1. Cerebrospinal fluid (csf) culture
D. M. M. Lab.

2. CSF culture Aim of the test Types of specimen CSF
Diagnosis of the bacteria or fungal meningitis by microscopic examination and culture with identification and susceptibility test of the isolated organism.
Types of specimen
CSF
Criteria of specimen rejection
Non sterile container and the general causes for rejection stated in the introduction as mislabeling, un-labeling, insufficient amount.
Specimen more than 30 minutes.

3. Common bacterial pathogen Microbes that cause chronic meningitis
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
Viruses
Enteroviruses(coxsackieviruses A and B, echoviruses)
Herpes simplex virus
Mumps virus
Arboviruses (togavirus,bugavirus and other)
Parasite
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.

4. Pre specimen processing
Specimen collection
Who will collect the specimen
Only physicians.
Quantity of specimen
Mini ml of CSF is recommended for culture.
Time relapse before processing the sample
CSF is an emergency specimen and should be processed immediately.
Storage
Room Temperature, Do not refrigerate.

5. Specimen processing

6. 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.

7. 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.

8. 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.

9. 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.

10. 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.

11. Post specimen processing
Interfering factors
Patient on antibiotic therapy.
Improper sample collection.
Result reporting
Results of the microscopy and all positive cultures of CSF are reported immediately to the treating physician.
Turn around time
Gram stain result is reported within 30 minutes of specimen receipt
Positive Culture results = 3- 5 days
Negative Culture results = 2-3 days

12. 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.

13. H. Influenzae On Chocolate agar

14. X and V Factor test Method Expected results:
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.
Method
Make avery light suspension ( 0.5 MacFarland )of the organism in sterile saline.
Dip a sterile swab in to the organism suspension. Roll the swab over the entire surfase of MH agar palte
Place X, V, and XV factors disks on the agar surface .
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. aprophilus)

15. H. parainfluenzae only V factor

16. H. influenzae both V and X factors

17. 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.