quality control (QC) strains should be tested along
with the unknown specimens. In addition to known reference strains for N. meningitidis, S. pneumoniae, and H. influenzae
, other reference strains that may be used includeStaphylococcus aureus for grampositive cocci and Escherichia coli for gram-negative rods.
Gram stain procedure for CSF
1. Centrifuge the CSF for 10-15 minutes at 1000 x g, if > 1 ml
is available (see above).
2. Divide a glass slide into two sections using a marker. Use one section
for the unknown CSF and the other section for a known organism for QC.
3. Prepare a smear by placing 1-2 drops of the well-mixed CSF sediment on
the slide, allowing the drop(s) to form one large slightly turbid, uniform
suspension.
-To prepare a smear using an isolate, add a small drop of sterile water or
physiological saline to the slide and create a slightly turbid, uniform
suspension of cells from an overnight culture.
4. Let the suspension air dry. The suspension MUST be completely dry before
proceeding.
5. Fix the smear by the flooding the slide with 95% methanol for a minimum
of 2 minutes. Rinse with distilled water. Shake off excess water.
-If methanol is not available, heat-fix the smears by quickly passing the
slide through a flame three times. Do not over-heat the slide as
over-heating will cause significant distortion or destruction of the cells.
-It is possible to use simple water (filet d’eau de robinet) if distilled
water is not available for the entire Gram stain procedure.
6. Flood the slide with crystal violet ammonium oxalate for 1 minute to
stain. Rinse with distilled water. Shake off excess water.
-Avoid touching the slide with the tip of the reagent bottle or applying
liquid directly
onto the smear.
7. Flood the slide with Gram’s iodine for 1 minute. The iodine acts as a
mordant as it binds the alkaline crystal violet dye to the cell wall. Rinse
with distilled water. Shake off excess water.
8. Decolorize with 95% ethanol until no more stain washes off (5-10 seconds
may be enough). Rinse with distilled water. Shake off excess water.
-It is essential to view decolorization closely: gram-positive bacteria can
be made to appear gram-negative by over-decolorization and gram-negative
bacteria can be made to appear gram-positive by under decolorization.
9. Counterstain with safranin for 30 seconds or with carbol-fuchsin for
10-15 seconds.
Rinse with distilled water. Shake off excess water.
10. Gently blot the slide using bibulous paper or a clean paper towel. Let
air dry.
11. When dry, examine the stained smear under a microscope with 100X oil
immersion objective.
Reading the Gram stain results (under microscopic examination):
-Gram-positive organisms will appear dark violet or purple.
-Gram-negative organisms will appear red or pink (from the counterstain).
N. meningitidis
may occur intracellularly or extracellularly in PMN leukocytes and will
appear as gram-negative, coffee-bean shaped diplococci.
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