An introduction to diagnostic

microbiology
Dr Patrick Kimmitt
HCPC registered Clinical
Microbiologist
1

Aims of this session

To give an overview of the principles and
practice of diagnostic microbiology
To contrast investigations of the 4 general
groups of pathogens
To consider the reasons why so many
different types of test are used
To use the respiratory tract to illustrate
different approaches to the investigation of
pathogens
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Recap
I am assuming you have read the
documents provided?
You should have a basic understanding of
the use of bacterial culture media, different
types of media and why different types are
needed
You should also understand approaches
to the diagnosis of viral pathogens and
why and how they are used
3

Some extra help

Check out my YouTube channel DrKimmitt
for podcasts to support your learning on
this module
These podcasts + more can be found at
www.drkimmitt.co.uk
Please use the module site discussion
board

We are making some assumptions

about you
That you know that there are 4 groups of human
pathogens!
That you know their biological characteristics!
That you know the important diseases that they
cause!
That you know the symptoms of these diseases
are due to the interaction between these
pathogens and our immune system!

Some general principles

The Standard Operating Procedures
employed by many laboratories for the
investigation of infectious diseases are
well established.
They are based on accumulated
experience about which pathogens are
most likely to cause a patients symptoms
and the predictable properties of these
pathogens
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Normal flora
Some parts of our bodies are sterile and
so the presence of any microorganism
may be significant
Other parts have a normal flora that may
make detection of a pathogen more
difficult
The site of infection may therefore
influence the laboratory approach
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Types of microbiological
investigations
Traditionally: M, C + S
Can try to see the pathogen microscopy
Can try to grow the pathogen isolation or
culture
Can investigate whether or not an isolated
pathogen is susceptible (sensitive) to an
antibiotic
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Bacteria
Can they be seen
using a microscope?
Can they be easily
grown in vitro?
Can sensitivity tests
be performed?

Yes, if stained light

microscopy
Yes, using solid agar
media
Yes, widely tested

Viruses
Can they be seen
microscopically?
Can they be easily
grown in vitro?
Can sensitivity tests
be performed?

Only with electron

microscopy
Only by using cell
culture
Not using typical
methods

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Fungi
Can they be seen
microscopically?
Can they be grown in
vitro?
Can sensitivity tests
be performed?

Yes, they are larger

than bacteria
Yes, on different solid
agar media
Yes, in a similar way
to testing bacteria

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Parasites
Can they be seen
with a microscope?
Can they be grown in
vitro?
Can sensitivity tests
be performed?

Yes, staining for

protozoa, not worms
Possible, but rarely
attempted
Rarely attempted.

12

Culture media
Please see notes in Blackboard and
YouTube podcasts

13

Immunological tests
Serology /serologic tests
Based on antibody-antigen reactions
Early tests were developed to detect the
presence (or absence) of antibodies in a
patients peripheral blood
Later tests can now also detect the presence of
antigens associated with particular pathogens
There are a whole range of these and they may
be available as commercial kits and be highly
automated
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Types of serological tests

Complement fixation test (CFTs): of great
historic importance but less so today
Agglutination tests: recognition of Ag by Ab
results in a visible clumping of the complex
Latex agglutination + Haemagglutination: by
attaching the Ag to latex particles or RBCs,
agglutination is easier to detect
Precipitation tests: Ab and Ag allowed to diffuse
towards each other in a gel; positive reaction
results in visible lines of precipitation
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Antibody vs antigen screening

You must know the difference between an
antibody and an antigen
Antibody screening was introduced first
but ultimately is of little value to patient
management in most cases why?
Detection of pathogen-specific antigen
indicates that a pathogen is present in a
patient sample
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Other serological tests

Enzyme-Linked Immunosorbent Assay
An example of a modern test that is
widely used:
Can detect antigen or antibody, can
distinguish different antibody classes e.g.
IgM or IgG (why might this be important?)
Can be automated
High specificity + sensitivity
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Other serological tests

Immunofluorescence
Uses a pathogen specific antibody to
detect the presence of antigen on the
surface of the pathogen
The antibody is labelled with a fluorescent
molecule at its non-binding end
Antigen-antibody complexes are detected
by fluorescence microscopy
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Clinical specimens
Successful microbiological investigations
rely heavily on the correct specimen being
taken from the patient
Must be transported rapidly to the
laboratory in a sterile container
Must be taken from the site at correct time
Some specimens are easier to collect than
others
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Sampling sites
Sites that should be
sterile
Blood
Cerebrospinal fluid
Tissues
Lower respiratory
tract
Bladder

Sites with normal flora

Mouth + nose
Upper respiratory
tract
Gastrointestinal tract
(except stomach)
Female genital tract
Urethra
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Specimens

Gastrointestinal tract
Urinary tract
L. respiratory tract
Skin
U. respiratory tract
Urethra
Cerebrospinal fluid
Tissues
Cardiovascular
Abscess

S*** a.k.a. faeces or stool

Midstream urine
Sputum
Skin swab
Swabs
Urethral swab
Lumbar puncture: CSF
Biopsy, aspirate
Blood
Pus
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Processing of specimens
On arrival, specimen is processed by type, e.g.
based on whether it is urine, faeces etc or on
what type of swab it is.
Processing really means deciding on the
appropriate tests to carry out
Microbiologists will know the type of pathogen,
or normal flora likely to be present
Also need some clinical information to help with
decisions
22

Likely pathogens
Early decision needs to be taken on the
likely pathogen
Is it likely to be a bacterium, fungus, virus
or parasite?
Is most likely to be a bacterium or virus
Depending on the decision, in UK,
specimen will be sent to Virology lab. or
Bacteriology lab. (will cover fungi +
parasites)
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Benches
Depending on type of investigation
selected, specimen is assigned to a
particular bench e.g. urine bench
There, the appropriate techniques are
performed by biomedical scientists who
will follow appropriate SOPs
They should also receive appropriate
clinical information
24

Typical benches/areas

Urine
Faeces
Respiratory for swabs
Wounds
Blood cultures
MRSA
Tuberculosis
25

Upper respiratory tract

Upper respiratory
tract
Mainly swabs
Mainly bacterial
pathogens
Some fungi
Extensive normal
flora

26

Lower respiratory tract

Lower respiratory
tract
Pneumonia or
Tuberculosis
High risk sputum
Must use a
specialised lab and
safety cabinet for
Category 3
containment
27

Some other clinical specimens

28

Swabs
Very widely used and
come in a variety of
types

29

Faeces (Stool)
The stool sample is
often improperly
formed

30

Pus

31

Cerebrospinal fluid by lumbar puncture

32

Blood cultures

34

Automation
A few years ago labs who could afford it
started using machines such as the VITEK
II system
This is semi-automated and can perform a
full ID and sensitivity test in 4 hours
Previously this took 24 hours or more

35

Automation
Companies such as Kiestra are
introducing fully automated microbiology
labs
Systems will inoculate, incubate and read
plates
MALDI-TOF is being used for rapid
identification of pathogens
A concern for the existing workforce!
36

Molecular testing
Detection of nucleic acid of pathogens
DNA or RNA
Polymerase Chain Reaction including
Real-time PCR and Reverse transcriptase
PCR
There are a number of other molecular
technologies also in use e.g. NASBA,
Reverse Hybridisation, SDA etc...
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Next generation sequencing

For labs who can afford it new sequencing
platforms can sequence the genome of a
microorganism within a few hours
In the not too distant future this may
become part of the routine work of the
Biomedical Scientist
Sequence the genome, ID, susceptibility
and epidemiological information available
in real-time
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Whatever the techniques, the end

results should be the same!
A report on presence or absence of a
pathogen/pathogens
The identification of the pathogen(s)
Some guidance given to clinician on
appropriate antibiotic to be used if
treatment is needed

39

Tuberculosis
Chronic respiratory infection (+ sometimes
other sites) where the causative organism
resides in the lungs, withstands
phagocytosis and causes fibrosis and
necrosis of lung tissue
Caused by Mycobacterium tuberculosis
Has a thick waxy cell wall containing
mycolic acids which resists phagocytosis

TB lab diagnosis
Microscopy is very useful
for diagnosis of TB
Acid/Alcohol Fast Stain
(Acid fast bacillus)
Stain with carbol fuchin
treat with acid/alcohol
Mycolic acids resist
decolourisation with acid/
alcohol so AFB appear
pink
Other bacteria lose their
colour
Must confirm with culture
Acid fast stain (ZN stain) of M. tuberculosis

TB - culture
Problem M. tuberculosis one of the
slowest growing bacteria mean
generation time 12-18 hrs (compare with
E. coli 20-30 mins)
Use specialist medium - LowensteinJensen (contains egg) and incubate for up
to 8 weeks
Use slopes not plates so the media
doesnt dry out

TB culture
Colonies are yellow and
look dusty
This plus clinical picture
is enough to confirm the
diagnosis as TB
Antibiotic susceptibility
tests performed at
specialist labs
There is a need for rapid
tests for TB e.g. PCR

Streptococcus pyogenes
Causes a number of different infections
including pharyngitis and Scarlet Fever (rare but
increasing)
Streptococcus contains many species, many
reside in the URT as part of the normal flora
others are pathogenic
Enterococcus is closely related and found in the
gut
We need to be able to distinguish between the
different species and also to differentiate
Streptococcus from Staphylococcus

Streptococcus spp
Gram positive coccus
often seen in chains
Lacks the enzyme
catalase and this is a
simple way to
differentiate Streps from
Staphs (catalase positive)
Add hydrogen peroxide
if positive see release of
oxygen (fizz)

Differentiation of Streptococcus
First stage is to look at
the type of haemolysis on
blood agar
Haemolysins are
produced which lyse red
blood cells in the agar
-haemolysis is a clear
zone around the colony
-haemolysis is
uncomplete haemolysis
and has a green tint
S. pygoenes is haemolytic

eta-haemolysis

Alpha-haemolysis

-Haemolytic Streptococcus
Rebecca Lancefield 1895-1981

This group can be further

divided on the basis of
their cell wall
polysaccharides
Known as Lancefield
grouping main groups
are A, B, C, D, F & G
Streptococcus pyogenes
is group A
Simple agglutination test
using group specific
antisera

-Haemolytic Streptococcus
Also known as the viridans group
Most are members of the URT flora but
can cause infections e.g. endocarditis
However there is one major pathogen in
this group Streptococcus pneumoniae
Causes bronchitis and pneumonia (and
meningitis)
How do we differentiate this species from
the other members of the viridans group?

Streptococcus pneumoniae
S. pneumoniae is
sensitive to a chemical
called optochin
All the other viridans
streps are resistant
This is the basis for a
simple test for S.
pneumoniae look for
green colonies and a
zone of no growth around
the disc
Optochin - 4,8,9R)-6'-Ethoxy-10,11-dihydrocinchonan-9-ol

Influenza
Causes flu which is primarily a disease of the
upper respiratory tract
In some cases it can cause pneumonia life
threatening
There are two major forms of flu
Seasonal
Pandemic (e.g. swine flu)

We need to be able to detect the presence of

influenza virus and then the type of strain
typically use a nasopharyngeal aspirate

Diagnostic tests for influenza

Diagnostic test

Time for result

Problems

Electron microscopy

2-3 h

Poor sensitivity, expensive instrument

Viral culture

3-10 days

Slow, cumbersome

Immunofluorescence

2-4 h

Insensitive, labour intensive

PCR

2-4 h

Lack of expertise/ validation

Antibody detection

2-6 weeks

Very slow

ELISA for virus antigen

2h

Uses expensive equipment

Immunochromatography

30 mins

Insensitive

Virus cell culture

Certain mammalian cells
are susceptible to certain
viruses the virus kills
the cells & produces a
specific cytopathic
effect (CPE)
This is the basis of an
important lab test for
viruses - clinical samples
are used to infect tissue
culture lines
Use a cells line that is
susceptible to the virus to
be detected
See virus induced CPEs here: http://www.microbiologybytes.com/video/virus.html

Virus cell culture

Virus cell culture

Immunofluorescence
Based on detection of
virus using specific
antibodies labelled with a
fluorescent molecule
Two methods, direct and
indirect (look up)
Fix sample onto a slide,
permeabilise the cells,
treat with labelled Ab,
allow to bind, wash off
excess and view by
fluorescence microscopy

IF of influenza infecting vero cells

Serology
Can determine the presence of infection by
demonstrating an increase in influenza
antibodies in patient serum
Two samples required one during the illness
(acute) and one 30 days later (convalescent)
Measure antibody levels in both samples using
an ELISA test
An increase in convalescent titre indicates
infection
Alternatively use a test which specifically
detected influenza IgM antibodies

Enzyme Linked Immunosorbant

Assay
Can be used for detection of influenza
antigen
Will be covered in the Immunology
module, please research this technology

Immunochromatography
A.k.a. Lateral flow test
Simple, easy to use test involving adding sample
plus reagents to a solid matrix or card
Can be used as a Point of Care test to be
performed in the lab, on the ward, in the field or
at home
Point of care testing is becoming increasingly
important and while attractive in principle there
are important implications to be considered with
this approach
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Immunochromatography

Immunochromatography
Patient sample dropped onto a membrane
Virus antigen forms an immunocomplex when it
comes into contact with antibody coated latex
particles
Capillary action moves the complex down the
membrane where it contacts a detection
antibody
Binding triggers an enzymatic detection system
an a visible colour change if the sample contains
the virus
e.g. QuickVue Influenza kit

Pneumocystis jiroveci
Formerly known as Pneumocystis carinii
this is a fungus which causes pneumonitis
(PCP) almost exclusively in patients with
HIV
It was practically unknown before the HIV
pandemic
Causes life-threatening infection in HIV
patient, not pathogenic in healthy
individuals

PCP
PCP is diagnosed by
collecting a sample of
sputum or bronchial
washing
Can perform
immunofluorescence
or staining with e.g.
Grocott silver and
observe characteristic
morphology

Aspergillus
The genus Aspergillus is a fungus which
causes respiratory infection only in
immunocompromised patients and special
cases e.g. cystic fibrosis
In the immunocompromised especially
those who have undergone transplants
can cause life-threatening infection.
Fungi have characteristic structures so
microscopy is useful in diagnosis

Aspergillus microscopy
Sample from
respiratory tract may
be bronchial washing
or biopsy
Can view direct or
stain e.g. with
lactophenol blue
Each genus has a
characteristic
structure

Aspergillus culture
Fungi are generally
more resistant to acid
than bacteria so
media with reduced
pH is used e.g.
Sabourauds agar
Incubate at lower
temperature (28C)
and for longer than for
bacteria
ID by microscopy

What you need to know...

You need to know what medical
microbiologists do and how the lab is
organised
To understand and contrast how
investigations of the four groups of
pathogens is performed with reasons
What types of samples are received and
what are the considerations before
samples are processed e.g. normal flora
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What you need to know...

Knowledge of the range of different tests
available and the reasoning behind why some
are used in SOPs but not others
An understanding of the use of culture media
including selective and enrichment media
An understanding of the applications of
immunological tests and their limitations
Knowledge of what tests are available to detect
bacterial, viral and fungal pathogens
78

Finally
What will the lab you may work in look
like?
Consider the impact of automation,
molecular technologies and point of care
testing

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