Cerebrospinal Fluid and Sterile Body Fluid Cultures

Cerebrospinal fluid (CSF) reaches the microbiology laboratory when meningitis is suspected, and infectious meningitis is a medical emergency. The specimen is therefore processed immediately on arrival, the direct Gram stain is read without delay, and a positive result goes to the treating clinician at once through the laboratory’s urgent-notification procedure.¹,² Fluid aspirated from the pericardial, pleural, or peritoneal cavity, from a joint space, or from inside the eye is handled with the same urgency but raises a different interpretive question. These spaces are normally sterile, so any organism recovered is either a true pathogen or a contaminant introduced during collection.² Counting chambers, cell-count calculations, and slide preparation for cell identification belong to body-fluid analysis; the counting technique is covered in Manual Cell-Counting Calculations, and examination reference values for CSF appear in ASCP BOC Examination Reference Ranges. General transport rules, rejection criteria, Gram-stain mechanics, media, incubation, and the staged reporting cycle are covered in Specimen Collection, Transport, and Primary Processing in Microbiology.

Cerebrospinal fluid collection and routing

CSF is obtained by lumbar puncture, occasionally by ventricular aspiration, or from a shunt or reservoir, with skin antisepsis performed as carefully as for a blood culture.¹ The specimen usually arrives in three or four sequential tubes. The first tube collected is the most likely to contain skin flora introduced during needle passage, so the Gram stain and bacterial culture are performed on a later tube, commonly the second or third. Exact tube allocation varies with the local ordering and routing policy, and a specimen collected into a single tube is sent to microbiology regardless of order because the workup cannot be repeated.²

At least 0.5 to 1.0 mL should reach the laboratory for Gram stain and bacterial culture. When mycobacterial or fungal culture is also requested, 5 to 10 mL is the target, because those organisms appear in low numbers and larger volumes raise culture yield.² A single small-volume tube cannot support every requested test, so when volume is limited the laboratory and the ordering clinician decide together which tests proceed.

CSF is processed as rapidly as possible. When a brief delay is unavoidable the specimen is held at room temperature, because organism viability for bacterial and fungal culture is the priority and cold injures sensitive organisms such as Neisseria meningitidis. A small aliquot (0.5 to 1.0 mL) intended for viral culture may be refrigerated briefly.²

Meningitis syndromes and laboratory expectations

The duration of symptoms and the patient’s age narrow the list of likely organisms and set expectations for the Gram stain and the culture plates.

Age is the strongest predictor of the causative organism in acute bacterial meningitis.¹

The likely causes also shift with vaccination status, immunocompromise, pregnancy, a cerebrospinal fluid leak, and recent surgery or neurosurgical devices, so the age table is a starting point rather than a rule.³

The CSF cell count and chemistry are interpreted alongside the Gram stain and culture. Acute bacterial meningitis in adults typically produces a neutrophil-predominant pleocytosis, often above 500 cells/µL, with elevated protein and low glucose. Tuberculous meningitis typically shows a lymphocyte-predominant count of 200 to 2000 cells/µL, again with elevated protein and low glucose. Enteroviral meningitis often begins with a neutrophil predominance that shifts to lymphocytes, and glucose is preserved.¹ A CSF glucose below the expected proportion of a paired plasma glucose supports bacterial, tuberculous, and fungal meningitis, while viral meningitis usually preserves the ratio; the physiology and measurement of CSF glucose are covered in Carbohydrate Metabolism and Glucose Testing.¹ Counting technique, dilution and correction calculations, and the examination reference values are covered in the body-fluid analysis pages.

Shunt and reservoir infections carry a different organism profile than community-acquired acute meningitis. Skin flora introduced at device placement dominates: coagulase-negative staphylococci, Staphylococcus aureus, Corynebacterium species, aerobic gram-negative rods, and Cutibacterium (formerly Propionibacterium) species.¹ Coagulase-negative staphylococci, usually dismissed as a contaminant in a peripheral blood culture, are treated as clinically significant when recovered repeatedly from CSF or from the hardware of a shunt patient.

Cerebrospinal fluid processing

Bacterial culture. When 1 mL or more is available, the specimen is concentrated by centrifugation and the supernatant is decanted to leave about 0.5 mL of sediment and fluid. That concentrate is mixed and used for both the cytocentrifuged Gram-stain smear and the culture inoculum. Cytocentrifugation of the concentrate raises the number of visible organisms on the smear by about 100-fold compared with a direct preparation.⁴

Chronic meningitis workups. Several agents require their own handling when they are specifically requested. Suspected brucellosis follows the routine bacterial workflow with prolonged incubation: direct plates are held up to 7 days, and when blood-culture bottles are used, automated systems hold them for 10 days with a blind or terminal subculture considered when brucellosis remains suspected after negative bottles.⁵ Suspected Brucella must be communicated to the laboratory in advance because it is a frequent cause of laboratory-acquired infection and triggers biosafety-cabinet work and public-health referral.⁵ Leptospirosis can be cultured from CSF during the first weeks of illness or referred for nucleic acid amplification. Neurosyphilis diagnosis rests on the combination of compatible neurologic findings, reactive serology, and CSF findings of pleocytosis, elevated protein, and a positive CSF-VDRL. Because the CSF-VDRL is highly specific but insensitive, a nonreactive result does not fully exclude neurosyphilis in a patient with a compatible clinical picture.⁶ Mycobacterial culture requires at least 5 mL of CSF with concentration to optimize smear and culture yield, and a negative nucleic acid amplification result does not exclude tuberculous meningitis because organism burden in CSF is very low.²,⁷

Fungal culture. Fungal processing parallels the bacterial workflow: concentration by centrifugation or filtration, a smear of the sediment, and inoculation of enriched, antibiotic-free media such as brain-heart infusion or SABHI agar. Cryptococcus grows on routine media but is inhibited by cycloheximide, so media selected for cryptococcal workup are cycloheximide-free.⁴

Rapid and adjunct cerebrospinal fluid tests

A commercially available multiplex molecular meningitis panel detects several bacterial, viral, and fungal targets simultaneously and returns results faster than culture. It does not detect every possible CSF pathogen, and it does not replace culture, which remains necessary for susceptibility testing.²

Cryptococcal capsular antigen detection by latex agglutination, enzyme immunoassay, or lateral-flow assay is the preferred rapid test for cryptococcal meningitis and has replaced the India ink stain in routine practice. Its sensitivity and specificity exceed 90%, and either unspun CSF or the supernatant of a centrifuged specimen can be tested, following the manufacturer’s instructions.² An India ink wet mount, prepared by mixing one drop of CSF sediment with one drop of India ink, allows direct visualization of encapsulated yeast. Its sensitivity is low outside high-burden disease such as advanced HIV infection, so it serves as an adjunct where antigen testing is unavailable.²

Viral and parasitic detection. Nucleic acid amplification is now the primary method for CNS viral diagnosis. Conventional cell culture remains useful mainly for enteroviruses, and serology supports diagnosis of the US arboviral encephalitides, including eastern equine, western equine, St. Louis, La Crosse, California serogroup, and West Nile viruses.² CSF is occasionally submitted for African trypanosomiasis (Trypanosoma brucei gambiense, T. brucei rhodesiense) or free-living ameba infection (Naegleria fowleri, Acanthamoeba species) and should be processed immediately on arrival. Because trypomastigotes and amebae adhere to the tube wall, the specimen is gently agitated before centrifugation at about 250 g for 10 minutes, and wet preparations of the sediment are examined with the condenser lowered or by phase contrast to improve visibility of motile organisms.⁴ Free-living amebae are cultured by spreading centrifuged sediment onto nonnutrient agar overlaid with a lawn of Escherichia coli or Klebsiella aerogenes, which the amebae feed on. Plates are incubated at 35 °C to 37 °C, with higher incubation up to 42 °C favoring thermophilic N. fowleri, and examined daily for up to 10 days.⁴ Heat fixation destroys amebae, so a Gram stain is avoided when amebic infection is suspected, and confirmation of any recovered ameba is by reference testing such as species-specific PCR.⁸

Sterile body fluids

Collection and transport

Fluid from the pericardial, pleural, or peritoneal cavity or from a joint space is aspirated by needle and syringe, transported in a sterile container, and delivered promptly.² A volume of 1 to 5 mL is adequate for recovering most bacteria, and 10 to 15 mL is preferred when mycobacteria or fungi are suspected, because those organisms are usually present in low numbers.²

Peritoneal fluid, including peritoneal dialysis effluent, may also be inoculated into blood culture bottles. This is the preferred technique for suspected peritoneal dialysis peritonitis: 5 to 10 mL of effluent into each of one aerobic and one anaerobic bottle at the bedside, with prompt transport at room temperature and no refrigeration.⁹ Bottle inoculation improves recovery of low-burden organisms, but a bottle alone supports neither direct microscopy nor an assessment of the original bacterial burden and mix, so the bottles are paired with a separate specimen or aliquot for direct Gram stain and plated media.² When more than one organism type is expected, as in secondary bacterial peritonitis from a gut source, bottle-only submission is avoided because broth enrichment can mask clinically important members of a mixed flora; a conventional anaerobic-transport specimen for Gram stain and plated media is sent instead.² An alternative concentration method starts from about 50 mL of effluent: the fluid is centrifuged, the sediment is resuspended in 3 to 5 mL, and that concentrate is inoculated to solid media and broth. This raises yield 5- to 10-fold over direct plating and is one option when bottle cultures remain negative.⁹

Enteroviruses, chiefly coxsackievirus B and less often coxsackievirus A, are classic causes of viral pericarditis, although many cases remain without a proven etiology. Pericardial fluid alone yields virus unreliably, so throat and stool specimens collected alongside the pericardial fluid improve detection of enterovirus, while the diagnosis rests on pericardial-fluid and biopsy nucleic acid amplification, culture, histology, and paired serology. A throat or stool positive result reflects shedding and is interpreted with the cardiac picture. Other viruses, including herpes simplex, varicella-zoster, cytomegalovirus, Epstein-Barr, mumps, and influenza viruses, are infrequent causes and are pursued when the syndrome points to them.²

Processing

For bacterial detection, the specimen is centrifuged, the supernatant is reduced to about 0.5 mL according to the laboratory’s validated procedure, and the sediment is used for both a Gram-stained smear and the culture inoculum. Alternatively, a small aliquot of nonviscous fluid can be cytocentrifuged directly for the smear.² Mycobacterial processing follows the concentration principles described for CSF, with tissue biopsy preferred when fluid yield is poor. Fungal processing concentrates the specimen, commonly to 1.5 to 2.0 mL, with a sediment smear stained by Gram stain or calcofluor white and 0.5 to 1.0 mL of sediment inoculated to primary fungal media where volume allows.⁴

Parasitologic examination. Body-cavity fluid is rarely collected specifically for parasites, but Entamoeba histolytica can appear in pericardial, pleural, or peritoneal fluid after rupture of a hepatic or pulmonary amebic abscess into an adjacent cavity, or after perforation of an amebic intestinal ulcer into the peritoneum.² Hydatid cyst rupture into a body cavity is another uncommon source of parasite-containing fluid, usually clear unless secondarily infected with bacteria. The rupture itself matters beyond the laboratory finding: spilled cyst contents can disseminate infection and provoke anaphylaxis, so suspected echinococcal cyst rupture is communicated immediately.¹⁰

Ocular specimens

Ocular specimens fall into two categories: surface specimens from the conjunctiva, and intraocular fluids and corneal tissue from normally sterile internal structures. Corneal scrapings are worked up as sterile-site specimens even though the cornea is exposed to the environment, because keratitis can destroy vision quickly.¹¹

Conjunctival specimens

Bacterial conjunctivitis in adults is caused most often by Staphylococcus aureus, Streptococcus pneumoniae, and Haemophilus influenzae; in children the leading causes are H. influenzae, S. pneumoniae, and Moraxella species. Chlamydia trachomatis causes trachoma, the leading infectious cause of blindness worldwide, and inclusion conjunctivitis in newborns and, less commonly, adults. Viruses account for most acute infectious conjunctivitis in US adults, most often adenoviruses, with herpes simplex virus less common.¹¹

Because ocular specimens are typically scant, the workup targets the most likely organisms for the clinical presentation. Conjunctival material is collected by swab from the superior and inferior tarsal conjunctiva, or with a sterile spatula when material is scraped for direct smears, fungi, or chlamydia. Where the laboratory and the ophthalmology service have set up a bedside workflow with controlled, in-date media, direct inoculation at collection preserves organism recovery from these low-volume specimens. Chocolate agar supports fastidious bacteria, additional media are added for specific suspected organisms, and separate fungal media are used when fungi are suspected.¹¹ Direct smears prepared at the same time aid preliminary identification.

When viral conjunctivitis is suspected, a second sample goes into the viral transport medium specified for the assay. Nucleic acid amplification is faster and more sensitive than viral culture for pathogens with a validated assay; one assay is FDA-cleared for herpes simplex virus detection from cutaneous and mucocutaneous lesion swabs, including ocular lesion specimens.¹² Direct fluorescent antibody staining of conjunctival cells offers a rapid alternative when a validated nucleic acid test is unavailable, with the caveat that fluorescence methods are less sensitive than amplification.¹¹

For C. trachomatis, a validated nucleic acid amplification test is the preferred method, and ocular use of genital-cleared assays is an off-label use validated by the laboratory; the CDC notes that direct fluorescent antibody testing is the only nonculture test FDA-cleared for conjunctival swabs.¹³ A Giemsa-stained smear showing epithelial cells with basophilic intracytoplasmic inclusions is suggestive, while direct fluorescent antibody staining with monoclonal antibody directed at the major outer membrane protein offers a specific confirmatory method. Comparative performance between Giemsa and fluorescence methods varies by study, so a smear result is interpreted with the clinical context.¹¹

Corneal specimens

In one recent series from a Midwestern US tertiary eye center, bacteria accounted for 73.5% of laboratory-positive microbial keratitis cases; coagulase-negative staphylococci, Staphylococcus aureus, and Cutibacterium acnes led the gram-positive isolates, and Pseudomonas aeruginosa and Moraxella species led the gram-negative isolates.¹⁴ Improper use or contamination of contact lens systems is a specific risk factor, associated in particular with outbreaks of Fusarium and Acanthamoeba keratitis.¹¹,¹⁵ Viral keratitis is most often recurrent HSV-1 or VZV; adenoviruses cause a superficial keratitis, and EBV-associated keratitis is uncommon and mainly seen with immunosuppression.¹¹ Postsurgical keratitis is typically caused by S. aureus, coagulase-negative staphylococci, or Cutibacterium acnes, and rare outbreaks caused by rapidly growing mycobacteria such as the Mycobacterium chelonae-M. abscessus group have followed contaminated water exposure. Keratitis following trauma should prompt culture for environmental organisms such as Nocardia species, which requires notification of the laboratory so that prolonged incubation and additional media can improve recovery.¹¹

Corneal scrapings are collected with a sterile spatula or blade and used both for direct smear preparation and for inoculation of chocolate agar for fastidious bacteria, brain-heart infusion agar with 10% sheep blood or the sheep-blood concentration in the laboratory’s validated protocol, and a fungal medium such as inhibitory mold agar.¹¹ If viral keratitis is suspected, scrapings go into the validated viral transport medium, and nucleic acid amplification is the preferred method, with viral culture and immunofluorescent staining of direct smears as alternatives when amplification is unavailable or awaits validation.¹¹

Vitreous and aqueous specimens

Vitreous fluid is obtained by vitreous tap or as part of a therapeutic vitrectomy; aqueous fluid is obtained by anterior chamber paracentesis. Both procedures yield small volumes, commonly under 0.5 mL for aqueous and diagnostic taps, with vitrectomy washings larger. The fluid is transported promptly to the laboratory or, where a bedside workflow exists, inoculated directly onto culture media or slides at collection. Concentration by cytocentrifugation or membrane filtration before smear preparation minimizes specimen loss from these limited volumes.²

The leading pathogens of endophthalmitis differ by mechanism of infection. Postoperative endophthalmitis is chiefly a gram-positive disease, with coagulase-negative staphylococci prominent and Cutibacterium acnes causing indolent infection weeks to months after cataract surgery. Endophthalmitis after intravitreal injection shows a similar gram-positive predominance, with documented outbreaks involving gram-negative rods and molds. Streptococci and Bacillus cereus follow penetrating trauma, with B. cereus endophthalmitis notable for its fulminant course, and Candida species and molds cause endophthalmitis from hematogenous spread or direct inoculation.² When the available fluid cannot support both a smear and a full panel of culture media, testing is prioritized and a molecular assay may be the only test the volume can support. A positive molecular result on this normally sterile fluid is still correlated with the smear, the clinical picture, and the known commensal status of organisms such as C. acnes.²

Separating pathogens from contaminants in sterile fluids

Fluid from the pericardial, pleural, peritoneal, synovial, vitreous, or aqueous space is normally sterile, so every isolate poses the same question: true pathogen, or contaminant introduced during needle passage through skin or an adjacent mucosal surface. The organisms that raise the question most often are the ones that colonize skin and mucosa: coagulase-negative staphylococci, Corynebacterium species and other aerobic actinomycetes, Cutibacterium species, viridans streptococci, Bacillus species, and Micrococcus species.²

No single finding settles the question, so several lines of evidence are weighed together. Heavy or pure growth supports infection, while scant growth mixed with other skin flora supports contamination, with the caveat that low-burden device infections can yield scant growth and prior antimicrobials can suppress it. Recovery of the identical organism from more than one independently collected specimen supports true infection, and concordant growth in more than one bottle from the same collection adds further support. Organisms and accompanying leukocytes seen together on the original direct Gram stain support infection. The collection route matters as well: a specimen obtained by direct surgical exposure of a normally sterile space generally carries a lower contamination risk than one obtained by percutaneous needle puncture through skin, and careful asepsis lowers the risk on either route.⁴

Isolates of coagulase-negative staphylococci, Corynebacterium, and Cutibacterium carry particular interpretive weight here because the same species appear as common blood culture contaminants. The characteristics of these organisms and their significance in blood cultures are covered in Blood Cultures and Gram-Positive Blood Isolates.

Molecular methods on sterile fluids

Broad-range 16S rRNA gene PCR with sequencing is used on culture-negative pleural, peritoneal, pericardial, synovial, or amniotic fluid, particularly from a patient already receiving antimicrobial therapy, since antibiotics can sterilize the fluid for culture while leaving amplifiable nucleic acid. The persistence of DNA does not prove viability, and availability is limited to validated reference laboratories.² A targeted multiplex panel is FDA-authorized for suspected joint infection, identifying the most common causative organisms and selected resistance markers directly from synovial fluid.¹⁶ Validated molecular panels for suspected pleural infection are laboratory-developed uses in most US laboratories.² Mycobacterium tuberculosis nucleic acid amplification is used on pleural and synovial fluid when tuberculous pleuritis or arthritis is suspected, as an adjunct to culture and tissue histology, since culture for M. tuberculosis can take weeks to become positive and a negative fluid result does not exclude disease.⁷ Metagenomic next-generation sequencing sequences the recoverable nucleic acid in a specimen without targeting a specific organism, which lets it detect unexpected or fastidious pathogens. Its interpretation is limited by background human and environmental nucleic acid, by the bioinformatic expertise required to run it, and by uncertainty about whether a given read count reflects infection or reagent and environmental background.²

Molecular results guide therapy, but culture remains the route to full susceptibility testing. Molecular panels return selected resistance-gene markers, which support therapy, while comprehensive phenotypic susceptibility testing still requires a viable isolate. Culture is set up in parallel whenever fluid remains, to recover that isolate.¹⁶

References

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14. Kang L, Lu MC, Niziol LM, et al. Microbial keratitis isolates at a Midwestern tertiary eye care center. Cornea. 2023;42(12):1488-1496. doi:10.1097/ICO.0000000000003198
15. Centers for Disease Control and Prevention. Acanthamoeba keratitis associated with contact lenses—United States. MMWR Morb Mortal Wkly Rep. 2007;56(21):532-534.
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