CEREBROSPINAL FLUID (CSF)
D. M. M. Lab.
Diagnosis of the bacteria or fungal meningitis by microscopic
examination and culture with identification and susceptibility test
of the isolated organism.
Non sterile container and the general causes for rejection stated
in the introduction as mislabeling, un-labeling, insufficient
Specimen more than 30 minutes.
Infection of CSF
Common bacterial pathogen
Haemophilus influenzae Salmonella (rare)
Neisseria meningitidis Brucella (rare)
Streptococcus pneumoniae Treponema pallidum (rare)
Group A & B streptococci Listeria monocytogenes
Gram negative bacilli
Enteroviruses(coxsackieviruses Herpes simplex virus
A and B, echoviruses)
Mumps virus Arboviruses (togavirus,bugavirus and
Free living amoebae Toxoplasma (rare)
Toxoplasma gondii Strongyloides strecoralis
Microbes that cause chronic meningitis
M. tuberculosis Blastomyces dermatitides
Cryptococcus neoformans Candida spp.
Coccidoides immitis Nocardia
Histoplasma capsulatum Actinomyces
Note: CSF is a sterile body fluid and does not contain any commensals; however, care should be
taken not to contaminate the specimen with skin normal flora during collection.
Pre specimen processing
Who will collect the specimen
Quantity of specimen
Mini. 5-10 ml of CSF is recommended for culture.
Time relapse before processing the sample
CSF is an emergency specimen and should be processed
Room Temperature, Do not refrigerate.
Initial processing of CSF for bacterial and fungal detection includes
centrifugation of all specimens greater than 1 mL in volume for at
least 15 minutes at 1500x g.
The supernatant is removed to a sterile tube, leaving approximately
0.5 mL of fluid in which to suspend the sediment before visual
examination or culture.
The supernatant can be used for to test for the presence of antigens
or for chemistry evaluations.
Note: if the specimen turbid it is not necessary to make the initial
processing as mentioned above.
Staining of CSF
After thoroughly mixing the sediment heaped drop is placed on the
surface on a sterile slide. The sediment should never spread out on the
slide surface, because this increases the difficulty of finding small
numbers of microorganisms, the drop of sediment is allowed to air dry,
and heated or methanol fixed and stained by gram stain, and another
slide stained by Methylene blue in parallel.
India ink stain
India ink stain: the large polysaccharide capsule of cryptococcus
neoformans allows these organisms to be visualized After mixing the CSF
and ink to make a smooth suspension, a coverslip is applied to the drop
and the preparation is examined under high power magnification for
characteristics encapsulated yeast cells and by oil immersion.
Wet mount preparation
Wet mount: amoebas are best observed by examining thoroughly mixed
sediment as a wet preparation under phase contrast microscopy or light
microscopy with the condenser closed slightly.
Amoebas are recognized by typical slow, methodical movement in one
direction by advancing pseudopodia.
After vortexing the sediment and preparing smears Several drops of
the sediment shoud be inoculated to each medium. Plates should be
incubated at 37oC for at least 72 hours. The broth should be
incubated in air for at least 5 days.
These media will support the growth of almost all bacterial pathogens
and several fungi.
Post specimen processing
Patient on antibiotic therapy.
Improper sample collection.
Results of the microscopy and all positive cultures of CSF are reported
immediately to the treating physician.
Gram stain result is reported within 30 minutes of specimen receipt
Positive Culture results = 3- 5 days
Negative Culture results = 2-3 days
Total and differential white blood cell count is essential particularly in
the differentiation of bacterial and non-bacterial meningitis.
In bacterial meningitis the glucose level is usually low and the protein
level is high, where in viral meningitis the glucose is within normal
value and might increase slightly.
Several antigen detection methods are available for the direct
detection of the polysaccharide capsular antigen of H. influenzae, N.
meningitidis, S.pneumoniae and Group B streptococci in CSF which
showed specificity and sensitivity of about 90-97%.
Direct detection of Cryptococcus antigen in CSF is also available
which replaced India ink in many laboratories.
The routine culture for CSF does not include all organisms mentioned
in the above table.
H. Influenzae On Chocolate agar
X and V Factor test
Members of the genus haemophilus require accessory growth factor in vitro
Some haemophilus spp. X factor (hemin) alone, V factor (NAD) alone or
acombination of both.
I. Make avery light suspension ( 0.5 MacFarland )of the organism in sterile saline.
II. Dip a sterile swab in to the organism suspension. Roll the swab over the entire
surfase of MH agar palte
III. Place X, V, and XV factors disks on the agar surface .
IV. Incubate overnight.
growth around the XV disks only shows required for both factors.( H. influenzae )
growth around the XV, X and no growth around the V disk shows required only
for X factor. ( H. ducreyi )
growth around the XV, V and no growth around the X disk shows required only
for V factor. ( H. parainfluenzae )
Growth on all entire plate indicate no need for any of these factors.(H.
H. parainfluenzae only V factor
H. influenzae both V and X factors
Sheep blood agar contains hemin but not NAD, Several bacterial species including
Staphylococcus aureus, produce NAD as a metabolic byproduct,therfore tiny colonies
of Haemophilus spp. May be seen growing on blood agar very close to colonies
that can produce V factor.