CSF Sample: Collection, Processing, Staining, and Culture

Skull meningitis

Meningitis is an inflammation of the membranes covering the brain and spinal cord known as the meninges. The most common etiologic agents of acute meningitis are enteroviruses (primarily echoviruses and coxsackieviruses) and bacteria (Streptococcus pneumoniae Neisseria meningitidis, Haemophilus influenzae), etc. Organisms expected to cause chronic meningitis (symptoms ≥4 weeks) include Mycobacterium tuberculosis, fungi, and spirochetes.

Viral infections usually get better without treatment but bacterial infection of the meninges is very serious and is a major cause of deaths and disability worldwide. Patients’ age and other factors (i.e, immune status, post neurosurgery, trauma) are associated with specific bacterial pathogens.

Common Causes of Meningitis

Common causes of bacterial meningitis vary by age group:

Age GroupCauses
NewbornsGroup B Streptococcus (Streptococcus agalactiae), Escherichia coli, Listeria monocytogenes
Infants and ChildrenStreptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae type b (Hib), Group B Streptococcus
Adolescents and Young AdultsNeisseria meningitidis, Streptococcus pneumoniae
Older AdultsStreptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae type b (Hib), Group B Streptococcus, Listeria monocytogenes
Collection of cerebrospinal fluid (CSF) by lumbar puncture
Collection of cerebrospinal fluid (CSF) by lumbar puncture

CSF Collection

Whenever possible, collect CSF (1 mL minimum, 3-4 mL if possible) prior to initiating antimicrobial therapy. CSF is generally obtained by lumbar spinal puncture (at the L4-L5 level) and is collected usually in three separate tubes:

  1. Tube 1: For cell count and differential stains
  2. Tube 2: For Gram’s stain and culture (A minimum of 0.5–1 mL of CSF should be sent to the microbiology laboratory in a sterile container for bacterial testing. )
  3. Tube 3: For protein and glucose or for special studies such as VDRL, cryptococcal antigen, or cytology depending on the clinical situation.
  • When the specimen volume is less than required for multiple test requests, prioritization of testing must be provided to the laboratory.
  • Two to four blood cultures should also be obtained if bacterial meningitis is suspected.
  • Inform the microbiology laboratory if unusual organisms are possible (such as Nocardia, Fungi, Mycobacteria, etc.) for which special procedures are necessary.
  • Do not refrigerate cerebrospinal fluid.
  • Attempt to collect as much sample as possible (approximately 5–10 ml of CSF should be collected) for multiple studies (minimum recommended is 1 ml); prioritize multiple test requests on small volume samples.

Note:

When CSF is collected into five calibrated sterile tubes,

  1. The first tube is sent to biochemistry (for total protein, glucose, immunoglobulin index, and/or oligoclonal banding),
  2. 2nd to microbiology for bacterial culture and sensitivity, viral culture, fungal culture, or detection of cryptococcal antigen.
  3. The third tube is also sent to microbiology for AFB culture and special stain.
  4. The fourth tube is sent to hematology for cell count, flow cytometry, and cytology.
  5. The fifth tube is used for the detection of special pathogens such as Acanthamoeba, West Nile virus, viral PCR, anaerobic culture (including gram stain), and Creutzfeldt-Jakob disease (14-3-3 protein).

If only one tube of CSF is available, it should be sent to the microbiology laboratory. If CSF sample is collected in multiple tubes, the first tube collected (which could contain contaminating blood from the lumbar puncture) should not be the tube sent to the microbiology laboratory.

CSF volume & Production in Human

Total CSF in adult 85-125 mL
Total CSF in neonate 10-60 mL
Normal rate of CSF production in Adult 20 mL/Hour

Processing of CSF Sample

CSF should never be refrigerated prior to culturing because fastidious organisms may not survive lowered temperatures. If bacterial culture cannot be set up immediately, store the CSF at 35°C to maintain body temperature or leave it at room temperature.

CSF from a patient suspected of meningitis is an emergency specimen that requires immediate processing to determine the etiologic agent. Common etiological agents of bacterial agents; N. meningitidis, S. pneumoniae, and H. influenzae are fastidious and fragile bacteria. For successful isolation of these bacteria, CSF  must be cultured within one hour of collection or inoculated into Trans-Isolate (T-I) medium for transport to the laboratory if processing within one hour is not feasible.

A delay in examining CSF:

  • Reduces the chances of isolating pathogens
  • Lowers cell count due to WBCs being lysed
  • Falsely shows low glucose value due to glycolysis

Laboratory Tests

Biochemical Testing

In the case of acute bacterial meningitis, CSF analysis shows

  • Presence of a large number of inflammatory cells (>1000/mm3) primarily polymorphonuclear neutrophils,
  • Decreased glucose level relative to the serum glucose level (the normal ratio of CSF to serum glucose is approximately 0.6) and
  • An increased protein concentration.

Interpretation of Biochemical Analysis of CSF Fluid

Leukocytes/mm3Predominant Cell TypeProteinGlucoseClinical Setting
0-5None15-50 mg/dL45-100 mg/dLNormal
2-2000 (mean of 80)MononuclearSlightly elevated (50-100 mg/dL) or normalNormalViral meningitis
5-20,000 (mean of 800)PMNElevated (>100 mg/dL)Low (<45 mg/dL), but may be normal early in the course of the diseaseAcute Bacterial Meningitis
5-2000 (mean of 100)MononuclearElevated (>50mg/dL)Normal or often low (>45 mg/dL)Meningitis caused by tuberculosis or fungi

Microscopy and Staining

The number of organisms in the CSF can be as low as 103 CFU/ml so CSF Gram stains should be prepared after cytocentrifugation. Report positive Gram stain results immediately to clinicians, usually within one hour of receipt.

Streptococcus pnuemoniae: Gram positive diplococci
Streptococcus pnuemoniae: Gram-positive diplococci (see arrow)
  1. N. meningitidis may occur intracellularly or extracellularly in PMN leukocytes and will appear as gram-negative, coffee-bean-shaped diplococci.
  2. S. pneumoniae may occur intracellularly or extracellularly and will appear as gram-positive, lanceolate diplococci, sometimes occurring in short chains.
  3. H. influenzae are small, pleomorphic gram-negative rods or coccobacilli with random arrangements.

If cryptococcal meningitis is suspected, India ink preparation should be done.

Culture and Sensitivity

As CSF is a sterile body fluid collected by transcutaneous aspiration, all organisms recovered from the culture are potential pathogens. Concentration for culture is unnecessary since the plate inoculum is sufficient to detect the usually low numbers of organisms.

Bacterial meningitis is mostly caused by aerobic bacteria though anaerobes may be present in CSF in a meningeal abscess or a similar infectious process adjacent to the meninges. Following media are routinely used in the diagnostic laboratory for the isolation of common bacterial agents of meningitis;

Chocolate Agar

On chocolate agar plate, H. influenzae appear as large colorless to grey, opaque colonies with no discoloration of the surrounding medium.

Blood Agar

N. meningitidis on blood agar plate
N. meningitidis on blood agar plate
  1. Overnight growth of N. meningitidis on blood agar plate appears as round, moist,
    glistening and convex colonies.
  2. S. pneumoniae appear as small greyish mucoid (watery) colonies with a greenish zone of
    alpha-hemolysis surrounding them on the blood agar plate.

MacConkey Agar

Summary of identification scheme (Gram staining and culture)

Growth on Chocolate Agar plateGrowth on Blood Agar plate Gram StainingPresumptive ID
++Gram-negative diplococciN.meningitidis 
++ (α-hemolysis)Gram-positive diplococciS. pneumoniae
+ (perform a test for X and V factor requirements)– (negative)Gram-negative pleomorphic coccobacilliH. influenzae

Antigen-Antibody Reactions 

  1. Antigen Testing: Cryptococcal antigen latex agglutination test is the preferred method in the suspected cases of cryptococcal meningitis. Bacterial antigen testing on CSF is not recommended.
  2. Serology: Serologic diagnosis is based on CSF to serum antibody index, 4- fold rise in acute to convalescent immunoglobulin G (IgG) titer, or a single positive immunoglobulin M (IgM). Submission of acute (3–10 days after onset of symptoms) and convalescent (2–3 weeks after acute) serum samples is recommended.

Molecular Diagnosis

Nucleic acid amplification tests (NAAT) are now available for most pathogens in developed countries but such facilities may not be available in developing countries or resource-poor settings. Molecular testing has replaced viral culture for the diagnosis of enteroviral meningitis.

Reference and further reading

  1. CDC meningitis home page
  2. Laboratory Methods for the Diagnosis of Meningitis, WHO Manual, 2nd Edition

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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