Essential microbiology
Suneeta Kochhar, Matthew Strutt, and John Philpott-Howard guide you through the key principles of
clinical microbiology
Microbiological investigations are crucial to
optimally manage infections and infectious
diseases. Depending on the results, medical
staff can give the appropriate treatment to
target the infective organism and minimise
the spread of resistant organisms. Staff may
also need to initiate public health and infec-
tion control measures. Here we provide a
brief overview of the techniques available in
microbiology laboratories and give exam-
ples of their application to different types of
specimen.
Specimen collection
You must provide full clinical information on
the request form for the laboratory to process
the specimen correctly. Microbiologists may
process the same specimen type differently in
the laboratory depending on the information
accompanying the sample. Ideally doctors
should not give antibiotics before specimens
are taken. However, empirical treatment may
be indicated if the patient is seriously ill and
waiting to collect specimens would lead to an
unacceptable delay. For example, patients
with suspected meningitis outside of hospital
should be treated with benzylpenicillin.
Take great care when collecting specimens
to minimise contamination with environmen-
tal organisms. You should put samples in leak
proof containers and enclose them in a plastic
bag during transport to the laboratory. You
should also note any particular infection haz-
ard such as blood borne viruses on the
request form, for example HIV, hepatitis B
and hepatitis C. If you have any doubt about
which investigations are required then discuss
the case with the local medical microbiologist.
Principles of investigation
Direct investigations
Direct macroscopic examination of the
specimen may be needed. For example, you
may see blood in sputum or cerebrospinal
fluid may appear cloudy.
Microscopy
Microscopy may involve the use of special
stains to identify organisms—for instance,
Ziehl-Nielsen stain may be used to identify
mycobacteria and Gram staining may be
used to classify bacteria according to the
composition of their cell walls. Gram posi-
tive bacteria include Staphylococcus aureus
and Streptococcus pyogenes. Gram negative
bacteria include Neisseria meningitidis and
Escherichia coli. Other techniques may
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involve antibodies directed against a
pathogen labelled with a fluorescent
marker. This technique may be used to
identify respiratory syncytial virus.
Culture
Microbiologists may culture organisms in
many liquid or solid media depending on
the type of specimen and the likely causative
organisms. Direct culture of blood confirms
the presence of bacteraemia. If an organism
is successfully cultured, the type and dura-
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tion of treatment may be altered after identi-
fication of the organism and antibiotic sensi-
tivity testing. Organisms are identified on the
basis of morphology, Gram stain, and bio-
chemical tests, such as coagulase and oxi-
dase. Coagulase is an enzyme which converts
fibrinogen in plasma to fibrin, thus produc-
ing clumping when coagulase positive
staphylococci are mixed with plasma. For
instance, Staphylococcus aureus has a “bunch
of grapes” morphology on Gram stain and is
coagulase positive and Escherichia coli is rod
shaped and Gram negative.
Most laboratories now use automated sys-
tems for culture of organisms. If endocarditis
is suspected, you should take three sets of
blood cultures to maximise organism yield.
In patients with indwelling venous catheters
you should take blood for culture both via
any lines present and from a peripheral vein.
Serology
Serology is useful in diagnosing viral infec-
tions and infections with difficult to culture
organisms. Other uses are for screening
before vaccination and antenatal screening.
Detection of antigens or antibodies in
serum may support the presence of infec-
tion. Enzyme linked immunosorbent assay
(ELISA) is the most commonly used tech-
nique, and increasing automation has
allowed many samples to be processed rap-
idly to identify different infectious agents.
The combination of various antibody and
antigen assays allows distinction between
acute and chronic hepatitis B. For example, if
the patient is a carrier there will be persist-
ence of the hepatitis B surface antigen for
more than six months and the “e antigen”
would be present up to two months after the
acute illness, suggesting high infectivity. For
many infections detection of IgM is indicative
of an acute infection. However, specific IgM
assays are not available for all infections and
in these cases paired acute and convalescent
(when the patient has recovered from the
acute phase of illness) sera should be tested; a
significant rise in antibody titres is diagnostic.
Molecular
Molecular techniques allow minute quanti-
ties of genetic material to be detected by
replication or amplification. For instance,
HIV viral load can be measured using a
polymerase chain reaction to monitor
response to HIV antiretroviral therapy.
Microbiologists can apply molecular tech-
niques to many types of specimen such as
blood, cerebrospinal fluid and sputum.
These techniques are of increasing impor-
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Some infections that may be
diagnosed by serology
Syphilis, Lyme disease, toxoplasmosis,
Weil’s disease, mycoplasma, legionnaires’
disease, rickettsia, measles, mumps, rubella,
viral hepatitis, HIV, varizella zoster virus,
parvovirus B19, and human T cell
leukaemia/lymphoma virus
tance in diagnosing meningitis, when
prompt antimicrobial treatment has
reduced the yield of organisms by conven-
tional culture. Molecular methods are also
available for typing or distinguishing
between organism subtypes to assess
whether transmission of infection has
occurred between individuals.
Different specimen types
Sputum
Culturing sputum is useful in the diagnosis
of respiratory tract infections. However
sputa are often contaminated with upper
respiratory tract flora, and results can be
difficult to interpret. Samples obtained by
bronchoscopic approaches such as bron-
choalveolar lavage—in which a known vol-
ume of buffered saline is added into the
distal airway and then aspirated—are better
at predicting the infective organism. How-
ever, these techniques are both invasive and
expensive and are usually reserved for
severe or non-responsive infections.
Other investigations that may be useful
include immunofluoresence of sputum for
Pneumocystis jiroveci. You may want to try to
detect a urinary antigen for Legionella pneu-
mophila or do a high resolution computed
tomography scan for Aspergillus lung disease.
When you suspect tuberculosis, you should
notify the laboratory so that Ziehl-Nielsen
staining can be done.
Cerebrospinal fluid
Examination of cerebrospinal fluid is critical
for diagnosing meningitis. However, it is vital
that investigations do not delay treatment and
that doctors give antibiotics and antivirals as
soon as possible. When cerebrospinal fluid is
taken, cell counts and Gram stains in conjunc-
tion with protein and glucose concentrations
may give an immediate diagnosis. In addition,
you may take throat swabs for bacterial cul-
ture, and stool samples for virus isolation, as
meningitis may be secondary to infection at
these sites. If you strongly suspect infection
and results are negative, you can send blood
and cerebrospinal fluid for nucleic acid ampli-
fication (where genetic material is copied to
enable detection). You may also want to send
samples for cryptococcal antigen latex agglu-
tination and toxoplasma polymerase chain
reaction if the patient is immunocompro-
mised, as this group of patients is more sus-
ceptible to such infections.
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Urine
To reduce the risk of contamination by
perineal organisms, mid-stream urine speci-
mens are needed for microbiological investi-
gation. Microscopy is important for
detecting pus cells and bacteria. In asympto-
matic, non-catheterised patients, bacteriuria
may be defined as more than 105 organisms
per millilitre or more than 104 organisms per
millilitre in the presence of symptoms, such
as increased urinary frequency, dysuria,
urgency, and fever. Infection is usually
caused by bacteria from the patient’s own
bowel flora with the most common causative
organism being Escherichia coli. Most Gram
negative organisms reduce nitrates to nitrites
and this forms the basis of a dipstick test that
you can perform at the bedside.
Stool
Salmonella sp and Camplyobacter jejuni are
the commonest causes of gastroenteritis
associated with contaminated food in the
United Kingdom. Clostridium difficile toxin
may be found in diarrhoea from hospitals’
tissue culture techniques. Electron
microscopy of the stool sample or an
enzyme linked immunosorbent assay
(ELISA) can identify rotaviruses and small
round structured viruses. Light microscopy
is done for ova cysts and parasites—for
example, the protozoan Giardia lamblia
causing giardiasis and Entamoeba histolytica
causing amoebiasis. An accurate travel and
food history is important to ensure that
specimens are investigated for all likely
organisms including those acquired abroad,
such as Vibrio cholerae, which causes cholera.
Swabs, pus, and tissue samples
You can swab wounds if you suspect infection.
However, taking a sample of pus or liquid is
preferable if possible. Wound sepsis is often
caused by Staphylococcus aureus and less com-
monly by Streptococcus pyogenes, the source of
infection usually being the patient’s own skin
or nasal flora. Wound infections after gas-
trointestinal surgery may be caused by anaer-
obes and coliforms from the gastrointestinal
tract. Infections may be iatrogenic—for exam-
ple, after inserting intravenous cannulas and
central venous lines as well as prosthetic
devices. Swabs of the skin may be useful in
making a diagnosis of cellulitis and impetigo,
since these infections are usually due to Strep-
tococcus pyogenes. A throat swab for group A
Streptococcus is usually diagnostic, you would
do this in patients presenting with sore throat,
dysphagia, and cervical lymphadenopathy.
Gram staining of swabs from the genital tract,
rectum, and throat may allow a presumptive
diagnosis of gonorrhoea to be made; how-
ever, confirmation should be got by culture.
Different genital infections need swabs from
different sites—for example, high vaginal
swabs for Candida albicans and bacterial vagi-
nosis, as opposed to cervical and urethral
swabs for Chlamydia trachomatis.
Molecular tests available
Blood—HIV, hepatitis C, Epstein-Barr virus,
cytomegalovirus, Meningococcus
Sputum—Tuberculosis, pneumocystis,
Aspergillus
Cerebrospinal fluid—Meningococcus, herpes
simplex virus, enterovirus, Toxoplasma
Genital tract—Chlamydia, Neisseria
gonorrhoeae, herpes simplex virus
Other specimens
Doctors may aspirate pleural effusions and
send them for microbiological investigation.
The exudate may be due to bacterial pneu-
monia and, less commonly, tuberculosis in
the United Kingdom. However, it is impor-
tant to remember that tuberculosis may be
more prevalent in other parts of the world.
Ascites is the presence of fluid in the peri-
toneal cavity, and you should do diagnostic
aspiration. Gram stain and culture are helpful
if peritonitis is suspected, and cytology with
biochemistry is useful to determine if there is
peritoneal seeding due to malignancy.
In renal patients with a permanent
catheter leading into the peritoneum for
continuous ambulatory peritoneal dialysis,
peritonitis is a problem, and you should send
cloudy fluid from the bags for microscopy
and culture.
For suspected septic arthritis, aspiration of
the joint space under ultrasound guidance
may be diagnostic. Septic arthritis should be
suspected if there are signs of inflammation
that have appeared acutely or if the patient
appears ill. It is most often caused by Gram
positive organisms, commonly staphylo-
cocci. Septic arthritis is relatively common in
children and should be excluded in those
presenting with a painful joint.
In addition to fluids, you may send skin
scrapings and nail clippings to the laboratory
for microscopy and fungal culture if microbi-
ological investigation is helpful in determin-
ing a diagnosis.
Microbiological investigations are an
essential part of the management of infec-
tious diseases, but to use them effectively it is
important to understand a little of the vari-
ous techniques and procedures available in
the microbiology laboratory.
Suneeta Kochhar final year medical student, Guy’s,
King’s, and St Thomas’s School of Medicine, London
suneeta.kochhar@kcl.ac.uk
Matthew Strutt specialist registrar in medical
microbiology, King’s College Hospital, London
John Philpott-Howard senior lecturer, King’s College
Hospital, London
Further reading
Shanson DC. Microbiology in clinical practice.
3rd ed. London: Butterworth-Heinemann,
1999
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