Examination of effusions

DR.DUNYA JAWAD .
An effusion is fluid which collects in a body cavity or 
joint. Fluid which collects due to an inflammatory
process is referred to as an exudate and that which
forms due to a non-inflammatory condition is
referred to as a transudate. When the effusion is an
exudate, it is important to investigate whether the
inflammatory process is an infective one (septic) or
caused by a non-infective process, e.g. malignancy.
When the fluid is a transudate, no further microbio-
logical testing is required.
Effusions sent to the laboratory for investigation
include
 FLUID Origin 
Synovial From a joint
Pleural From the pleural cavity
(Space between the lungs
and the inner chest wall)
Pericardial From the pericardial sac
(Membranous sac
surrounding the heart)
Ascitic (peritoneal) From the peritoneal
(abdominal) cavity
Hydrocele Usually from the sac
surrounding the testes
 pathogensPossible 
SYNOVIAL FLUID
(Synovitis and Infective arthritis)
Gram positive Gram negative
Staphylococcus aureus Neisseria gonorrhoeae
Streptococcus pyogenes Neisseria meningitidis
Streptococcus pneumoniae Haemophilus influenzae
Anaerobic streptococci Brucella species
Actinomycetes S almonella serovars
Escherichia coli
Pseudomonas aeruginosa
Proteus
Bacteroides
Also Mycobacterium tuberculosis 
PLEURAL AND PERICARDIAL FLUIDS 
(Empyema and Purulent Pericarditis)
Gram positive Gram negative
Staphylococcus aureus Haemophilus influenzae
Streptococcus pneumoniae Bacteroides
Streptococcus pyogenes Pseudomonas aeruginosa
Actinomycetes Klebsiella strains
Other enterobacteria
Also Mycobacterium tuberculosis, fungi, and viruses
especially coxsackie B virus.
ASCITIC FLUID 
(Ascites and Peritonitis)
Gram positive Gram negative
Enterococcus species Escherichia coli
Streptococcus pneumoniae Klebsiella strains
Staphylococcus aureus Other enterobacteria
Streptococcus pyogenes Pseudomonas aeruginosa
Streptococcus agalactiae Bacteroides
Viridans streptococci
Clostridium perfringens
Also Mycobacterium tuberculosis and Candida species.
HYDROCELE FLUID
Occasionally Wuchereria bancrofti microfilariae and
rarely Brugia species can be found in hydrocele fluid.
● Synovitis means inflammation of the synovial membrane
(lining of a joint capsule). It can be caused by bacteria,
rheumatic disorder, or injury. Infective synovitis is usually
secondary to bacteraemia. Patients with existing or
previous joint disorders are most at risk.
● Inflammation of a joint is called arthritis. The term
polyarthritis is used when many joints are affected.
Arthritis can be caused by bacteria (infective arthritis),
rheumatoid arthritis, gout and pseudogout, osteoarthriti
Gout a Calcium Pyrophosphate Deposition
Disease
The term pleural effusion is used to describe a non- 
purulent serous effusion which sometimes forms in
pneumonia, tuberculosis, malignant disease, or pul-
monary infarction (embolism). It may also occur with
systemic lupus erythematosus, lymphoma, rheumatoid
disease, or amoebic liver abscess. The commonest cause
of pericardial effusion in developing countries is pericar-
dial tuberculosis (often HIV-related).
Empyema is used to describe a purulent pleural
effusion when pus is found in the pleural space. It can
occur with pneumonia, tuberculosis, infection of a
haemothorax (blood in the pleural cavity), or rupture of
an abscess through the diaphragm.
Note: The transfer of fluid (transudate) into the pleural
cavity is called hydrothorax. It occurs in cardiac failure,
nephrotic syndrome, severe malnutrition, and advanced
cirrhosis. The collapse of a lung brought about by air in
the pleural space is called pneumothorax
● Causes of acute pericarditis other than infecting micro- 
organisms, include myocardial infarction, rheumatic
fever, malignant disease, systemic lupus erythematosus,
uraemia, and trauma.
● Peritonitis means inflammation of the peritoneum, which
is the serous membrane that lines the peritoneal cavity.
Ascites refers to the accumulation of fluid in the peri-
toneal cavity causing abdominal swelling.
Peritonitis can be caused by the rupture of an abdom-
inal organ, or as a complication of bacteraemia. Causes of
ascites include tuberculosis, advanced schistosomiasis,
portal hypertension, cardiac failure, and malignancy
especially of the ovary, stomach, colon, and liver. A
chylous ascites can develop as a complication of advanced
filariasis
Commensals 
The small amounts of fluid which surround the joints
and can be found in the pleural cavity, pericardial
sac, and peritoneal cavity, have no normal microbial
flora.
C OLLECTION AND TRANSPORT OF EFFUSIONS 
Collection of synovial, pleural, pericardial, peritoneal,
In a hospital with a microbiology laboratory
1 After aspiration, aseptically dispense the fluid as
follows:
– 2–3 ml into a dry, sterile, screw-cap tube or
bottle to observe for clotting.
– 9 ml into a screw-cap tube or bottle which
contains 1 ml of sterile tri-sodium citrate . Mix the fluid with the
anticoagulant.
*Tri-sodium citrate prevents clotting, especially of
exudates. The sterile citrated sample can be used to
estimate cell numbers, protein concentration, and for
microscopy and cultureor hydrocele fluid is carried out by a medical
officer
2 Label, and as soon as possible deliver the 
samples with a completed request form to the
laboratory.
In a health centre for dispatch to the
laboratory
1 After aspiration, aseptically dispense the fluid as
follows:
– 5 ml into a bottle of sterile thioglycollate
broth and mix.
– 9 ml into a screw-cap tube or bottle which
contains 1 ml of sterile tri-sodium citrate . Mix the fluid with the
anticoagulant.
– If any fluid remains, dispense into a dry,
sterile, screw-cap tube or bottle, and observe
for clotting
2 Label each container with the date and the 
patient’s name, number, and health centre.
3 Send the samples with a completed request form
to reach the microbiology laboratory within a few
hours. The inoculated thioglycollate broth should
be kept in a warm environment, but not over
37 C or in direct sunlight
LABORATORY EXAMINATION OF
EFFUSIONS Day 1
1 Describe the appearance of the specimen 
Report:
– Colour of the effusion
– Whether it contains blood
– Whether it is clotted (sample without anti-coagu-
lant)
Purulent effusion: When the specimen is pus or
markedly cloudy, examine and report a Gram
stained smear as soon as possible.
Whether it is clear, cloudy, or purulent (like pus) 
Blood-stained effusion: Culture the specimen 
and examine a Gram stained smear .
Note: When the specimen is not pus or a blood-
stained effusion, transfer about 1 ml of the
well-mixed citrated sample to a separate tube or
bottle (need not be sterile).
.
2 Examine the fluid for cells 
Estimate the number of white cells in the fluid using
the technique described for c.s.f.
Report whether the cells are mainly polymorpho-
nuclear neutrophils (pus cells) or lymphocytes.
Tuberculous effusions contain mainly lymphocytes
and often plasma cells (it is rare to find AFB in the
fluid).
Note: A transudate may contain a few cells, whereas
an exudate usually contains many cells
3 Estimate the protein 
Dilute the fluid 1 in 100 in physiological saline (0.1
ml effusion mixed with 9.9 ml saline).

Note: A transudate usually contains less than 30 g/l
(3 g/dl) of protein whereas an exudate contains
more than 30 g/l.
Note: An exudative pleural effusion containing lym-
phocytes with no organisms seen in the Gram smear
is found with tuberculosis.
to differentiate transudates from exudates
Appearance Clear, pale yellow Purulent, cloudy or blood- 
stained,
Clotting Does not clot Often clots
Cells Few cells Purulent: Many cells, mostly neutrophils
Non-purulent:
Few or many cells, mostly lymphocytes
Protein Less than 30 g/l More than 30 g/l
*When the sample is a transudate there is no need to
examine it further.
4 Culture the specimen 
Culture the fluid when it contains more than a few
white cells and more than 30 g/l of protein, or when
it appears blood-stained
Centrifuge the citrated sample in a sterile tube at
high speed for about 20 minutes to sediment the
bacteria. Remove the supernatant fluid (do not
discard) and resuspend the sediment. Culture the
sediment as follows
Chocolate agar, blood agar and MacConkey 
agar
● Inoculate the sediment on chocolate (heated
blood) agar, blood agar, and MacConkey agar

● Incubate the chocolate agar plate in a carbon

dioxide enriched atmosphere at 35–37 C for up
to 48 hours , checking for
growth after overnight incubation.
Incubate the blood agar plate and MacConkey
agar plate aerobically at 35–37 C for up to 72
hours, examining for growth after overnight
incubation.
ADDITIONAL 
Culture of specimen when tuberculosis is
suspected
Isolation, identification, and sensitivity testing of
M. tuberculosis and other mycobacteria require the
facilities of a Tuberculosis Reference Laboratory.
5 Examine the specimen microscopically
Gram smear
Make a thin evenly spread smear of a purulent
effusion or sediment from a centrifuged non-
purulent sample (use the citrated specimen). When
 fix the smear with methanol for 2 minutes, and 
stain it by the Gram technique ).
Examine the smear for pus cells and bacteria using
the 40 and 100 objectives.
Look especially for:
● Gram positive cocci that could be S. aureus.
● Gram positive streptococci that could be
S. pyogenes, or possibly enterococci.
● Gram positive diplococci or short chains that
could be pneumococci.
● Gram negative rods that could be enterobacteria,
Pseudomonas, or H. influenzae especially if the
rods are pleomorphic.
● Gram negative intracellular diplococci that could
be gonococci when the fluid is from a joint.
● Gram positive branching threads that could be
actinomycetes
Ziehl-Neelsen smear 
Make a smear on a slide using several drops of
sediment from the centrifuged fluid. Fix the dried
smear and stain by the Ziehl-Neelsen
. AFB are usually few
and therefore a careful search of the smear is
required. Frequently, no AFB can be found.
The appearance of M. tuberculosis in a Ziehl-
Neelsen stained smear
Fluorochrome smear 
Tubercle bacilli in effusions can rapidly be detected in a fluo-
rochrome stained smear examined by fluorescence
microscopy. The fluorescing rods can be detected using the
objective. The auramine staining
Note: The commonest cause of pericardial effusion in devel-
oping countries is pericardial tuberculosis (PCTB) often
linked to HIV infection.
ADDITIONAL 
Wet preparation to detect crystals when
gout or pseudogout is suspected (usually in
men)
When the fluid is from a joint, transfer a drop of the
sediment from the centrifuged fluid (citrated
sample) to a slide, and cover with a cover glass.
Examine the preparation using the 10 and 40
objectives with the condenser iris closed sufficiently
to give maximum contrast. Look for colourless,
extracellular and intracellular (inside white cells)
crystals:
– Monosodium urate crystals are needle-like in 
form and measure 8–10 m in length. They can
be found in effusions from patients with gout.
– Calcium pyrophosphate crystals measure up to
25 m in length, are rod-shaped, and may have 
a line running through them. They can be found
in effusions from patients with pseudogout.
Gout: High serum urate levels are usually found in patients
with gout. Normal levels are found in patients with pseudo-
gout.
Cytology smear when malignancy is
suspected
Make two thin smears of effusion sediment and
while still wet, fix the smears in a container of 95%
v/v ethanol for 20 minutes. Send the smears to a
Cytology Laboratory for special staining and exam-
ination for malignant cells
Day 2 and Onwards
6 Examine and report the cultures 
Chocolate agar, blood agar, and MacConkey
agar cultures
Look especially for colonies that could be:
Staphylococcus aureus,
Streptococcus pyogenes,
Streptococcus pneumoniae,
Haemophilus influenzae,
Enterobacteria,
Pseudomonas aeruginosa,
s Neisseria species,
Diagnostic Methods Culture 1. Specimens of choice: - CSF - 
sputum - joint fluid - pleural fluid - middle ear aspirates 2. Media:
- Chocolate Agar Plate - Blood Culture 3. Conditions: - 35-37°C,
- ~ 5% CO 2, 18-24 hrs 4. Colony Morphology:"water droplets"

Diagnostic Methods (Cont’d) Gram Stain 1. Pink, pleomorphic, 

G- Biochemical Reactions 1. Oxidase + 2. Catalase +

Diagnostic Methods (Cont’d) Growth Factor Requirements 

1.X/V strips 2. Quad Plate H. influenzae will only grow H.
influenzae will only grow around the disk containing in the
quadrant containing Hemin (X) and NAD (V). Hemin + NAD &
quadrant containing 5% horse blood, but will not hemolyze it.

Diagnostic Methods (Cont’d) Satellitism on BAP (Streaked with

known Staph. Aureus) Serological Typing (Slide Agglutination)
- 