COLLECTION

AND
PROCESSING
OF CSF
OUTLINE
• Possible pathogens

• Commensals

• Collection & transport

• Lab examination : Day 1

• Lab examination : Day 2 & onwards

• Summary of lab examination

 POSSIBLE PATHOGENS
BACTERIA GRAM NEGATIVE
GRAM POSITIVE • Neisseria meningitidis
• Streptococcus pneumoniae • Haemophilus influenzae type b
• Streptococcus agalactiae • Escherichia coli
• Listeria monocytogenes
• Pseudomonas aeruginosa
• Streptococcus suis
• Proteus species
• Salmonella serovars
• Flavobacterium meningosepticum

• Also M. tuberculosis & T. pallidum

• Bacteria ~ also found in CSF when there is a brain abscess e.g.
Bacteroides sp. & other anaerobes
POSSIBLE
VIRUSES
PATHOGENS
• Particularly coxsackie viruses, echovirus, & arboviruses
• Also, HSV 2 ,VZV & lymphocytic choriomeningitis virus (LCM)
• Rarely polioviruses
FUNGI
• C. neoformans (mainly in AIDS patients) & less commonly Aspergillus sp.
PARASITES
• Trypanosoma sp. & Naegleria fowleri
• Rarely the larvae of Angiostrongylus cantonensis & Dirofilaria immitis (CSF ~
usually contains eosinophils)
• Also Toxoplasma gondii (mainly in AIDS patients)
POSSIBLE PATHOGENS
• Inflammation of the meninges (membranes that cover the brain & spinal
cord) = MENINGITIS
• Pathogens reach the meninges in the blood stream or occasionally by
spreading from nearby sites (middle ear or nasal sinuses)

• Fever, headache, neck stiffness & intolerance of light ~ typical symptoms

of ACUTE BACTERIAL MENINGITIS
• In children~ vomiting, convulsions & lethargy
• A haemorrhagic rash is associated with MENINGOCOCCAL
MENINGITIS
POSSIBLE PATHOGENS
Meningitis is described as:
• PYOGENIC (PURULENT)
– CSF contains mainly PMN neutrophils (pus cells)
– Acute meningitis caused by N. meningitidis, H. influenzae & S.
pneumoniae
– Pus also found in CSF ~ Acute Amoebic Meningoencephalitis
• LYMPHOCYTIC
– CSF contains mainly lymphocytes
– Meningitis caused by viruses, M. tuberculosis & C. neoformans
– Lymphocytes also found in CSF ~ Trypanosomiasis
meningoencephalitis & neurosyphilis
POSSIBLE PATHOGENS
• Developing countries ~ Meningitis epidemics are caused by N.
meningitidis serogroups A & C & occasionally by gp B & other
serogroups
• Outbreaks are common in SUB-SAHARAN AFRICA (MENINGITIS
BELT) with most GP A MENINGOCOCCAL MENINGITIS
epidemics ~ hot dry season
• Recently, Meningococcal meningitis has risen to epidemic proportions
in some countries of South America, the Middle East & Asia

• Rarely, epidemic meningitis is caused by S. pneumoniae but

ENDEMIC pneumococcal meningitis is common & has a high fatality
rate
POSSIBLE PATHOGENS
• In developing countries ~ Neonatal meningitis is caused mainly by
S. pneumoniae (about 1/3 of cases), Salmonella serovars & other
Enterobacteria, N. meningitidis & H. influenzae
– Streptococcus agalactiae is a rare cause

• Haemophilus meningitis ~ mainly in infants & young children

below 5 yrs with a high incidence below 2 yrs
POSSIBLE PATHOGENS
• C. neoformans ~ opportunistic pathogen, causing life-threatening
Meningoencephalitis in those with AIDS & other conditions
associated with immunosuppression
– Areas of high HIV prevalence~ Cryptococcosis in up to 30% of AIDS
patients

• Syphilitic meningitis may occur in secondary syphilis ~ usually a

complication of late syphilis

• N. fowleri causes primary amoebic meningoencephalitis~ rare &

fatal disease
COMMENSALS
• CSF HAS NO NORMAL MICROBIAL FLORA
COLLECTION AND TRANSPORT
• CSF must be collected by an experienced
MEDICAL OFFICER OR HEALTH WORKER
– Must be collected ASEPTICALLY to prevent
organisms being introduced into the CNS
• The fluid is usually collected from the
ARACHNOID SPACE
– A sterile wide-bore needle is inserted
between the 4TH & 5TH LUMBAR
VERTEBRAE & CSF is allowed to drip
into a dry sterile container
– A ventricular puncture is sometimes
performed to collect CSF from infants
COLLECTION AND TRANSPORT
Collection of CSF from patient with suspected
TRYPANOSOMIASIS
• CSF to be examined for Trypanosomes
– Is collected after treatment to kill the trypanosomes in the blood
has started
• To avoid the accidental introduction of the parasites into
the CNS should the lumbar puncture be traumatic
(bloody)
COLLECTION AND TRANSPORT
In a hospital with a MB lab
• Advise the lab before performing a lumbar puncture so that staff are
prepared to receive & examine the specimen IMMEDIATELY

• A delay in examining CSF reduces the chances of isolating a

pathogen
– Also result in a lower cell count due to WBCs being lyzed & to a
falsely low glucose value due to glycolysis
– When trypanosomes are present, they will be difficult to find b/c
they are rapidly lyzed once the CSF has been withdrawn
COLLECTION AND TRANSPORT
In a hospital with a MB lab
Collection of CSF
1. Take 2 sterile, dry, screw-capped containers & label one No. 1 (1ST
sample collected~ used for CULTURE) & the other No. 2 (2ND
sample collected~ used for OTHER INVESTIGATIONS)

2. Collect about 1 ml of CSF in container No. 1 & about 2–3 ml in

container No. 2

3. Immediately deliver the samples with a request form to the lab

COLLECTION AND TRANSPORT
In a health centre
• Patients with SUSPECTED MENINGITIS usually receive emergency
treatment in a health centre & are TRANSFERRED to the nearest
hospital for lab investigations & care
LAB EXAMINATION OF CSF:
DAY 1
LAB EXAMINATION OF CSF: DAY 1
• CSF must be examined W/O DELAY & the results of tests reported to
the medical officer as soon as they become available, especially a Gram
smear report

• The fluid should be handled with special care b/c a lumbar puncture is
required to collect the specimen
1. REPORT THE APPEARANCE OF THE CSF
• As soon as the CSF reaches the lab ~
note its appearance

• Report whether the fluid :

– Is clear, slightly turbid, cloudy or
definitely purulent (looking like pus)
– contains blood
– contains clots

• Normal CSF ~ clear & colourless

1. REPORT
• Purulent THE APPEARANCE
or cloudy CSF OF THE CSF
– Indicates presence of pus cells ~ acute pyogenic bacterial meningitis
• Blood in CSF
– Due to a traumatic (bloody) lumbar puncture or less commonly
to haemorrhage in the CNS
– When due to a traumatic lumbar puncture, sample No. 1 will usually
contain more blood than sample No. 2
– Following a subarachnoid haemorrhage, the fluid may appear
XANTHROCHROMIC, i.e. yellow-red (after centrifuging)
• Clots in CSF
– Indicates a high protein conc. with increased fibrinogen ~ occur
with pyogenic meningitis or when there is spinal constriction
2. TEST THE CSF
• Depending on the appearance of the CSF proceed as follows:

Purulent or cloudy CSF

• Suspect PYOGENIC MENINGITIS & test as follows:
– Immediately make & examine a Gram stained smear for bacteria
& PMN neutrophils (pus cells)
• Issue the report w/o delay
– Culture the CSF
2. TEST THE CSF
Slightly cloudy or clear CSF
Test as follows:
• Perform a cell count ~ whether there is an increase in white cells &
whether the cells are mainly pus cells or lymphocytes

When cells predominantly pus cells:

• Examine a Gram stained smear for bacteria
• Examine a wet prep (sediment from centrifuged CSF) for motile
amoebae which could be Naegleria (rare)
• Culture the CSF
2. TEST THE CSF
Slightly cloudy or clear CSF
When cells predominantly lymphocytes:
• May indicate :
– viral meningitis
– tuberculous meningitis
– cryptococcal meningitis
– trypanosomiasis encephalitis or
– other condition in which lymphocyte No. in the CSF are increased
2. TEST THE CSF
Slightly cloudy or clear CSF
When cells predominantly lymphocytes:
Perform the following tests:
• Measure the conc. of protein or perform a Pandy’s test ~ CSF
protein is raised in most forms of meningitis & meningoencephalitis
• Measure the conc. of glucose~ differentiating viral meningitis (CSF
glucose is normal) from TB meningitis & other conditions (CSF
glucose is reduced)
• Examine a wet prep for encapsulated yeast cells ~ C. neoformans
• Examine a wet prep for trypanosomes & a Giemsa stained smear
for morula (Mott) cells when late stage trypanosomiasis is suspected
2. TEST THE CSF
Report the CSF as ‘Normal’

• When it appears CLEAR, contains no more than 5 WBC x 106/1 & the
PROTEIN CONC. IS NOT RAISED (or Pandy’s test is -ve)

• A CSF begins to appear turbid when it contains about :

200 WBC X 106/1
2. TEST THE CSF
GRAM SMEAR
• Required when the CSF contains pus cells (neutrophils)

• 1ST investigation to be performed & reported when the CSF appears

purulent or cloudy (suggestive of acute pyogenic meningitis)

• Provide useful information when a CSF is unsuitable for cell

counting or biochemical testing (e.g. when it is heavily blood stained
or contains clots)
 2. TEST THE CSF
GRAM SMEAR: MAKING A SMEAR OF CSF
1. Mix No. 2 sample & centrifuge most of it at approx. 1000 g for 5–10 mins
(leave a small amount of uncentrifuged CSF~ if a cell count is required)
• Purulent CSF: Do not centrifuge ~ Gram staining is best prepared
from the uncentrifuged CSF
2. Transfer the supernatant fluid to another tube (used for glucose &
protein tests if required)
3. Mix the sediment. Transfer several drops of the sediment to a slide, but
do not make the prep too thick b/c this will make it difficult to decolorize
adequately. Allow the prep to air-dry in a safe place
4. Alcohol-fix the prep & stain it by the Gram technique
2. TEST THE CSF
EXAMINING A CSF GRAM SMEAR
• Examine the smear microscopically for pus cells & bacteria

Pus cells:
• Report as many, moderate or few

• Found mainly in PYOGENIC BACTERIAL MENINGITIS & in amoebic

meningoencephalitis (rare)
2. TEST THE CSF
EXAMINING A CSF GRAM SMEAR
Bacteria:
Look in well stained (not too thick) areas for:
• GN intracellular diplococci ~ possibly N.
meningitidis
• GP diplococci or short streptococci~
possibly S. pneumoniae
– Often possible to see the capsules as
unstained areas around the bacteria
2. TEST THE CSF
EXAMINING A CSF GRAM SMEAR
Bacteria:
Look in well stained (not too thick) areas for:
• GN rods~ possibly H. influenzae, esp. if filamentous or
other polymorphic forms are seen
• GN rods~ possibly E. coli or other coliforms, esp. when
the CSF is from a newborn infant
• Unevenly stained irregular in size yeast cells
(some showing budding)~ suggestive of C. neoformans
– The large capsule that surrounds the cell DOES
NOT stain
– Best seen in an India ink prep
– The smear will usually contain LYMPHOCYTES
2. TEST THE CSF
EXAMINING A CSF GRAM SMEAR
Bacteria:
2. TEST THE CSF
EXAMINING A CSF GRAM SMEAR
• Advise the medical officer immediately if the Gram smear contains bacteria,
pus cells, or yeast cells (confirmed as capsulated in India ink prep)

• When bacteria & pus cells are seen in the Gram smear, culture the CSF

• NO need to perform a cell count or measure the protein or glucose

• When the patient has been given antibiotics (as emergency treatment) it will
be more difficult to detect bacteria in the Gram smear & to isolate
pathogens in culture
2. TEST THE CSF
CULTURING CSF
• Culture when bacteria are seen in the Gram smear & or cells are present
or the protein conc. is raised
• Use CSF sample No. 1.
– When the CSF is clear or slightly cloudy, centrifuge the sample in
a sterile capped tube for about 15 mins & use the sediment to
inoculate the culture media
• CSF must be cultured ASAP after collection
– When a delay is unavoidable ~ the fluid should be kept at 35–37 °C
(not refrigerated)
2. TEST THE CSF
CULTURING CSF
Chocolate (heated blood) agar & blood agar
• Inoculate the specimen on CA & BA
– When GP diplococci are seen in the Gram smear, add an optochin disc
to the BA plate to assist in the ID of S. pneumoniae

• Incubate both plates in a C02 enriched atm at 35–37 °C for up to 48 hrs,

checking for growth after o/n incubation

When patient is a newborn infant:

• Inoculate the specimen also on MAC agar
• Incubate aerobically at 35–37 °C o/n
2. TEST THE CSF
CELL COUNT
• A white cell count with an indication whether the cells are pus cells or
lymphocytes ~ when the CSF appears slightly cloudy or clear or
when the Gram smear DOES NOT indicate pyogenic bacterial meningitis

• Samples that are heavily blood stained or contain clots are

unsuitable for cell counting
– Make a Gram smear & report the presence of pus cells & bacteria
2. TEST THE CSF
CELL COUNT: METHOD
• To identify whether white cells in the CSF are PMN neutrophils (pus cells) or
lymphocytes, dilute the CSF in a fluid which stains the cells
• Isotonic 0.1% TB is recommended
– Stains lymphocytes & the nuclei of pus cells BLUE
– C. neoformans yeast cells stain pink
– Red cells remain unstained
• The motility of trypanosomes is NOT affected by the dye
• When TB is unavailable, ISOTONIC MB can be used
– Stain the nuclei of leucocytes
• If preferred, leucocytes can be differentiated by examining a Leishman, Giemsa or
rapid Field’s stained smear (sediment from centrifuged CSF) after counting the
cells
2. TEST THE CSF
CELL COUNT: METHOD
1. Mix the CSF (sample No. 2 uncentrifuged) & dilute the fluid 1 in 2
• 1 drop of CSF with1 drop of TB diluting fluid (drops must be of equal
volume)
2. Assemble a modified Fuchs-Rosenthal ruled counting chamber
making sure the chamber & cover glass are completely clean
• Recommended b/c it has twice the depth (0.2 mm) & is more suitable for
counting WBCs in CSF
• When unavailable, an improved Neubauer chamber can be used
3. Using a fine bore Pasteur pipette or capillary tube, carefully fill the
counting chamber with the. well-mixed diluted CSF
• Fluid must NOT overflow into the channels on each side of the chamber
2. TEST THE CSF
CELL COUNT: METHOD
4. Wait about 2 mins for the cells to settle & count the cells microscopically

5. Focus the cells & rulings using the x10 objective w/ condenser iris closed
• Use x40 objective to check that the cells are white cells & not red cells &
note whether the white cells are mainly PMN neutrophils or lymphocytes
• If a mixture of both, estimate approx. the % of each type of cell
• When yeast cells are seen~ examine an India ink prep
• When red cells are seen~ mention this in the report
– When many red cells are present ~ CSF is unsuitable for WBC cell
counting
2. TEST THE CSF
CELL COUNT: METHOD
6. Count the cells in 5 of the large squares
• When the cells are too many to count, dilute the CSF 1 in 10 (1 drop
CSF mixed with 9 drops of DILUTING FLUID), refill the chamber &
count the cells
2. TEST THE CSF
CELL COUNT: METHOD
Using 1 in 2 CSF dilution & Fuchs- Rosenthal chamber:

• Multiply the cells counted by 2

• Report the no. of cells per litre of CSF
E.g. If 240 cells are counted in 5 squares: 240 X 2 = 480
• Report as 480 X 106 cells/1 OR 480 cells/mm3 or 480 cells/l
(FORMERLY)
2. TEST THE CSF
CELL COUNT: METHOD
When using an Improved Neubauer
chamber:
• Count the cells in 4 of the large
squares
• Multiply the cells counted by 5
• Report the no. of cells per litre of CSF
E.g. If 64 cells are counted in 4 squares:
64 X 5 = 320
• Report as 320 X 106 cells/1
2. TEST THE CSF
CELL COUNT: Cal factors when using 1 in 10 CSF dilution
Fuchs-Rosenthal chamber:
• Multiply cells counted in 5 squares by 10
• Report number of cells per litre of CSF
Improved Neubauer chamber:
• Multiply cells counted in 4 squares by 25
• Report number of cells per litre of CSF
Normal CSF:
• Contains up to 5 x106 cells/litre (higher in neonates)
• When no WBCs are seen, report the count as: < 5 cells x 106/1
2. TEST THE CSF
BIOCHEMICAL TESTING OF CSF
• Biochemical CSF tests which may be required include the measurement
of protein & glucose
• When the Gram smear shows organisms & pus cells, little additional info
will be provided by testing for protein & glucose
• When NO bacteria are seen in the Gram smear & the cell count is raised,
testing for protein & glucose can help to differentiate those
conditions in which lymphocytes are found in CSF
– E.g.Viral meningitis (slightly raised protein, normal glucose) from
Tuberculous meningitis (high protein, low glucose)
2. TEST THE CSF
BIOCHEMICAL TESTING OF C.S.F.
Measurement of CSF glucose
• Glucose must be measured within 20 mins of the CSF being withdrawn
otherwise a falsely low result will be obtained due to glycolysis

• Use the supernatant fluid from centrifuged CSF or uncentrifuged CSF if

the sample appears clear

• Glucose can be measured in CSF using a colorimetric technique or a

simpler semi quantitative technique using Benedict’s reagent
2. TEST THE CSF
BIOCHEMICAL TESTING OF C.S.F.
Measurement of CSF glucose
Normal CSF glucose:
• About 1/2 to 2/3 that found in blood i.e. 2.5–4.0 mmol/1 (45–72 mg%)
Raised CSF glucose:
• In hyperglycaemia & sometimes with encephalitis
Low CSF glucose:
• In most forms of meningitis (except viral meningitis)
• In pyogenic bacterial meningitis it is markedly reduced & may even be
undetectable
2. TEST THE CSF
BIOCHEMICAL TESTING OF C.S.F.
Measurement of CSF total protein & globulin test
• Use the supernatant fluid from centrifuged CSF or uncentrifuged CSF
when the sample appears clear

• Total protein can be measured in CSF using a colorimetric technique

or a visual comparative technique

• Pandy’s test is a screening test which detects rises in CSF globulin

– It is of value when it is not possible to measure CSF total protein
2. TEST THE CSF
BIOCHEMICAL TESTING OF CSF
Measurement of CSF total protein & globulin test
Normal CSF protein:
• Total CSF protein is normally 0.15–0.40 g/l (15–40 mg%)
• The range for ventricular fluid is slightly lower.
• Values up to 1.0 g/l (100 mg%) are normal for newborn infants
• Only traces of globulin are found in normal CSF , insufficient to give a +ve
Pandy’s test
2. TEST THE CSF
BIOCHEMICAL TESTING OF CSF
Measurement of CSF total protein & globulin test

Increased CSF total protein with +ve Pandy’s test:

• Occurs in all FORMS OF MENINGITIS ~ amoebic & trypanosomiasis
meningoencephalitis, cerebral malaria, brain tumors, cerebral injury, spinal
cord compression, poliomyelitis, Guillain-Barre syndrome & polyneuritis

• Also occur in diseases which cause changes in plasma proteins

(myelomatosis)
2. TEST THE CSF
BIOCHEMICAL TESTING OF CSF
Measurement of CSF total protein & globulin test
• When the total protein exceeds 2.0 g/l (200 mg%), the fibrinogen level is
increased sufficiently to cause the CSF to clot
– may occur in severe pyogenic meningitis, spinal block, or following
haemorrhage

• In diseases of the nervous system such as multiple sclerosis, neurosyphilis,

& some connective tissue disorders it is possible to find a +ve Pandy’s
test for gobulin with only a slight rise or even normal total protein
2. TEST THE CSF
Ziehl-Neelsen smear when tuberculous meningitis is suspected

• Examine a Ziehl-Neelsen stained CSF smear for acid fast bacilli (AFB)
when tuberculous meningitis is clinically suspected or the CSF contains
lymphocytes & the glucose conc. is low and the protein raised

• AFB are difficult to detect in CSF

2. TEST THE CSF
Ziehl-Neelsen smear when tuberculous meningitis is suspected
• The following technique increases the chances of finding the bacteria:

1. Centrifuge the CSF at high speed for 20–30 mins. Remove the supernatant
fluid & mix the sediment. Transfer several drops of the sediment to a slide,
allowing each drop to dry before adding the next

2. Fix the dry prep with methanol & stain by the Ziehl-Neelsen technique

3. Examine the smear with x40 objective to see the distribution of material
and then with then x100 objective to detect the AFB. Examine the entire
prep
2. TEST THE CSF
India ink prep when cryptococcal
meningitis is suspected

• When cryptococcal meningitis is clinically

suspected, e.g. patient with HIV disease, or
when yeast cells are detected when performing
a cell count or examining a Gram smear,
examine an India ink prep or a wet prep by
dark-field microscopy for encapsulated yeasts
2. TEST THE CSF
India ink prep when cryptococcal meningitis is suspected
1. Centrifuge the CSF for 5–10 mins; remove the supernatant fluid & mix
the sediment
2. Transfer a drop of the sediment to a slide, cover with a cover glass &
examine by DF microscopy or add a drop of India ink; mix & cover with a
cover glass
3. Examine the prep using the 40 objective

• Look for oval or round cells, some showing budding, irregular in size,
measuring 2–10 m in dia & surrounded by a large unstained capsule
• When encapsulated yeasts are detected in CSF, a presumptive diagnosis of
cryptococcal meningitis can be made
2. TEST THE CSF
Wet prep & Giemsa smear when trypanosomiasis
meningoencephalitis is suspected
• Fresh CSF is required to detect trypanosomes

• About 15 mins after the fluid is withdrawn, the trypanosomes begin to

lose their motility & are rapidly lyzed

• The trypanosomes are usually few & therefore a careful search of a wet
prep is required to detect the motile flagellates
2. TEST THE CSF
Wet prep & Giemsa smear when
trypanosomiasis meningoencephalitis is
suspected
1. Centrifuge the CSF at about 1000 g for 10 mins.
Remove the supernatant fluid & mix the sediment

2. Transfer a drop of sediment to a slide & cover with a

cover glass

3. Examine for motile trypanosomes using x40

objective with the condenser iris closed . Alternatively
examine the prep by dark field microscopy
2. TEST THE CSF
Wet prep & Giemsa smear when trypanosomiasis meningoencephalitis is
suspected
Giemsa smear to detect morula cells
• When NO trypanosomes are seen in the wet prep, remove the cover glass & allow
the prep to air-dry
• Fix the smear with methanol & stain it using Giemsa technique or Field’s rapid stain as
used for thin films
• Examine the prep for morula cells (IgM producing cells) using x40 objective.
• The cells are easily recognized
– They are LARGER THAN LYMPHOCYTES WITH A DARK MAUVE STAINING
NUCLEUS & CHARACTERISTIC VACUOLES IN THEIR CYTOPLASM
• Finding morula cells in CSF of a person with African trypanosomiasis indicates CNS
involvement
2. TEST THE CSF
Wet prep to detect amoebae
• Examine a wet prep for motile amoebae when PAM is clinically
suspected or the CSF contains pus cells with raised protein & low
glucose, but NO bacteria are seen in the Gram smear
• Red cells may also be present

1. Transfer a drop of uncentrifuged purulent CSF or a drop of sediment

from a centrifuged specimen to a slide & cover with a cover glass
2. Examine the prep using 10 & 40 objectives, with the condenser closed
2. TEST THE CSF
Wet prep to detect amoebae
• Look for small, clear, motile,
elongated forms among the pus cells
• The amoebae often contain vacuoles
but NOT red cells
• When amoebae are seen, immediately
notify the medical officer attending the
patient ~ Amoebic meningocephalitis is
a rapidly fatal condition
CSF MB WET PREP
SHOWING PAM
LAB EXAMINATION OF CSF:
DAY 2 & ONWARDS
 EXAMINE & REPORT THE CULTURES
CA & BA cultures
Look especially for colonies that
could be:
• Neisseria meningitidis (grow on
CA & BA, oxidase +ve)
• Streptococcus pneumoniae
(sensitive to optochin)
• Haemophilus influenzae (grow C. neoformans on BA
ONLY on CA) H. influenzae

• Cryptococcus neoformans (Gram

stain the colonies)
 EXAMINE AND REPORT THE CULTURES
MacConkey agar culture
Look especially for colonies that could be:
• E. coli or other coliform
• S. agalactiae
• L. monocytogenes
• Other bacteria that cause neonatal meningitis
Streptococcus suis ID
• A non-haemolytic GPC belonging to Lancefield Group D
• CAMP negative, hydrolyzes aesculin & is able to grow on bile agar
 EXAMINE & REPORT THE CULTURES
Antimicrobial susceptibility testing
• Test isolates of S. pneumoniae for susceptibility to chloramphenicol
& penicillin (use 1g oxacillin disc)

• Test H. influenzae for beta-lactamase production & susceptibility to

chloramphenicol (using chocolate agar)

• Perform susceptibility testing on Gram negative rods as indicated

SUMMARY OF THE EXAMINATION OF CSF
DAY 1

1. Report Describe whether CSF

Appearance – Clear, slightly turbid, cloudy, purulent
– Contains blood
– Contains clots
 DAY 1
2.Test CSF

Purulent or cloudy CSF Slightly turbid or clear CSF

• Suspect pyogenic bacterial meningitis Perform cell count
• Gram smear ~ Report: Note whether pus cells or lymphs
– No. of pus cells
– Bacteria
• Culture CSF PUS CELLS LYMPHS
– Blood agar & chocolate agar Gram smear Measure protein
Incubate in CO2 Culture CSF Measure glucose
– If neonate: Zn: For AFB
Also MacConkey agar ADDITIONAL TESTS India Ink: For
Incubate aerobically Wet prep: encapsulated yeasts
For motile amoebae Wet prep & Giemsa smears:
For trypanosomes & morula cells
 SUMMARY OF THE EXAMINATION OF CSF
DAY 2 & ONWARDS

3. Examine and Chocolate agar & blood ADDITIONAL

Report Cultures agar cultures -Perform antimicrobial
Look particularly for: susceptibility tests as indicated
-N. meningitidis - Beta-lactamase test.
-S. pneumoniae H. influenzae isolates
-H. influenzae (chocolate agar)

MacConkey agar culture

Look especially for bacteria
that cause neonatal meningitis
THAT’S ALL FOLKS