6.1 CSF Sources and Acute Meningitis Pathogens

Why CSF Is a Separate Bacteriology Problem

Cerebrospinal fluid is normally sterile. Outline II.B of the M(ASCP) Examination Content Guideline (revised September 25, 2025) treats it as its own analytic stream because a missed meningococcus, a refrigerated aliquot, or a discarded "diphtheroid" can cost a patient hours of untreated meningitis. Bacteriology is 45–55% of M; CSF items sit inside that block and mix preanalytic technique with colony identification.

Three sources appear on the outline: lumbar puncture (LP), shunt, and reservoir. LP samples lumbar subarachnoid fluid, usually at L3–L4 or L4–L5. A typical numbered set sends chemistry first, microbiology second, and cell count third; some laboratories send the last tube to microbiology because it is the least likely to carry skin flora from the needle tract. Either practice is acceptable on the exam if the aliquot is dedicated, labeled, and processed STAT. Ventricular shunts (ventriculoperitoneal, ventriculoatrial, ventriculopleural) and external ventricular drains yield ventricular fluid through a tap of tubing or a dome. An Ommaya or similar reservoir is an implanted port used for chemotherapy or repeated sampling. Device fluid is still CSF, but the organism list shifts toward biofilm skin flora (Section 6.2). Do not process a shunt tap as community-acquired meningitis and then discard coagulase-negative staphylococci without clinical correlation.

Record volume and appearance on receipt: crystal-clear, cloudy or turbid, xanthochromic, or bloody. Cloudy fluid in a febrile patient is a STAT cytospin until proven otherwise. Traumatic taps show red cells that decrease in successive tubes; that finding does not cancel the bacterial workup.

STAT Processing and the Refrigerator Rule

Acute bacterial meningitis is a laboratory emergency. Inoculate media as soon as the tube arrives. Never refrigerate CSF intended for bacterial culture. Neisseria meningitidis is cold-sensitive and dies in the refrigerator. Streptococcus pneumoniae is also labile and autolyses, so a delay plus cold storage can produce a negative culture after a positive Gram stain—an exam classic. If a short delay is unavoidable, hold the bacterial aliquot at room temperature. Viral PCR aliquots may be refrigerated or frozen per the assay insert, and mycobacterial or fungal portions tolerate cold better than meningococcus does. The M rule is organism-specific: bacterial culture of CSF → do not refrigerate.

Minimum volume is laboratory-defined; 1–2 mL is a common floor for stain plus culture. Acid-fast and fungal cultures need more. When the tube is nearly empty, prioritize cytospin Gram stain and chocolate plus blood agar over optional plates.

Cytospin Gram Stain

An unconcentrated smear of clear CSF misses organisms that a cytocentrifuge (cytospin) will catch. Cytospin deposits cells and bacteria onto a small area, raising Gram-stain sensitivity. Read it immediately. A positive smear is a critical value: notify the clinician or designated caregiver, then document who was called and the time.

A negative cytospin does not exclude meningitis. Antibiotic pretreatment, low organism load, and Listeria (which stains poorly and can look like cocci or debris) all produce false-negative smears. Plates and, when indicated, multiplex PCR still go forward.

Primary Media and Capnophilic Incubation

The standard bacterial set is chocolate agar (CHOC), 5% sheep blood agar (BAP), and an enrichment broth (thioglycollate, TSB, or BHI). Chocolate supplies X (hemin) and V (NAD) so H. influenzae can grow. Blood agar shows hemolysis for pneumococcus, Listeria, GBS, and S. aureus. Broth rescues very small numbers. Add MacConkey agar when Gram-negative rods are seen or the patient is a neonate. N. meningitidis grows on both BAP and CHOC; N. gonorrhoeae does not grow on unsupplemented BAP. That distinction is heavily tested.

Incubate plates at 35–37 °C in 5% CO2. Pneumococcus, Haemophilus, and meningococcus are capnophilic. Hold at least 72 hours (many laboratories hold shunt fluid 4–5 days). Ambient-air-only incubation is how Haemophilus and meningococcus are missed.

Age-Based Epidemiology

Match the host to the organism before the plates are fully identified. Neonates (about the first month of life) are dominated by group B Streptococcus (Streptococcus agalactiae), K1-encapsulated Escherichia coli, other Enterobacteriaceae, and Listeria monocytogenes. Intrapartum GBS prophylaxis lowered early-onset disease but did not erase it, so GBS remains the leading exam answer for neonatal meningitis with sepsis or pneumonia. Unvaccinated infants and young children historically developed Haemophilus influenzae type b meningitis; Hib conjugate vaccine collapsed that disease, so type b on a current exam almost always travels with an unvaccinated or incompletely vaccinated child. Adolescents and young adults are the meningococcal group: dormitories, military barracks, complement (terminal MAC) deficiency, and complement-blocking drugs such as eculizumab. Adults are led by Streptococcus pneumoniae, especially with asplenia, cochlear implants, alcoholism, or a skull fracture with CSF leak. Elderly, pregnant, and cell-mediated immunocompromised patients add Listeria back onto a pneumococcal background. Soft cheeses and deli meats are the classic food exposures, not a required history for the isolate to count.

Exam scenario. A 19-year-old college student is found febrile and confused in a dormitory. CSF is cloudy. Cytospin shows intracellular kidney-bean Gram-negative diplococci. The organism that must survive transport is N. meningitidis: room temperature, rapid plating on BAP and CHOC, 5% CO2. Refrigerating that tube to "preserve it overnight" is the wrong action.

Exam scenario. A 3-week-old infant has fever and a bulging fontanelle. Gram-positive cocci in pairs and chains on cytospin, beta-hemolytic gray colonies, CAMP-positive and hippurate-positive: GBS. A second neonate with Gram-negative bacilli and lactose-fermenting colonies is E. coli until proven otherwise; K1 capsule is the virulence factor the outline wants. A third neonate with a "contaminating diphtheroid" that is catalase-positive and weakly beta-hemolytic is Listeria until you have motility and CAMP results.

Colony Morphology and Identification of Outline Pathogens

Streptococcus pneumoniae. Alpha-hemolytic, often mucoid when heavily encapsulated. Older colonies become umbilicate ("draughtsman" or checker) as the center autolyses. Catalase-negative. Optochin (P disk) susceptible and bile soluble (10% sodium deoxycholate dissolves the colony). Lancet-shaped diplococci match the cytospin. Viridans streptococci are optochin-resistant and bile-insoluble; do not report them as pneumococcus from CSF without those tests.

Haemophilus influenzae. Tiny gray translucent colonies on CHOC and no growth on BAP unless satelliting around a S. aureus streak that leaks V factor. Requires both X and V. Encapsulated type b was the meningitis serotype; nontypeable strains dominate otitis and COPD, so meningitis in an unvaccinated child still points to type b on M.

Neisseria meningitidis. Gray, glistening colonies on BAP and CHOC. Oxidase-positive Gram-negative diplococci. CTA carbohydrates: glucose and maltose oxidized (gonococcus oxidizes glucose only). Hold the plates in CO2 and never let the specimen sit in a refrigerator while you "batch Gram stains at 07:00."

Escherichia coli and other Enterobacteriaceae. Large gray colonies, often lactose-fermenting on MacConkey, oxidase-negative. Neonatal meningitis strains frequently carry the K1 capsule, which is antiphagocytic and structurally related to meningococcal group B capsule—the reason latex kits historically cross-reacted. Klebsiella, Enterobacter, and Serratia appear in neonates, immunocompromised adults, and post-neurosurgical patients.

Listeria monocytogenes. Small gray colonies with narrow beta-hemolysis that is easy to miss if you do not move the colony. Catalase-positive Gram-positive rods. Tumbling motility on a wet mount at 25 °C, umbrella motility in semisolid agar at 25 °C but not 37 °C, esculin hydrolysis, and a block or shovel CAMP reaction with S. aureus. Cold enrichment is a food-microbiology trick, not a reason to refrigerate the CSF tube. Never report "diphtheroids, probable contaminant" from neonatal or elderly CSF without ruling out Listeria.

Staphylococcus aureus. Creamy, usually beta-hemolytic, catalase-positive, coagulase-positive clusters. Community meningitis from S. aureus is uncommon; think neurosurgical hardware, trauma, or concomitant bacteremia. It still belongs on the II.B pathogen list.

Beta-hemolytic streptococci, especially GBS. Gray colonies with a wider, softer zone of beta-hemolysis than many group A isolates. CAMP-positive, hippurate-positive, PYR-negative. GBS is the neonatal chain-former the outline names; group A Streptococcus is more often a sterile-site or toxin-mediated isolate than a classic community CSF pathogen, but any beta-hemolytic streptococcus from CSF is significant.

Capsule is the shared virulence theme for pneumococcus, Hib, meningococcus, and K1 E. coli: antiphagocytic polysaccharide that also lets the organism survive in the bloodstream long enough to seed meninges. Identification methods in this section stay phenotypic because that is how overnight plates still confirm a STAT smear. Molecular panels that run in parallel are the next section.