Bacteriological examination of CSF

CSF should be collected in sterile containers. These are provided by the Bacteriology
department.
CSF samples are high priority samples. As soon as the CSF is obtained from the lumbar
puncture, it must be IMMEDIATELY transported to the laboratory (within one hour) and
cultured. Any delay in the cultivation of the sample (more than an hour) will lead to a drop in
sensitivity of the test and possible loss of bacteria.
The CSF sample should never be refrigerated.
Procedures
1. The appearance of the CSF is noted as follows: clear, violated, cloudy, purulent, yellow,
tinged with blood, or with a fibrous film.
2. After centrifugation at high speed for 5 to 10 minutes, the CSF centrifugation pellet/base
will be used for staining of gram and a culture on a preheated medium (centrifugation is
not recommended when less than 1 ml is available or if the CSF is purulent).
If bacteria was observed in the smear colored by gram, it is advisable to inoculate the culture
medium appropriate and incubate at 35-37°C, with the agar plates in an atmosphere enriched
with carbon dioxide.

The best medium of culture is chocolate agar containing sheep blood or horse. This medium
allows the growth of three microorganisms: Hib, S. pneumoniae and N. meningitidis.
These microorganisms are identified with the following tests:
1. Haemophilus influenzae: oxidase test and requirement factors X and V;
2. Streptococcus pneumoniae: optochin test and solubility through bile:
3. Neisseria meningitidis: oxidase test.

REMARKS
Since more than 95% of Haemophilus infuenzae isolated from LCR are Haemophilus influenza
type b, we can assume that H. influenzae are actually H. influenzae type b. The Presumed
Neisseria will be assumed to be Neisseria meningitidis.
SPECIES IDENTIFICATION SCHEME
Haemophilus (influenzae), Neisseria and Streptococcus
Chocolate Agar growth

GRAM STAIN (-) GRAM STAIN (+)

OXYDASE OXYDASE
TEST (-) TEST (+)
OPTOCHIN
TEST + BILE
(+)

/
FACTOR
X & V IN
AGAR

PRESUME PRESUME
HAEMOPHILUS NEISSERIA
influenza type B
PRESUME
STREPTOCOCCUS
pneumoniae

``
OPTOCHIN SENSITIVITY
This test makes it possible to differentiate pneumococci from others streptococci.
A plate of blood agar is inoculated as for carrying out an antibiogram, with the germ to be
studied. We places a disk impregnated with 5 ug of optochin (hydrochloride of
ethylhydrocupreine).
After incubation at 37°C for 18 hours, we look for the existence or absence of a zone of
inhibition around the disc. For pneumococci, we observe an inhibition zone greater than 15 mm.
Other streptococci are not inhibited.
BACITRACIN SENSITIVITY
Group A B-hemolytic streptococci (S. pyogenes) are sensitive to a low concentration of
bacitracin. On blood agar, culture the sample or strain to be tested, place a disk of commercial
available bacitracin on top. After incubation of 24 hours, sensitivity to bacitracin manifests by a
growth inhibition zone around the disc. If the presence of yeast fungus is suspected
Cryptococcus neoformans, mix the contents of a loop of Indian ink on a slide, cover with a
coverslip and examine with microscope looking for typical, encapsulated shapes, spherical, of
budding yeasts.
If it is an enterobacterium, proceed by identification on the small gallery (Kligler, Citrate, SIM or
MIU) and possibly API 20.