Cerebrospinal Fluid and Sterile Body Fluid Cultures

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.^{11 Tunkel AR, et al. Practice guidelines for the management of bacterial meningitis. 2004.} 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.^{22 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

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.^{22 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.} 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 to perform.

Process CSF as rapidly as possible. When a brief delay is unavoidable, keep the specimen 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.^{22 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

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.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Age is the strongest predictor of the causative organism in acute bacterial meningitis.^{11 Tunkel AR, et al. Practice guidelines for the management of bacterial meningitis. 2004.}

The likely causes also shift with vaccination status, immunocompromise, pregnancy, a cerebrospinal fluid leak, and recent surgery or neurosurgical devices, so use the age table as a starting point.^{44 Centers for Disease Control and Prevention. About bacterial meningitis.}

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 (an increased CSF white-cell count), often above 500 cells/µL, with elevated protein and low glucose. Tuberculous meningitis typically shows a lymphocyte-predominant count of 200 to 2,000 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. A CSF glucose below the expected proportion of a paired plasma glucose supports bacterial, tuberculous, and fungal meningitis. In viral meningitis the ratio is usually preserved. The physiology and measurement of CSF glucose are covered in Carbohydrate Metabolism and Glucose Testing.^{11 Tunkel AR, et al. Practice guidelines for the management of bacterial meningitis. 2004.} Counting technique, dilution and correction calculations, and the examination reference values are covered in Body Fluid Cell Counts and Cerebrospinal Fluid.

Shunt and reservoir infections have a different organism profile from community-acquired acute meningitis. Skin flora introduced at device placement predominates: coagulase-negative staphylococci, Staphylococcus aureus, Corynebacterium species, aerobic gram-negative rods, and Cutibacterium (formerly Propionibacterium) species.^{11 Tunkel AR, et al. Practice guidelines for the management of bacterial meningitis. 2004.} Coagulase-negative staphylococci are common blood-culture contaminants but can cause device-associated infection, and they 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, concentrate the specimen by centrifugation and decant the supernatant to leave about 0.5 mL of sediment and fluid. Mix that concentrate and use it 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.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Chronic meningitis workups. Several agents require their own handling when they are specifically requested. Most bloodstream and body-fluid isolates need only the routine continuous-monitoring incubation period of about 5 days. Suspected brucellosis is an exception. Direct plates are incubated up to 7 days, and when blood-culture bottles are used, current American Society for Microbiology (ASM) and Association of Public Health Laboratories (APHL) sentinel-laboratory guidance directs incubating them for 10 days, with a blind or terminal subculture considered when brucellosis remains suspected after negative bottles.^{55 American Society for Microbiology and… Brucellosis-causing Brucella species (BBS). 2025.} Brucella is a biosafety level 3 (BSL-3), laboratory-acquired-infection risk organism. The laboratory must be told of the suspicion in advance so that the isolate is handled under biological safety cabinet containment and referred through the public-health sentinel-laboratory procedure.^{55 American Society for Microbiology and… Brucellosis-causing Brucella species (BBS). 2025.} Leptospirosis can be cultured from CSF during the first weeks of illness or referred for nucleic acid amplification. The diagnosis of neurosyphilis is based on the combination of compatible neurologic findings, reactive serology, and CSF findings of pleocytosis, elevated protein, and a positive CSF Venereal Disease Research Laboratory (VDRL) test. 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.^{66 Centers for Disease Control and Prevention. Syphilis: CDC STI treatment guidelines.} 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.^{2,72 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.7 Lewinsohn DM, et al. Official American Thoracic Society/Infectious Diseases Society… 2017.}

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 Sabouraud brain-heart infusion (SABHI) agar. Cryptococcus grows on routine media but is inhibited by cycloheximide, so choose cycloheximide-free media for a cryptococcal workup.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

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 culture remains necessary for susceptibility testing.^{22 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

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. 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 is an adjunct where antigen testing is unavailable.^{22 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

Viral and parasitic detection. Nucleic acid amplification is now the primary method for central nervous system (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.^{22 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.} CSF is occasionally submitted for African trypanosomiasis (Trypanosoma brucei gambiense, T. brucei rhodesiense) or free-living ameba infection (Naegleria fowleri, Acanthamoeba species). Process it immediately on arrival. Because trypomastigotes (the flagellated bloodstream and CSF stage of the trypanosome) and amebae adhere to the tube wall, gently agitate the specimen before centrifugation at about 250 × g for 10 minutes, and examine wet preparations of the sediment with the condenser lowered or by phase contrast to improve visibility of motile organisms.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.} 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. Incubate the plates at 35 to 37 °C, with higher incubation up to 42 °C favoring thermophilic N. fowleri, and examine them daily for up to 10 days.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.} Heat fixation destroys amebae, so skip the Gram stain when amebic infection is suspected, and confirm any recovered ameba by reference testing such as species-specific polymerase chain reaction (PCR).^{88 Centers for Disease Control and Prevention. Clinical and laboratory diagnosis for Naegleria fowleri…}

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. 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.^{22 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

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.^{99 Li PK-T, et al. ISPD peritonitis guideline recommendations. 2022.} Bottle inoculation improves recovery of low-burden organisms. Bottles cannot be used for direct microscopy or to assess the original bacterial burden and mix, so pair the bottles with a separate specimen or aliquot for direct Gram stain and plated media. When more than one organism type is expected, as in secondary bacterial peritonitis from a gut source, avoid bottle-only submission, because broth enrichment can mask clinically important members of a mixed flora. Send a conventional anaerobic-transport specimen for Gram stain and plated media.^{22 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.} An alternative concentration method starts from about 50 mL of effluent: centrifuge the fluid, resuspend the sediment in 3 to 5 mL, and inoculate that concentrate to solid media and broth. This raises yield 5- to 10-fold over direct plating and is one option when bottle cultures remain negative.^{99 Li PK-T, et al. ISPD peritonitis guideline recommendations. 2022.}

Enteroviruses, chiefly coxsackievirus B and less often coxsackievirus A, are classic causes of viral pericarditis, although no cause is proven in many cases. Virus is recovered unreliably from pericardial fluid, so throat and stool specimens collected with the pericardial fluid improve detection of enterovirus. The direct laboratory evidence comes from pericardial-fluid and biopsy nucleic acid amplification, culture, histology, and paired serology. A positive throat or stool result reflects shedding and is interpreted with the cardiac findings. Other viruses, including herpes simplex, varicella-zoster, cytomegalovirus, Epstein-Barr, mumps, and influenza viruses, are infrequent causes and are tested for when the clinical picture suggests them.^{22 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

Processing

For bacterial detection, centrifuge the specimen, reduce the supernatant to about 0.5 mL according to your laboratory's validated procedure, and use the sediment for both a Gram-stained smear and the culture inoculum. Alternatively, a small aliquot of nonviscous fluid can be cytocentrifuged directly for the smear.^{22 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.} Mycobacterial processing follows the concentration principles described for CSF, with tissue biopsy preferred when fluid yield is poor. For fungal culture, the specimen is concentrated, commonly to 1.5 to 2.0 mL. A sediment smear is stained by Gram stain or calcofluor white (a fluorescent stain that binds fungal cell walls), and 0.5 to 1.0 mL of sediment is inoculated to primary fungal media where volume allows.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

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.^{22 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.} Hydatid cyst rupture into a body cavity is another uncommon source of parasite-containing fluid, usually clear unless secondarily infected with bacteria. Spilled cyst contents can disseminate infection and provoke anaphylaxis, so communicate a suspected echinococcal cyst rupture immediately.^{1010 Centers for Disease Control and Prevention. Echinococcosis.}

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.^{1111 Leal SM Jr, et al. Practical guidance for clinical microbiology laboratories. 2021.}

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.^{1111 Leal SM Jr, et al. Practical guidance for clinical microbiology laboratories. 2021.}

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.^{1111 Leal SM Jr, et al. Practical guidance for clinical microbiology laboratories. 2021.} 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 cleared by the Food and Drug Administration (FDA) for herpes simplex virus detection from cutaneous and mucocutaneous lesion swabs, including ocular lesion specimens.^{1212 US Food and Drug Administration. Simplexa HSV 1 and 2 Direct.} Direct fluorescent antibody staining of conjunctival cells is a rapid alternative when a validated nucleic acid test is unavailable. Fluorescence methods are less sensitive than amplification.^{1111 Leal SM Jr, et al. Practical guidance for clinical microbiology laboratories. 2021.}

For C. trachomatis, a validated nucleic acid amplification test is the preferred method. Ocular use of genital-cleared assays is an off-label use that requires laboratory validation. The Centers for Disease Control and Prevention (CDC) notes that direct fluorescent antibody testing is the only nonculture test FDA-cleared for conjunctival swabs.^{1313 Workowski KA, et al. Sexually transmitted infections treatment guidelines, 2021. 2021.} A Giemsa-stained smear showing epithelial cells with basophilic intracytoplasmic inclusions is suggestive. Direct fluorescent antibody staining with monoclonal antibody directed at the major outer membrane protein is a specific confirmatory method. Comparative performance between Giemsa and fluorescence methods varies by study, so interpret a smear result with the clinical context.^{1111 Leal SM Jr, et al. Practical guidance for clinical microbiology laboratories. 2021.}

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.^{1414 Kang L, et al. Microbial keratitis isolates at a Midwestern tertiary eye care… 2023.} Improper use or contamination of contact lens systems is a specific risk factor, associated in particular with outbreaks of Fusarium and Acanthamoeba keratitis.^{11,1511 Leal SM Jr, et al. Practical guidance for clinical microbiology laboratories. 2021.15 Centers for Disease Control and Prevention. Acanthamoeba keratitis associated with contact lenses—United… 2007.} Viral keratitis is most often recurrent herpes simplex virus type 1 (HSV-1) or varicella-zoster virus (VZV). Adenoviruses cause a superficial keratitis, and Epstein-Barr virus (EBV)-associated keratitis is uncommon and mainly seen with immunosuppression. 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 requires culture for environmental organisms such as Nocardia species. The laboratory needs this request in advance so that prolonged incubation and additional media can be set up to improve recovery.^{1111 Leal SM Jr, et al. Practical guidance for clinical microbiology laboratories. 2021.}

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. 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.^{1111 Leal SM Jr, et al. Practical guidance for clinical microbiology laboratories. 2021.}

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.^{22 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

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, and B. cereus endophthalmitis is notable for its fulminant course. Candida species and molds cause endophthalmitis from hematogenous spread or direct inoculation. When the volume is too small for both a smear and a full panel of culture media, prioritize testing. A molecular assay may be the only test the volume allows. Correlate a positive molecular result on this normally sterile fluid with the smear, the clinical picture, and the known commensal status of organisms such as C. acnes.^{22 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

Separating pathogens from contaminants in sterile fluids

Fluid from the pericardial, pleural, peritoneal, synovial, vitreous, or aqueous space is normally sterile, so each isolate is assessed as either a true pathogen or a contaminant introduced during needle passage through skin or an adjacent mucosal surface. The organisms that most often require this assessment are those that colonize skin and mucosa: coagulase-negative staphylococci, Corynebacterium species and other aerobic actinomycetes, Cutibacterium species, viridans streptococci, Bacillus species, and Micrococcus species.^{22 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

No single finding distinguishes infection from contamination, so weigh several findings together. Heavy or pure growth supports infection. Scant growth mixed with other skin flora supports contamination. Low-burden device infections can also yield scant growth, and prior antimicrobials can suppress growth. 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 also affects contamination risk. A specimen obtained by direct surgical exposure of a normally sterile space generally has a lower contamination risk than one obtained by percutaneous needle puncture through skin. Careful asepsis lowers the risk with either route.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Isolates of coagulase-negative staphylococci, Corynebacterium, and Cutibacterium need particular care in interpretation because the same species are 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, because antibiotics can make the fluid culture-negative and leave amplifiable nucleic acid. Detected DNA does not prove that viable organisms are present. The test is offered only by validated reference laboratories.^{22 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.} 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.^{1616 US Food and Drug Administration. bioMérieux BioFire Joint Infection Panel.} Validated molecular panels for suspected pleural infection are laboratory-developed uses in most US laboratories.^{22 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.} 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.^{77 Lewinsohn DM, et al. Official American Thoracic Society/Infectious Diseases Society… 2017.} Metagenomic next-generation sequencing sequences the recoverable nucleic acid in a specimen without targeting a specific organism, so it can 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.^{22 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

Molecular results can guide therapy, and panels report selected resistance-gene markers. Complete phenotypic susceptibility testing requires a viable isolate, so set up culture in parallel whenever fluid remains.^{1616 US Food and Drug Administration. bioMérieux BioFire Joint Infection Panel.} At the bench, interpret every sterile-fluid isolate with the direct smear, the amount of growth, and the collection route before you report it.

Practice