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 D. M. M. Lab.
    CSF culture
   Diagnosis of the bacteria or fungal meningitis by microscopic
    examination and culture with identification and susceptibility test
    of the isolated organism.


      CSF

   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
             Entamoeba histolytica
                             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
   Only physicians.

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.
Specimen processing
    Initial Processing

   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.
Blind Culturing

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
    Additional information
   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.
Expected results:
  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
Satellitism phenomenon

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.

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