Collection , transportation

and examination of specimens

Stool
Describe the appearance of the specimen

• Colour of the specimen.

• Whether it is formed, semiformed, unformed or fluid.
• Presence of blood, mucus or pus – Presence of worms, e.g.
Enterobius vermicularis, Ascaris lumbricoides, or tapeworm
segments e.g. Taenia species.
Normal faeces: Appear brown and formed or semiformed.
Infant faeces are yellow-green and semiformed.
Causes of inflammatory diarrhoeal disease
• Shigella species
• Campylobacter species
• Salmonella (non-typhoid serovars)
• E. histolytica EIEC
Less common:
• B. coli
• Y. enterocolitica
• C. difficile
• C. perfringens (causing pigbel)
• Aeromonas species
Microscopical Examination
• Saline and eosin preparations to detect E. histolytica and other parasites
– Place a drop of fresh physiological saline on one end of a slide and a drop
of eosin stain (Reagent No. 36) on the other. Using a piece of stick or wire
loop, mix a small amount of fresh specimen (especially mucus and blood)
with each drop. Cover each preparation with a cover glass. Important: The
eosin preparation must not be too thick otherwise it will not be possible to see
amoebae or cysts.
– Examine the preparations using the 10 and 40 objectives with the
condenser iris closed sufficiently to give good contrast.
– Look especially for motile E. histolytica trophozoites containing red cells,
motile G. lamblia trophozoites, motile Strongyloides larvae, and the eggs and
cysts of parasitic pathogens.
Note: Read about the microscopical appearance of E. histolytica, G. lamblia
and other protozoal parasites
Methylene blue preparation to detect faecal
leucocytes when the specimen is unformed
– Place a drop of methylene blue stain on a slide. Mix a small amount of specimen
with the stain, and cover with a cover glass.
– Examine the preparation for faecal leucocytes using the 40 objective with the
condenser iris closed sufficiently to give good contrast.
– Report also the presence of red blood cells (RBC) as these are often present with
pus cells in inflammatory invasive diarrhoeal disease
Faecal leucocytes (WBCs)
• Look for mononuclear cells and polymorphonuclear cells (pus cells).
• Mononuclear cells contain a nucleus which is not lobed whereas
polymorphonuclear cells contain a nucleus which has two or more lobes.
Sometimes the cells are too damaged to be recognized (do not attempt to identify).
• Pus cells are associated with bacteria that cause inflammation of the large
intestine.
• Often red cells are also found. Mononuclear cells are found mainly in typhoid and
in some parasitic infections, including amoebic dysentery.
 Basic fuchsin smear to detect campylobacters
• Prepare when the specimen is unformed and, or, contains mucus, pus, or
blood and is from a child under 2 y.
– Make a thin smear of the specimen on a slide. When dry, gently heat-fix.
Stain by covering the smear with 10 g/l basic fuchsin* for 10–20 seconds.
Wash well with water and allow to air dry. *Dissolve 1 g basic fuchsin in
100 ml of water, and filter.
– Examine the smear for campylobacters using the 100 oil immersion
objective.
Campylobacter organisms: Look for abundant small, delicate, spiral
curved bacteria (often likened to gull wings), S-shapes, and short
spirochaetal forms. Note: Examination of stained faecal smears for
campylobacters has been shown to be a sensitive method for the
presumptive diagnosis of campylobacter enteritis. Culture of
Campylobacter species
Motility test and Gram stained smear
• when cholera is suspected Examine an alkaline peptone water culture
(sample from the surface of the culture) for vibrios showing a rapid
and darting motility.
• The preparation is best examined using dark-field microscopy but the
vibrios can also be seen using transmitted light.
• Techniques for detecting motile bacteria.
• Experience is required to identify the characteristic motility of V.
cholerae.
• Examine also a Gram-stained smear of the culture for Gram negative
vibrios (use 1 in 10 dilution of carbol fuchsin as the counterstain
instead of neutral red).
Culture the specimen
• When the specimen is formed or semiformed, make a thick suspension of it in about 1 ml of sterile
peptone water. Xylose lysine deoxycholate (XLD) agar
• Inoculate a loopful of fresh emulsified faeces or a fluid specimen on XLD agar .
• Incubate the XLD agar plate aerobically at 35–37 °C overnight. XLD agar:
-This selective medium is recommended for the isolation of salmonellae and particularly shigellae
from faecal specimens.
- It contains the indicator phenol red which is red at an alkaline pH (medium is pH 7.4) and yellow
at an acid pH.
- Shigellae form pink-red colonies because they do not ferment xylose, lactose, or sucrose (except
some S. sonnei strains).
- Salmonellae also form pink-red colonies even though they ferment xylose with acid production.
- This is because they break down the amino acid lysine which gives an alkaline reaction.
- Hydrogen sulphide (H2S) producing salmonellae form red colonies with black centres.
- Some Proteus strains and Edwardsiella species form pink red colonies with black centres.
- Escherichia coli, Enterobacter species, and some other enterobacteria produce yellow colonies
due to carbohydrate fermentation.
Note: Some workers also recommend the use of a less selective medium such as MacConkey agar in
addition to XLD agar.
Alkaline peptone water and TCBS agar when
cholera is suspected
– Inoculate several loopfuls of specimen in alkaline (pH 8.6) peptone
water and incubate at 35–37 °C for 5–8 hours.
Note:
• Prior enrichment in alkaline peptone water is not necessary if the
specimen is likely to contain large numbers of vibrios (e.g. in acute
cholera).
• Alkaline peptone water is a useful transport medium for V. cholerae.
• – Subculture several loopfuls of the peptone water culture (taken from
the surface) on thiosulphate citrate bile-salt sucrose (TCBS) agar .
• Incubate aerobically at 35–37 °C overnight. TCBS medium:
Sorbitol MacConkey agar, when an outbreak of
E. coli 0157 is suspected
– Inoculate a loopful of specimen on sorbitol MacConkey agar.
– Incubate the plate aerobically at 35–37 °C overnight. Sorbitol
MacConkey agar This MacConkey medium contains the carbohydrate
sorbitol instead of lactose.
• E. coli 0157 produces colourless colonies on the medium because it
does not ferment sorbitol.
• Most other E. coli strains and other enterobacteria ferment sorbitol,
producing pink colonies.
• Sorbitol MacConkey agar is therefore a useful way of screening for E.
coli 0157 (reported as having a specificity of 85% and sensitivity of
100%).
Examine and report the cultures XLD agar
culture
• Look for colonies that could be Shigella or Salmonella.
• Shigella and H2S negative strains of Salmonella produce 1–2 mm
diameter red colonies on XLD agar.
• Red colonies with black centres are produced by H2S positive
salmonellae, e.g. strains of S. Typhimurium.
• Proteus, Providencia and Pseudomonas organisms may also produce
red colonies on XLD agar.
• Some Proteus strains are also H2 S producing and form red colonies
with black centres.
Note: On MacConkey agar, shigellae, and salmonellae and other non-
lactose fermenting organisms, produce colourless colonies. E. coli and
other lactose fermenting organisms produce pink colonies.
Identification of suspect Salmonella and
Shigella isolates
• Perform a urease test using urea broth or a Rosco urease identification
tablet.
• A positive urease test within 2–4 h indicates that the organism is
probably Proteus. No further tests are required.
• When the urease test is negative at 4 hours, proceed as follows:
1. Perform indole and lysine decarboxylase (LDC) tests.
2. Inoculate a tube of Kligler iron agar.
3. Use a sterile straight wire, stab first the butt and then streak the
slope.
4. Close the tube with a loose-fitting cap and incubate at 35–37 °C
overnight.
Results
LDC
Shigella are LDC negative.
Salmonella serovars are LDC positive except S. Paratyphi A which is LDC
negative.
Indole
• S. sonnei is indole negative.
• Other shigellae give variable indole reactions Salmonella serovars are indole
negative.
KIA
• Salmonella and Shigella organisms produce a pink-red slope and yellow butt.
• Many salmonellae also produce blackening due to hydrogen sulphide production
and cracks in the medium due to gas production from glucose fermentation.
• Salmonella Typhi produces only a small amount of blackening and no cracks in the
medium. KIA reactions
STOOL
CSF
Possible Pathogens
BACTERIA
Gram negative
Gram positive

• Streptococcus pneumoniae • Neisseria meningitidis

• Streptococcus agalactiae (Group • Haemophilus influenzae type b
B)* • Escherichia coli*
• Listeria monocytogenes* • Pseudomonas aeruginosa*
• Streptococcus suis • Proteus species*
• Salmonella serovars
• Flavobacterium
• meningosepticum*
*Mainly isolated from neonates * *S. suis is a pathogen of pigs. Mycobacterium tuberculosis and Treponema pallidum. Note: Bacteria may also be
found in the c.s.f. when there is a brain abscess, e.g. Bacteroides species and other anaerobes.
VIRUSES FUNGI
• Particularly coxsackieviruses, • Cryptococcus neoformans (mainly
echovirus in AIDS patients) and less
• arboviruses commonly Aspergillus species.
• herpes simplex 2 virus PARASITES
• varicella zoster virus • Trypanosoma species
• lymphocytic choriomeningitis virus • Naegleria fowleri.
(LCM). • Rarely the larvae of
• Rarely polioviruses may be Angiostrongylus cantonensis
isolated from cerebrospinal fluid. • Dirofilariaimmitis(c.s.f.usuallycont
ainseosinophils).
• Toxoplasma gondii (mainly in
AIDS patients)

Commensals: Cerebrospinal fluid has no normal microbial flora.

Meningitis
• Inflammation of the meninges (membranes that cover the brain and spinal cord) is
called meningitis.
• Pathogens reach the meninges in the blood stream or occasionally by spreading
from nearby sites such as the middle ear or nasal sinuses. Fever, headache, neck
stiffness, and intolerance of light are typical symptoms of acute bacterial
meningitis.
• In children, vomiting, convulsions and lethargy are common. A haemorrhagic
rash is associated with meningococcal meningitis.
• Meningitis is described as:
– Pyogenic (purulent), when the c.s.f. contains mainly polymorphonuclear
neutrophils (pus cells), as in acute meningitis caused by N. meningitidis, H.
influenzae, and S. pneumoniae. Pus cells are also found in the c.s.f. in acute amoebic
meningo-encephalitis.
– Lymphocytic, when the c.s.f. contains mainly lymphocytes, as in meningitis
caused by viruses, M. tuberculosis, and C. neoformans. Lymphocytes are also found
in the c.s.f. in trypanosomiasis meningoencephalitis, and neurosyphilis.
• Meningitis epidemics are usually caused by N. meningitidis serogroups A
and C and only occasionally by group B and other serogroups.
• neonatal meningitis is caused mainly by S. pneumoniae (about one third of
cases), Salmonella serovars and other enterobacteria, N. meningitidis, and
H. influenzae. Streptococcus agalactiae (Group B) is a rare cause.
• Haemophilus meningitis occurs mainly in infants and young children below
5 y with a high incidence below 2 y.
• C. neoformans is mainly an opportunistic pathogen, causing life-threatening
meningo-encephalitis in those with AIDS and other conditions associated
with immunosuppression.
• Syphilitic meningitis may occur in secondary syphilis but it is usually a
complication of late syphilis .
• N. fowleri causes primary amoebic meningoencephalitis, a rare and usually
fatal disease.
COLLECTION AND TRANSPORT OF CSF
• Cerebrospinal fluid must be collected by an experienced medical officer or
health worker.
• It must be collected aseptically to prevent organisms being introduced into
the central nervous system.
• The fluid is usually collected from the arachnoid space.
• A sterile wide-bore needle is inserted between the fourth and fifth lumbar
vertebrae and the c.s.f. is allowed to drip into a dry sterile container.
• A ventricular puncture is sometimes performed to collect c.s.f. from infants.
• When the c.s.f. is to be examined for trypanosomes, it is usually collected
after treatment to kill the trypanosomes in the blood has been started. This
will avoid the accidental introduction of the parasites into the central
nervous system should the lumbar puncture be traumatic (bloody).
In a hospital with a microbiology
laboratory
IMPORTANT:
• Advize the laboratory before performing a lumbar puncture so that
staff are prepared to receive and examine the specimen immediately.
Note:
• A delay in examining c.s.f. reduces the chances of isolating a
pathogen.
• It will also result in a lower cell count due to WBCs being lyzed, and
to a falsely low glucose value due to glycolysis.
• When trypanosomes are present, they will be difficult to find because
they are rapidly lyzed once the c.s.f. has been withdrawn.
Collection of c.s.f.
1 . Take two sterile, dry, screw-capped containers and label one No. 1
(first sample collected, to be used for culture), and the other No. 2
(second sample collected, to be used for other investigations).
2. Collect about 1 ml of c.s.f. in container No. 1 and about 2–3 ml in
container No. 2.
3. Immediately deliver the samples with a request form to the
laboratory.
Examination of CSF
1. Report the appearance of the c.s.f.
• As soon as the c.s.f. reaches the laboratory, note its appearance.
• Report whether the fluid: – is clear, slightly turbid, cloudy or definitely purulent
(looking like pus), – contains blood, – contains clots.
• Normal c.s.f. Appears clear and colourless.
• Purulent or cloudy c.s.f. Indicates presence of pus cells, suggestive of acute
pyogenic bacterial meningitis. Blood in c.s.f.
• This may be due to a traumatic (bloody) lumbar puncture or less commonly to
haemorrhage in the central nervous system.
• 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 (seen after centrifuging).
• Clots in c.s.f. Indicates a high protein concentration with increased fibrinogen, as
can occur with pyogenic meningitis or when there is spinal constriction
2. Test the c.s.f
Depending on the appearance of the c.s.f., proceed as follows:
• Purulent or cloudy c.s.f. Suspect pyogenic meningitis and test the c.s.f. as follows:
-Immediately make and examine a Gram stained smear for bacteria and
polymorphonuclear neutrophils (pus cells).
• Issue the report without delay.
Culture the c.s.f
• Slightly cloudy or clear c.s.f. Test the c.s.f. as follows:
-Perform a cell count and note whether there is an increase in white cells and whether
the cells are mainly pus cells or lymphocytes.
• When cells predominantly pus cells:
– Examine a Gram stained smear for bacteria.
– Examine a wet preparation (sediment from centrifuged c.s.f.) for motile amoebae
which could be Naegleria (rare).
– Culture the c.s.f. When cells predominantly lymphocytes: This could indicate viral
meningitis, tuberculous meningitis, cryptococcal meningitis, trypanosomiasis
encephalitis, or other condition in which lymphocyte numbers in the c.s.f. are increased.
Perform the following tests
– Measure the concentration of protein or perform a Pandy’s test. The
c.s.f. protein is raised in most forms of meningitis and
meningoencephalitis.
– Measure the concentration of glucose. This is helpful in
differentiating viral meningitis in which the c.s.f. glucose is usually
normal from tuberculous meningitis and other conditions in which the
c.s.f. glucose is reduced .
– Examine a wet preparation for encapsulated yeast cells that could be
C. neoformans.
– Examine a wet preparation for trypanosomes and a Giemsa stained
smear for morula (Mott) cells when late stage trypanosomiasis is
suspected.
Report the c.s.f. as ‘Normal’
• when it appears clear, contains no more than 5 WBC 106/1, and the
protein concentration is not raised (or Pandy’s test is negative).
Note: A c.s.f. begins to appear turbid when it contains about 200 WBC
106/1.
GRAM SMEAR
• A Gram smear is required when the c.s.f. contains pus cells (neutrophils).
• It should be the first investigation to be performed and reported when the c.s.f. appears
purulent or cloudy (suggestive of acute pyogenic meningitis).
• A Gram smear may also provide useful information when a c.s.f. is unsuitable for cell
counting or biochemical testing (e.g. when it is heavily blood stained or contains clots).
Making a smear of c.s.f. for Gram staining
1. Mix No. 2 sample c.s.f. and centrifuge most of it at approximately 1000 g for 5–10
minutes (leave a small amount of uncentrifuged c.s.f. for a cell count should this be
required).
2. Purulent c.s.f. Do not centrifuge a purulent fluid.
3. A smear for Gram staining is best prepared from the uncentrifuged c.s.f.
4. Transfer the supernatant fluid to another tube (to be used for glucose and protein tests
should these be required).
5. Mix the sediment. Transfer several drops of the sediment to a slide, but do not make
the preparation too thick because this will make it difficult to decolorize adequately.
Allow the preparation to air-dry in a safe place.
6. Alcohol-fix the preparation and stain it by the Gram technique
Examining a c.s.f. Gram smear
• Examine the smear microscopically for pus cells and bacteria using the 40 and 100
objectives.
Pus cells: Report as many, moderate number, or few. Pus cells will be found mainly in
pyogenic bacterial meningitis and in amoebic-meningoencephalitis (rare). Bacteria: Look
in well stained (not too thick) areas for:
• Gram negative intracellular diplococci that could be N. meningitidis . Gram positive
diplococci or short streptococci, that could be S. pneumoniae.
• It is often possible to see the capsules as unstained areas around the bacteria
• Gram negative rods, possibly H. influenzae, especially if filamentous or other
polymorphic forms are seen .
• Gram negative rods could also be E. coli or other coliforms, especially when the c.s.f. 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. It is best seen in an
India ink preparation .
• The smear will usually contain lymphocytes.
Important: Advize the medical officer immediately if the Gram smear contains bacteria,
pus cells, or yeast cells (confirmed as capsulated in India ink preparation).
Note: When bacteria and pus cells are seen in the Gram smear, culture
the c.s.f. There is no need to perform a cell count or measure the protein
or glucose. When the patient has been given antibiotics (usually as
emergency treatment) it will be more difficult to detect bacteria in the
Gram smear and to isolate pathogens in culture.
Acridine orange stained smear to detect bacteria in c.s.f.
• When facilities for fluorescence microscopy are available, examine an
acridine orange (A0) stained smear .
• Bacteria especially when few, are more easily detected in A0 smears.
• They stain bright orange and cells and debris stain green or yellow.
• The organisms can be detected using the 40 objective.
Immunological diagnosis of acute bacterial
meningitis
• Direct antigen testing of c.s.f. may provide a rapid diagnosis of acute
bacterial meningitis, particularly when the patient has been treated
with antimicrobials and bacteria cannot be detected in a Gram smear
or by culture.
• Tests are available to detect N. meningitidis groups A, B, C, Y and
W135 (Group B reagent cross-reacts with E. coli K1 antigen), H.
influenzae type b, S. pneumoniae, and S. agalactiae.
CULTURING C.S.F.
• Culture the c.s.f. when bacteria are seen in the Gram smear and, or,
cells are present, or the protein concentration is raised.
• Use c.s.f. sample No. 1. When the c.s.f. is clear or slightly cloudy,
centrifuge the sample in a sterile capped tube for about 15 minutes,
and use the sediment to inoculate the culture media.
Important: Cerebrospinal fluid must be cultured as soon as possible
after collection. When a delay is unavoidable, the fluid should be kept at
35–37 °C (not refrigerated).
Chocolate (heated blood) agar and blood
agar
– Inoculate the specimen on chocolate agar and blood agar . When Gram
positive diplococci are seen in the Gram smear, add an optochin disc to
the blood agar plate to assist in the identification of S. pneumoniae.
– Incubate both plates in a carbon dioxide enriched atmosphere at 35–37
°C for up to 48 hours, checking for growth after overnight incubation.
When patient is a newborn infant: Inoculate the specimen also on
MacConkey agar. Incubate aerobically at 35–37 °C overnight.
CELL COUNT
• A white cell count with an indication whether the cells are pus cells or
lymphocytes, is required when the c.s.f. appears slightly cloudy or
clear or when the Gram smear does not indicate pyogenic bacterial
meningitis.
Note: Samples that are heavily blood stained or contain clots are
unsuitable for cell counting. Make a Gram smear and report the
presence of pus cells and bacteria
Method
• To identify whether white cells in the c.s.f. are polymorphonuclear
neutrophils (pus cells) or lymphocytes, dilute the c.s.f. in a fluid which
stains the cells.
• Istonic 0.1% toluidine blue is recommended because it stains
lymphocytes and 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 toluidine blue is unavailable, isotonic methylene blue can be
used which will also 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 c.s.f.) after counting the cells.
Procedure
1. Mix the c.s.f. (sample No. 2 uncentrifuged c.s.f.). Dilute the fluid 1 in 2, i.e. mix 1 drop of
c.s.f. with 1 drop of toluidine blue diluting fluid (Reagent No. 84).
2. * *The drops must be of equal volume, therefore use Pasteur pipettes of the same bore size
for both fluids and hold the pipettes vertically when dispensing the drops.
3. Assemble a modified Fuchs-Rosenthal ruled counting chamber,* making sure the chamber
and cover glass are completely clean.
4. *When unavailable, an improved Neubauer (preferably Bright-Line) chamber can be used.
A Fuchs-Rosenthal chamber is recommended because it has twice the depth (0.2 mm) and is
more suitable for counting WBCs in c.s.f.
5. Using a fine bore Pasteur pipette or capillary tube, carefully fill the counting chamber with
the well-mixed diluted c.s.f. The fluid must not overflow into the channels on each side of
the chamber.
6. Wait about 2 minutes for the cells to settle. Count the cells microscopically.
7. Focus the cells and rulings using the 10 objective with the condenser iris closed sufficiently
to give good contrast. Before starting the count, use the 40 objective to check that the cells
are white cells and not red cells (unstained smaller cells without a nucleus)
Note: whether the white cells are mainly polymorphonuclear neutrophils (with lobed nucleus)
or lymphocytes. If a mixture of both, estimate approximately the percentage of each type of cell.
• When yeast cells are seen, examine
an India ink preparation .
Note: When red cells are seen,
mention this in the report.
• When many red cells are present,
the c.s.f. is unsuitable for WBC cell
counting.
• Count the cells in 5 of the large
squares
Note: When the cells are too many
to count, dilute the c.s.f. 1 in 10 (1
drop c.s.f. mixed with 9 drops of
diluting fluid), refill the chamber and
count the cells.

Modified Fuchs-Rosenthal ruled chamber. Cells are counted in the squares marked W1, W2, W3, W4, W5.
• Multiply the cells counted by 2.
• Report the number of cells per litre (1) of c.s.f.
Example (Using 1 in 2 c.s.f. dilution and FuchsRosenthal chamber)
• If 240 cells are counted in 5 squares:
-240 2 480 Report as 480 106 cells/1*
• *Formerly, 480 106 cells/1 would have been reported as 480
cells/mm3 or 480 cells/l.
• When using an Improved Neubauer chamber: Count the cells in 4 of
the large squares. Multiply the cells counted by 5.
• Report the number of cells per litre of c.s.f. Example If 64 cells are
counted in 4 squares:
-64 5 320 Report as 320 106 cells/1.
Calculation factors when using 1 in 10 c.s.f.
dilution
Fuchs-Rosenthal chamber:
Multiply cells counted in 5 squares
by 10. Report number of cells per
litre of c.s.f.
Improved Neubauer chamber:
Multiply cells counted in 4 squares
by 25. Report number of cells per
litre of c.s.f.
Normal c.s.f.: Contains up to 5 106
cells/litre (higher in neonates). When
no WBCs are seen, report the count
as: Below 5 cells 106/1.
BIOCHEMICAL TESTING OF C.S.F.
• Biochemical c.s.f. tests which may be required include the measurement of
protein and glucose.
Note: When the Gram smear shows organisms and pus cells, little additional
information will be provided by testing for protein and glucose.
• When however no bacteria are seen in the Gram smear and the cell count is
raised, testing for protein and glucose can help to differentiate those
conditions in which lymphocytes are found in c.s.f., e.g. viral meningitis
(slightly raised protein, normal glucose) from tuberculous meningitis (high
protein, low glucose).
• Measurement of c.s.f. glucose Glucose must be measured within 20 minutes
of the c.s.f. being withdrawn otherwise a falsely low result will be obtained
due to glycolysis.
• Use the supernatant fluid from centrifuged c.s.f. or uncentrifuged c.s.f. if
the sample appears clear.
• Glucose can be measured in c.s.f. using a colorimetric technique or a
simpler semiquantitative technique using Benedict’s reagent.
Normal c.s.f. glucose: This is about half to two thirds that
found in blood i.e. 2.5–4.0 mmol/1 (45–72 mg%).
Raised c.s.f. glucose: Occurs when the blood glucose level is
raised (hyperglycaemia) and sometimes with encephalitis.
Low c.s.f. glucose: The c.s.f. glucose concentration is reduced
in most forms of meningitis, except viral meningitis. In
pyogenic bacterial meningitis it is markedly reduced and may
even be undetectable.
 Measurement of c.s.f. total protein and
globulin test
• Use the supernatant fluid from centrifuged c.s.f. or uncentrifuged c.s.f. when the sample
appears clear.
• Total protein can be measured in c.s.f. using a colorimetric technique or a visual comparative
technique.
• Pandy’s test is a screening test which detects rises in c.s.f. globulin. It is of value when it is not
possible to measure c.s.f. total protein.
Normal c.s.f. protein: Total c.s.f. 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 c.s.f., insufficient to give a positive Pandy’s test.
Increased c.s.f. total protein with positive Pandy’s test: Occurs in all forms of meningitis, in
amoebic and trypanosomiasis meningoencephalitis, cerebral malaria, brain tumours, cerebral
injury, spinal cord compression, poliomyelitis, the Guillain-Barré syndrome (often the only
abnormality), and polyneuritis.
Increases in c.s.f. protein also occur in diseases which cause changes in plasma proteins such as
myelomatosis. When the total protein exceeds 2.0 g/l (200 mg%), the fibrinogen level is usually
increased sufficiently to cause the c.s.f. to clot. This may occur in severe pyogenic meningitis,
spinal block, or following haemorrhage.
Note: In diseases of the nervous system such as multiple sclerosis, neurosyphilis, and some connective tissue disorders it is possible to find a positive Pandy’s
test for gobulin with only a slight rise or even normal total protein.
Ziehl-Neelsen smear when tuberculous
meningitis is suspected
• Examine a Ziehl-Neelsen stained c.s.f. smear for acid fast bacilli (AFB)
when tuberculous meningitis is clinically suspected or the c.s.f. contains
lymphocytes and the glucose concentration is low and the protein raised.
AFB, however, are difficult to detect in c.s.f. The following technique
increases the chances of finding the bacteria:
1. Centrifuge the c.s.f. at high speed for 20–30 minutes. Remove the
supernatant fluid and 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 preparation with methanol and stain by the Ziehl-Neelsen
technique .
3. Examine the smear first with 40 objective to see the distribution of
material and then with the 100 objective to detect the AFB. Examine the
entire preparation.
India ink preparation 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 preparation or a wet preparation by dark-field microscopy for
encapsulated yeasts.
1. Centrifuge the c.s.f. for 5–10 minutes. Remove the supernatant fluid and mix the
sediment.
2. Transfer a drop of the sediment to a slide, cover with a cover glass and examine by
dark-field microscopy or add a drop of India ink (Pelikan black drawing ink is
suitable*), mix and cover with a cover glass. *When ink is not available, use nigrosin
200 g/l (20% w/v) solution.
Note: Do not make the preparation too thick otherwise the cells and capsules will not be
seen. 3 Examine the preparation using the 40 objective. Look for oval or round cells, some
showing budding, irregular in size, measuring 2–10 µm in diameter and surrounded by a
large unstained capsule. Very occasionally capsules are absent.
Important: When encapsulated yeasts are detected in c.s.f., a presumptive diagnosis of
cryptococcal meningitis can be made.
Wet preparation and Giemsa smear
when trypanosomiasis meningoencephalitis is suspected Fresh c.s.f. is
required to detect trypanosomes. About 15 minutes after the fluid is
withdrawn, the trypanosomes begin to lose their motility and are rapidly
lyzed. The trypanosomes are usually few and therefore a careful search
of a wet preparation is required to detect the motile flagellates.
1. Centrifuge the c.s.f. at about 1000 g for 10 minutes. Remove the
supernatant fluid and mix the sediment.
2. Transfer a drop of sediment to a slide and cover with a cover glass.
Examine for motile trypanosomes using the 40 objective with the
condenser iris closed sufficiently to give good contrast. Alternatively
examine the preparation by darkfield microscopy
Double centrifugation technique
• When a microhaematocrit centrifuge is available, a more sensitive
method of detecting trypanosomes in c.s.f. is to use a double
centrifugation technique.
• This involves transferring the sediment from centrifuged c.s.f. to a
capillary tube, and centrifuging it for a further 1 minute in a
microhaematocrit centrifuge.
• The capillary tube is mounted on a slide and the preparation examined
for motile trypanosomes
Note: African trypanosomiasis and the investigations which are used to
diagnose late stage disease including testing for IgM in c.s.f.
Giemsa smear to detect morula cells
• When no trypanosomes are seen in the wet preparation, remove the
cover glass and allow the preparation to air-dry. Fix the smear with
methanol and stain it using Giemsa technique or Field’s rapid stain as
used for thin films.
• Examine the preparation for morula cells (IgM producing cells) using
the 40 objective. The cells are easily recognized.
• They are larger than lymphocytes with a dark mauve staining nucleus
and characteristic vacuoles in their cytoplasm .
• Finding morula cells in the c.s.f. of a person with African
trypanosomiasis, indicates central nervous system involvement.
Wet preparation to detect amoebae
• Examine a wet preparation for motile amoebae when primary amoebic
meningoencephalitis is clinically suspected (rare condition caused by N. fowleri)
or the c.s.f. contains pus cells with raised protein and low glucose, but no bacteria
are seen in the Gram smear. Red cells may also be present.
1. Transfer a drop of uncentrifuged purulent c.s.f. or a drop of sediment from a
centrifuged specimen to a slide and cover with a cover glass.
2. Examine the preparation using the 10 and 40 objectives, with the condenser
closed sufficiently to give good contrast. Look for small, clear, motile,
elongated forms among the pus cells. Use the 40 objective to identify the
amoebae (even if not first seen using the 10 objective, always examine the
preparation with the 40 objective).
3. The amoebae often contain vacuoles but not red cells. Further information on
amoebic meningoencephalitis.
Important: When amoebae are seen in the c.s.f., immediately notify the medical
officer attending the patient. Amoebic meningocephalitis is a rapidly fatal condition.
Examining and reporting the cultures
Chocolate agar and blood agar cultures
• Look especially for colonies that could be: Neisseria meningitidis (growing on chocolate agar and blood agar,
oxidase positive.
• Streptococcus pneumoniae (sensitive to optochin)
• Haemophilus influenzae (growing only on chocolate agar)
• Cryptococcus neoformans (Gram stain the colonies)
MacConkey agar culture
• Look especially for colonies that could be: Escherichia coli or other coliform, Streptococcus agalactiae,
Listeria monocytogenes.
• Other bacteria that cause neonatal meningitis .
Streptococcus suis identification:
S. suis is a non-haemolytic Gram positive coccus, belonging to Lancefield Group D.
It is CAMP negative, hydrolyzes aesculin and is able to grow on bile agar.
Antimicrobial susceptibility testing Test isolates of S. pneumoniae for susceptibility to chloramphenicol and
penicillin (use 1µg oxacillin disc).
Test H. influenzae for beta-lactamase production and susceptibility to chloramphenicol (using chocolate agar).
Perform susceptibility testing on Gram negative rods as indicated.
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