Laboratory Diagnosis of

Cerebrospinal Fluid

MM Cajucom
Microbiology
I. Specimen Collection

ØPrimary reason for CSF collection:

Ø Diagnosis of meningitis and
Ø Shunt infections
ØCSF can be obtained by:
Ø Trancutaneous aspiration (lumbar puncture)
Ø Ventricular Shunts/ Reservoir
LUMBAR PUNCTURE

ØIt is a medical procedure that is performed by a physician

ØDone by aseptically inserting a needle into subarachnoid space at the
level of lumbar spine (L3-L4, L4-L5, L5-S1)
LUMBAR PUNCTURE

ØFluid is drained into 3 leakproof containers with a minimum volume

of 2 ml in each tube.
Ø I. Cell Count and differential stains
Ø II. GS and culture (MICROBIOLOGY)
Ø III. Chemistry and special studies
ØSubmit appropriate amount of fluid depending on the requested
examinations.
VENTRICULAR SHUNTS

ØCSF collected in a reservoir in patients with ventricular

shunts drainage.
ØVS are used for drainage in patients who overproduce
CSF and it is usually submitted in the laboratory in large
volume
ØCollected aseptically by removal of fluid via the
reservoir and placing it in a sterile tube.
II. Specimen Collection and Transport

ØMinimum amount of required: 2ml for each examinations requested.

Ø Submit appropriate amount depending on the requested examinations.
Ø Too little sample lowers sensitivity
ØFor insufficient volumes- call physician to prioritize request based on
the patients clinical condition.
Specimen Collection and Transport

ØSpecimen containing traces of blood is accepted and

repeat of LP is not necessary. Bloody sample is indicative of
trauma to the lumen of SC.
ØSpecimen containing clots of blood is also accepted
pending a repeat for LP. Clot is homogenized prior to
plating
ØHand delivered, processed immediately and never
refrigerate.
ØTransport at RT except for viral culture (-70)
III. Cerebrospinal Fluid

ØCommonly collected thru LP

ØNormally water clear
ØHas no more than 5 leukocytes (0-5)
ØHas glucose concentration bet 45-100 mg/dl
ØHas protein concentration bet 15-45 mg/dl
ØSterile
ØAny organism recovered from culture are potential pathogens
ØActive disease is present if there are chemical and cellular changes
in the CSF.
 IV. CSF Bacterial Etiologies

ØAcute – onset of symptoms within 24hours

ØStrep pneumoniae
ØN. meningitidis
ØS. agalactiae
ØH. influenzae
ØS. aureus
ØL. monocytogenes
ØG (-) B
ØChronic - symptoms lasting for weeks
ØMycobacteria
ØLeptospira
ØTreponema
ØNocardia
ØBrucella
ØCryptococcus
 V. Laboratory Processing

ØProcess specimen as soon as received in a BSC

ØRecord Volume
ØRecord gross appearance (clean /cloudy/bloody/xantho)
ØRecord Collection procedure (LP/VS)
ØFor Volume:
Ø<0.5mL (at least 1-5 gtts)
- Make a direct smear (GS)
- Dilute the remaining fluid into 0.5mL of broth mix the contents & inoculate to
the media
Ø>1mL
- Inoculate 1mL to broth
- Remaining fluid concentrate by centrifugation for 15min at 1500rpm
- Save supernate and inoculate sediment to media & make a GS
* Cultures are more sensitive when fluid is concentrated
VI. Laboratory Examination of CSF

Standard Policy:
ØThe following tests in Microbiology should be ordered
routinely on CSF
Ø Gram Stain
Ø Routine Bacterial Culture
Øanaerobic culture is not routinely done and usually indicated only for
brain abscess.
Ø Cryptococcal Ag Test
Øfungal pathogens in CSF are best and most rapidly diagnosed by serologic
methods
Øindia ink staining is not indicated bec of low sensitivity (50%); it should be
requested in combination with culture and or serology
 Laboratory Examination of CSF

Standard Policy:
ØThe following test will be done only by special request after initial
specimen testing:
ØPCR for Mtb and HSV
ØAFB Culture
ØFungal Culture
ØViral Culture
 VII. Gram Staining

ØA drop of CSF is placed on the surface of a sterile slide

- Concentrate if > 1mL
- Direct inoculation if < 0.5mL
ØDo not spread the fluid for easier reading
- Spreading of fluid, increases the difficulty of finding low number of organism
ØRead and Interpret GS immediately
- Usually within an hour of receipt
- Any bacteria seen are considered significant
- Notify the physician immediately
Gram Staining (continued)

GS reporting
Ø Semiquantitative enumeration of predominant cell
type bacterial morphology

Numerical Descriptive Comments

+1 < 1/ OIF Rare Confirm low
+2 1-5/OIF Few numbers only
seen in 1 or 2
+3 5-10/OIF Moderate
fields with a
+4 >10/OIF Many second smear
VIII. Culture of Etiologic Agents

Ø Bacterial
- Inoculate sediments into approriate culture media
Chocolate Agar
5% SB Agar
Culture Broth (BH1/TSB)
- Streaked into Quadrants using sterile loop
- Incubate:
Plates at 35-37C in 5% CO2
Broth at35-37C in an ambient air
Ø Fungal
- Inoculate sediments into appropriate Culture Media
Sabaroud’s Dextrose Agar
BHIA with 5% of SB
- Incubate at RT for 4W with weekly monitoring
IX. CULTURE EXAMINATION

ØExamine all plated and broth media for macroscopic evidence of

broth after 24h
ØIf no visible growth observed on culture media
- Reincubate plated media – 4 days
- Subculture broth media – 5 to 7 days
ØFor cultures with growth:
- Correlate plated culture results with those of direct GS and subcultured broth
medium.
- Identify all organisms
- Consider contaminants if there is 1 or 2 colonies on subcultured broth medium
but there is no growth on primary plated media and no organism seen on GS.
- Perform ID only for organism isolated from broth medium (NO-AST)
- Notify the physician of (+) culture readings.
 CULTURE EXAMINATION

ØIdentify all potential pathogens of meningitis. Determine the probable genus and
the species identification.
ORGANISM GS ISOLATION COLONIAL KEY ID TEST SEROLOGY
MORPHOLOGY MEDIA MORPHOLOGY (capsular ag)
Haemophilus G(-) coccobacilli CA in 5% CO2 Small transparent XV factor test
influenzae at 35C for with characteristic Satellitism Test
24hours mousy odor

Streptococcus G(+) cocci in 5%SB in 5% Small, pinpoint, Catalase (-)

agalactiae pairs and in CO2 at 35C for beta hemolytic Esculin (-)
chains 24hours Bacitracin (R)
Lancefield Grpng
Neiserria G (-) diplococci CA in 5% CO2 Grayish, convex Catalase (+)
meningitidis both inside and at 35C for and mucoid Oxidase (+)
outside PMN 72hours colonies CHO utilization
G/M (+)
S/L (-)
Listeria Short G (+) 5% SB at 35C Small, pinpoint, Catalase (+)
monocytogenes bacilli ambient narrow zone beta Motility (+)
hemolytic Esculin (+)

Cryptococcus Yeast cells SDA atRT for 4 Mucoid colonies India Ink (+) CN polysaccha-
neoformans which does not weeks and forms a long Urease (+) ride Ag
produce string when Caffeic acid (+)
pseudohyphae retracted from the
surface of the agar
Fungal Culture
Key ID Test for CN
CULTURE EXAMINATION

ØPerform AST per CLSI Policy

- Enteric GNB
- NF-GNB
- Staph. aureus
- Strep. pneumo
- Enterococci
- HI
*Except Listeria (universally sensitive to PG.
S.agalactiae Resistance is rare)
N.meningitis
ØHold (+) culture plates and tubes for 7 days or freeze isolates for
retrieval.
CULTURE EXAMINATION

ØReporting of results:

Ø When reporting (-) results indicate the incubation

time.
eg. “NG after 4 days of incubation”

Ø When reporting (+) results indicate the

quantitation of growth and the genus and species of
the organism.

eg. “Heavy Growth of Haemophilus influenzae”