CSF Sample: Collection, Processing, Staining, and Culture

CSF from a suspected meningitis patient is a true emergency specimen. Two facts govern everything that follows. The organisms that cause acute bacterial meningitis (Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae) are fastidious and fragile, so the sample must never be refrigerated and must be processed within about an hour. And because the first drops of CSF can carry skin and blood from the puncture, the order in which the tubes are filled decides which test can trust its result. Get these two right and the rest is routine.

Meningitis is inflammation of the membranes covering the brain and spinal cord, the meninges. The most common causes of acute meningitis are enteroviruses (mainly echoviruses and coxsackieviruses) and bacteria (Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae). Organisms that cause chronic meningitis (symptoms lasting 4 weeks or more) include Mycobacterium tuberculosis, fungi, and spirochetes.

Viral meningitis usually resolves without treatment, but bacterial meningitis is a medical emergency and a major cause of death and disability worldwide. The likely pathogen depends on the patient's age and on other factors such as immune status, recent neurosurgery, and trauma.

Common causes of meningitis by age

CSF Collection

CSF is obtained aseptically by lumbar puncture, usually at the L3 to L4 or L4 to L5 interspace, from the subarachnoid space. Collect a minimum of 1 mL, ideally 3 to 4 mL, and up to 5 to 10 mL when multiple studies are requested (AFB and fungal cultures in particular need larger volumes). Collect before starting antimicrobial therapy whenever possible.

Figure: Collection of cerebrospinal fluid (CSF) by lumbar puncture

CSF is collected into several sterile tubes, filled in order. The guiding principle is that the first tube may contain skin fragments and blood introduced by the needle, so the first tube is never the one sent for culture. A common allocation is:

• Tube 1: chemistry (total protein, glucose). Any puncture blood here does not affect these results.
• Tube 2: microbiology (Gram stain and culture). Send at least 0.5 to 1 mL. Because it is not the first tube, it is the least contaminated by the puncture.
• Tube 3: hematology (cell count and differential). Comparing the red cell count in the first and last tubes helps tell a traumatic tap from a true bleed.
• Tube 4 (if collected): additional microbiology or special studies (AFB culture, fungal culture, cryptococcal antigen, viral PCR, VDRL, cytology), depending on the clinical question.

If only one tube can be obtained, it goes to microbiology, because a culture cannot be repeated without another lumbar puncture, whereas chemistry and cell count can often be run on very small volumes.

A few more points at collection:

• If the volume is smaller than the requests need, tell the laboratory so testing can be prioritized. The default priority on a tiny sample is microbiology.
• Collect two to four blood cultures as well if bacterial meningitis is suspected.
• Warn the microbiology laboratory if unusual organisms are possible (such as Nocardia, fungi, or mycobacteria), so the special methods they need can be set up.
• Do not refrigerate CSF.

Collection and transport at a glance

The one rule that outranks the rest: do not refrigerate CSF. Every other specimen in this cluster has a "refrigerate if delayed" option. CSF is the exception, because Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae die at low temperature. Keep it warm, and if it cannot be processed within an hour, use Trans-Isolate transport medium.

When CSF is collected into five tubes (advanced variant): Tube 1 to biochemistry (total protein, glucose, immunoglobulin index, oligoclonal banding); Tube 2 to microbiology (bacterial, viral, and fungal culture, cryptococcal antigen); Tube 3 to microbiology (AFB culture and special stain); Tube 4 to hematology (cell count, flow cytometry, cytology); Tube 5 for special pathogens (Acanthamoeba, West Nile virus, viral PCR, anaerobic culture, and the 14-3-3 protein for Creutzfeldt-Jakob disease). The principle is unchanged: the first tube, which may carry puncture blood, is not the one sent for culture.

CSF volume and production

Processing of CSF Sample

CSF must never be refrigerated before culture, because the fastidious organisms may not survive lowered temperatures. If culture cannot be set up at once, hold the CSF at 35°C to keep it near body temperature, or leave it at room temperature.

CSF from a suspected meningitis patient is an emergency specimen that needs immediate processing. The common bacterial causes, Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae, are fastidious and fragile. To recover them, culture the CSF within one hour of collection, or inoculate it into Trans-Isolate (T-I) medium for transport if processing within an hour is not possible.

A delay in examining CSF:

• Reduces the chance of isolating the pathogen
• Lowers the cell count as white cells lyse
• Falsely lowers the glucose through ongoing glycolysis

Laboratory Tests

Biochemical testing

CSF findings by type of meningitis

The key discriminator: bacterial meningitis gives many neutrophils, high protein, and low glucose; viral gives mononuclear cells with normal glucose; TB and fungal give mononuclear cells with low glucose. The CSF-to-serum glucose ratio (normally about 0.6) falls in bacterial, TB, and fungal meningitis, because the organisms and inflammatory cells consume glucose.

Microscopy and staining

The number of organisms in CSF can be as low as 10³ CFU/mL, so CSF Gram stains should be prepared after cytocentrifugation, which concentrates cells and bacteria onto the slide. Report a positive Gram stain to the clinician immediately, usually within one hour of receipt.

Figure: Neisseria meningitidis appearing as Gram negative diplococci in CSF Gram stain

• Neisseria meningitidis appears as gram-negative, coffee-bean-shaped diplococci, inside or outside the PMN leukocytes.
• Streptococcus pneumoniae appears as gram-positive, lanceolate diplococci, sometimes in short chains, inside or outside cells.
• Haemophilus influenzae appears as small, pleomorphic gram-negative rods or coccobacilli with a random arrangement.

Figure: Streptococcus pneumoniae appearing as Gram positive diplococci in CSF Gram stain

If cryptococcal meningitis is suspected, the cryptococcal antigen test (lateral flow assay or latex agglutination) is the preferred and more sensitive method. India ink preparation can show the encapsulated yeast but misses many cases, so a negative India ink does not exclude cryptococcal meningitis.

Culture and sensitivity

CSF is collected aseptically by lumbar puncture from a normally sterile site, so any organism recovered is a potential pathogen. Concentration before culture is unnecessary, because the plate inoculum is enough to detect the usually low numbers of organisms.

Bacterial meningitis is mostly caused by aerobes, though anaerobes may appear in CSF when there is a meningeal abscess or an adjacent focus. The media routinely used are:

Chocolate agar: Haemophilus influenzae forms large, colorless to grey, opaque colonies with no discoloration of the surrounding medium.

Blood agar: Neisseria meningitidis forms round, moist, glistening, convex colonies overnight. Streptococcus pneumoniae forms small greyish, mucoid colonies with a greenish zone of alpha-hemolysis.

Figure: N. meningitidis on blood agar plate

MacConkey agar: Most meningitis pathogens do not grow on MacConkey. It is used to detect or identify gram-negative bacilli, which matter in neonatal and post-neurosurgical meningitis (for example Escherichia coli or Klebsiella).

Antigen and antibody tests

Antigen testing: The cryptococcal antigen test (latex agglutination or lateral flow) is preferred when cryptococcal meningitis is suspected. Bacterial antigen testing on CSF is not recommended, because it adds little to a good Gram stain and culture.

Serology: Serologic diagnosis rests on a CSF-to-serum antibody index, a 4-fold rise in IgG between acute and convalescent samples, or a single positive IgM. Collect acute serum 3 to 10 days after onset and convalescent serum 2 to 3 weeks later.

Molecular diagnosis

Nucleic acid amplification tests (NAAT) are available for most pathogens in well-resourced settings but may not be available in resource-poor ones. Molecular testing has replaced viral culture for diagnosing enteroviral meningitis.

How to Remember

CSF breaks the cluster's refrigeration rule. Every other specimen says "refrigerate if delayed." CSF says never. The meningitis trio (Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae) is fragile and dies cold. Keep CSF warm, and use Trans-Isolate if it must travel.

Microbiology never gets the first tube. The first drops carry skin and puncture blood. Chemistry can tolerate that, culture cannot. First tube to chemistry, second to microbiology. If you get only one tube, culture wins, because you cannot repeat a lumbar puncture on demand.

Bacterial meningitis: high cells, high protein, low sugar. Neutrophils flood in (high cells), the inflamed barrier leaks protein through (high protein), and bacteria plus cells eat the glucose (low sugar). Viral keeps the sugar normal. TB and fungal look like viral on cells but drop the sugar like bacterial.

Low glucose means something is eating it. Bacteria, TB, and fungi consume glucose; viruses mostly do not. A low CSF sugar points away from a simple viral cause.

The three morphologies: Neisseria meningitidis is a gram-negative coffee-bean diplococcus, Streptococcus pneumoniae a gram-positive lance-shaped diplococcus, Haemophilus influenzae a tiny gram-negative coccobacillus. Shape plus Gram color names the organism on the smear.

Key exam facts in one table

Where Students Get Confused

"Why can't CSF be refrigerated when almost everything else can?" The common bacterial causes of meningitis are fastidious and cold-sensitive. Refrigeration kills Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae, lowering the chance of growing them. Hold CSF at body or room temperature and process it fast.

"Which tube should go to the microbiology lab?" Not the first one. The first drops can carry skin and blood from the needle, which contaminates a culture. Send the second tube to microbiology. Only when a single tube is available does the first tube go to micro, and then culture is the priority.

"If only a tiny volume is obtained, which test comes first?" Microbiology. A culture cannot be repeated without another lumbar puncture, and it identifies the organism and guides treatment. Cell count and chemistry can often run on very small volumes.

"Bacterial and TB meningitis both have low glucose, so how do I tell them apart?" By the cells. Bacterial meningitis is dominated by neutrophils; TB and fungal meningitis are mononuclear. Both drop the glucose, but the cell type separates them.

"Is India ink enough to rule out cryptococcal meningitis?" No. India ink misses many cases. The cryptococcal antigen test is more sensitive and is the preferred method, so a negative India ink does not exclude the diagnosis.

"Why prepare the Gram stain by cytocentrifugation?" Because CSF can contain very few organisms (as low as 10³ CFU/mL). Cytocentrifugation concentrates the cells and bacteria onto the slide, which greatly raises the chance of seeing them.

Reference and further reading

1. World Health Organization. Laboratory Methods for the Diagnosis of Meningitis. 2nd ed. Geneva: WHO.
2. Tille PM. Bailey & Scott's Diagnostic Microbiology. 15th ed. St. Louis: Elsevier; 2022.
3. Leber AL, editor. Clinical Microbiology Procedures Handbook. 4th ed. Washington, DC: ASM Press; 2016. DOI: 10.1128/9781683670438.CMPH
4. Centers for Disease Control and Prevention. Meningitis. https://www.cdc.gov/meningitis/