DIAGNOSTIC MICROBIOLOGY

AND LABORATORY METHODS

BY :
KARLINA HARDJAWINATA
DIAGNOSTIC MICROBIOLOGY
 The study of specimens taken from
patients suspected of having
infections.
 The end result is a report, which
should assist the clinician in reaching a
definitive diagnosis and a decision
on antimicrobial therapy.
 The clinicians should be acquainted
with the techniques behind laboratory
analysis.
 THE DIAGNOSIS OF AN
INFECTIOUS DISEASE:
 Entails a number of decisions and
actions by many people.
 The diagnostic cycle begins when the
clinician takes a microbiological
sample and ends when the clinician
receives the laboratory report and
uses the information to manage the
condition.
The steps in the diagnostic cycle area:

 Clinical request and provision of

clinical information
 Collection and transport of
appropriate specimen(s)
 Laboratory analysis
 Interpretation of the microbiology
report and use of the information
 Clinical request:
 The first stage in the diagnosis cycle
comprises the specimen and the
accompanying request form.
 The following, which influence the quality
of the specimen, should be noted:
1. The clinical condition of the patient:
2. Antibiotic therapy will alter the quality and
quantity of the organisms.
Provision of clinical information:
 The appropriate tests for each specimen
have to be selected by the microbiologist
according to clinical information given in
the accompanying request form.
 Informations are important for the
rationalization of investigation and should
be supplied with the specimen, such as:
age, main clinical condition, date of onset
of illness, recent / current antibiotic
therapy, antibiotic allergies, and history of
previous specimens.
 SPECIMENS COLLECTION
 Always collect appropriate specimens.
 Specimen should be as fresh as possible:
many organisms (e.g anaerobes) do not
survive for long in specimens at room
temperature. Others, such as coliforms and
staphylococci, may multiply at room
temperature and subsequent analysis of such
specimens will give misleading results.
 TRANSPORT SPECIMENS
 Transport specimens in an appropriate
medium, otherwise dehydration and/or
exposure of organisms to aerobic conditions
occurs, with the resultant death and reduction
in their numbers.
 The transport medium should be compatible
with the organisms that are believed to be
present in the clinical sample.
 Transport specimens in safe, robust containers
to avoid contamination.
 LABORATORY ANALYSIS OF A PUS
SPECIMEN FROM THE DENTAL ABSCESS:
 Make a smear of the specimen, Gram stain and examine by microscopy.
 Inoculate the specimen on two blood agar plates for culture under
aerobic and anaerobic conditions (the primary plates).
 Incubate the blood agar plates for 2-3 days at 37oC (for isolating
aerobes an 18-hour incubation period is adequate)
 Inspect plates for growth. Note the shape, and size of different colony
types for subculture.
 Isolate the putative pathogen(s) by subculturing on to fresh blood agar
plate(s) and incubating at 37oC for 24-48 hours.
 Harvest a pure culture of the pathogen and identify using biochemical
reactions, selective media or specific antibody reactions.
 Antibiotic sensitivity tests can be performed on the mixed growth
obtained from pus (primary antibiotic tests) or on the pure organism(s)
obtained (secondary antibiotic tests).
 INTERPRETATION OF THE
MICROBIOLOGY REPORT AND
USE OF INFORMATION

 Interpretation of most microbiology

reports may be straightforward
 The clinician should contact the
microbiologist, e.g. for guidance in
relation to antibiotic therapy and the
necessity for further sampling.
 Good collaboration between the clinician
and the microbiologist is essential to
achieve optimal therapy
 LABORATORY METHODS
 Non-cultural methods.
 Cultural methods
 Immunological methods
 NON-CULTURAL METHODS
These are many and varied, and
include:
- microscopic methods (light
microscopy, electron microscopy)
- detection of microbes by probing
into for their genes
 CULTURAL METHODS
Classic methods of diagnosis, in which:
- solid or liquid media are used for
bacterial and fungal growth
- Cultural cells derived from animals
and humans are used for viral
growth.
IMMUNOLOGICAL METHODS
These are used to:
- identify organisms
- detect antibodies in a patient’s body
fluids (e.g. serum, saliva),
especially when the organism
cannot be cultured in laboratory
media.
 MICROSCOPIC METHODS
 Light microscopy
 Bright-field or standard microscopy
 Dark-ground microscopy
 Phase-contrast microscopy
 Fluorescence microscopy
 Electron microscopy
Bright-field (standard) microscopy.
 Routinely used in diagnostic
microbiology, stained smears from
lesions are examined with the oil
immersion objective (x100) using the
x10 eye-piece, yielding a
magnification of x1000.
 Wet films are examined with a dry
objective (x40) e.g. to demonstrate
motility of bacteria
 Dark-ground microscopy:
 The specimen is illuminated obliquely
by a special condenser so that the
light rays do not enter the objective
directly.
 Instead the organisms appear bright,
as the light rays hit them, against
the dark background.
 Phase-contrast microscopy:
Although rarely employed in
diagnostic microbiology, this
technique may be used to define the
detailed structure of unstained
microbes.
 Fluorescence microscopy:
 Fluorescence techniques are widely used,
especially in immunology.
 This method employs the principle of emission of a
different wavelength of light when light of one
wavelength strikes a fluorescence object.
 Ultraviolet light normally used, and the bacteria or
cells are stained with fluorescence dyes such as
auramine; for example, to detect microbial
antigen in a specimen the latter is ‘stained’ with
specific antibodies tagged with fluorescent dyes
(immunofluorescence).
 Electron microscopy:
 In electron microscopy light waves are
replaced by a beam of electron, which
allows resolution of extremely small
organisms such as virion, e.g. 0,001
μm.
 Electron microscopy can be used in
diagnosis virology, for instance for
direct examination of specimens.
 LIGHT MICROSCOPY
AND STAINS
In light microscopy bacterial stains are used:
- to visualize bacteria clearly
- to categorize them according to staining
properties
- The most commonly used stain in diagnostic
microbiology is the Gram stain.
 GRAM STAIN TECHNIQUE
1. After heat-fixing the dry film (by passing through a
flame), flood with crystal violet for 15 seconds.
Then wash the excess.
2. Flood with Lugol’s iodine for 30 seconds (to fix the
stain), wash the excess.
3. Critical step: Decolorize with acetone or alcohol for
about 5 seconds. When no blue colour comes off
the smear, wash immediately with water.
4. Counterstain will dilute carbolfuchsin for 30
seconds (for neutral red for 2 minutes).
5. Wash with water, blot dry.
 ZIEHL-NEELSEN TECHNIQUE
 Some bacteria, such as tubercle bacilli, are difficult to
stain by the Gram method because they possess a
thick, waxy outer cell wall.
 Instead, the Ziehl-Neelsen technique is used.
 The organisms are exposed to hot, concentrated
carbolfuchsin for about 5 minutes, decolorized with
acid and alcohol (hence the term acid- and alcohol-fast
bacilli), and finally counterstained with methylenblue or
malachite green.
 The bacilli will stain red against a blue background.
 OTHER STAINS
 A number of other stains are used in
microbiology to demonstrate flagella,
capsules and granules, and for
staining bacteria in tissue sections.
 CULTURAL METHODS
 Bacteria grow well on artificial media, unlike viruses which
require live cells for growth.
 Blood agar is the most widely used bacterial culture medium.
It is an example of a non-selective medium as many
organisme can grow on it.
 When chemicals are incorporated into media to prevent the
growth of certain bacterial species and to promote the growth
of others, selective media can be developed (e.g. the addition
of bile salts help isolation of enterobacteria from a stool
sample by suppressing the growth of most gut commensals).
Some selective media used in microbiology
 BACTERIOLOGICAL MEDIA
The main constituents of bacteriological media are:
1. Water
2. Agar: a carbohydrate obtained from seaweed (as agar
melts at 90oC) and solidifies at 40oC, heat-sensitive
nutrients can be added to the agar base before the
medium solidifies)
3. Growth-enriching constituents: e.g. yeast extract, meat
extract (these contain carbohydrates, proteins, inorganic
salts and growth factors for bacterial growth)
4. Blood: defibrinated horse blood or sheep blood.
 PREPARATION OF SOLID MEDIA AND
INOCULATION PROCEDURE
When all the necessary ingredients
have been added to the molten agar,
it is dispensed, while still warm, into
plastic or glass petri dishes.
The agar will gradually cool and set at
room temperature, yielding a plate
ready for inoculation of the
specimen.
bacteria on to a solid medium is
to obtain discrete colonies of
organisms after appropriate
incubation.
Hence a standard technique
should be used.
 Solid media are more useful than
liquid media as they facilitate:
 Discrete colony formation, allowing single, pure colonies to be
picked from the primary plate for subculture on a secondary
plate. The pure growth from the secondary culture can then
be used for identification of the organism using biochemical
tests, etc.
 Observation of colonial characteristics helpful in identification
of organisms
 Quantitation of organisms as colony forming units (CFU). This
is valuable both in research and in diagnostic microbiology
(e.g. if a urine specimen yields more than 105 CFU/ml the
patient is deemed to have a urinary tract infection, a mixed
saliva sample with more than 106 CFU/ml of Streptococcus
mutans indicates high cariogenic activity).
 Liquid media

Liqud media are used in microbiology to:

 Promote growth of small numbers of bacteria

present in specimens contaminated with antibiotics.

The antibiotic is diluted in the fluid medium,
thereby promoting growth of the organism.
 Preferentially promote the growth of a specific

bacterium while suppressing other bacterial

commensals present in the sample. These are
called enrichment media (e.g. selenite F broth).
 Test the biochemical activities of bacteria or

identification purposes
Transport media: Specimens are
transported from the clinic to the
laboratory in a transport medium, which
helps to maintain the viability of the
organisms in transit.
Bacteriological transport media: A semisolid
, non-nutrient agar, such as Stuart
transport medium is widely used. It also
contains thioglycolic acid as agent, and
electrolytes.
 ATMOSPHERIC REQUIREMENTS
AND INCUBATION
Once inoculated the agar plates may be incubated:
 Aerobically: but addition of 10% carbon dioxide

enhances the growth of most human pathogens.

 Anaerobically: most bacteria, especially the oral

pathogens are strict anaerobes and grow only in

the absence of oxygen. Anaerobic condition can be
produced in a sealed jar or in large anaerobic
incubators. In either case the environmental
oxygen is replaced by nitrogen, hydrogen and
carbon dioxide.
 At body temperature: 37oC ( a few bacteria grow

well at a higher or a lower temperature, fungi

usually grow at ambient temperature).
 BACTERIAL IDENTIFICATION
When the putatives pathogen from the
clinical specimen is isolated as a pure
culture it is important to identify the
organism(s).
 BACTERIAL IDENTIFICATION
INITIALLY ENTAILS:
1. Inspection of the colonial characteristics:
size, shape, elevation (flat,
conves,umbonate) margin (entire, undulate,
filamentous), colour, smell and texture,
effect on blood ( α-, β-, or non-haemolytic)
2. Examination of microscopic morphology and
staining characteristics: a stained film of the
colony helps identification
3. Identification a growth condition: aerobic
conditions: aerobic, anaerobic, capnophilic
(I.e. grows well in carbon-dioxide excess),
growth on selective and enrichment media.
 BIOCHEMICAL TESTS
Each bacterial species has a
characteristic biochemical profile
valuable for its identification. These
include:
 Sugar fermentation and assimilation

profile. The pure culture is incubated

with specific sugars and checked for
the production of acid and gas or both.
 Enzyme profile
 Enzyme profile:
The organism is incubated with an appropriate
enzyme substrate.
If the enzyme is secreted by the organism this
will react with the substrate and cause a colour
change. In addition, some bacteria can be
identified primarily by production of a
characteristic enzyme
Thus, coagulase produced by Staphylococcus
aureus clots (or coagulates) plasma and is a
specific enzyme for this organisms.
 Commercial identification kits
 Definitive identification of an organism
requires testing for a spectrum of enzymes
as well as its ability to ferment (anaerobic
breakdown) or assimilate (aerobic
breakdown) a number of carbohydrates.
 This is facilitated by commercially
available kits, such as the API and AnIdent
systems, which incorporate a wide range
of the foregoing tests (usually 20) in a
single kit system.
 Method of identification:
 A pure culture of the test organism is
inoculated into each small well (cupule)
containing the appropriate carbohydrate or
the chemical and incubated overnight. The
resultant colour or turbidity change for each
test is then compared with a standard colour
chart (provided by the manufacturers) and
scored.
 The numerical profile thus obtained for the
organism is compared with a profile
compiled from the type cultures, and the
degree of concordance between the profiles
of the two organisms enables identification
of the test bacterium.
 Bacterial typing
Sometimes the process of identifying
an organism has to be extended
further than speciation (I.e.
identifying the bacteria beyond the
species level) decribed above, this is
called bacterial typing.
 Typing of bacteria
It is important to realize that bacteria of the same
species may have different characteristics (just
as individual members of the species Homo
sapiens vary in characteristics, such as skin
colour, stature, etc).
This is especially important when tracing the
epidemic spread of an organism either in the
community or in a hospital ward (like tracing a
criminal in a vast population)
Tracing such an organism can be performed by
strain differentiation.
 Strain differentiation using the
following typing procedures:
 Serotyping: differentiates bacteria
according to antigenic structure
 Biotyping: differentiates bacteria according
to the biochemical reactivity
 Phagetyping: differentiates bacteria on the
basis of susceptibility to a panel of known
bacteriophages (viruses that kill bacteria)
 Bacteriocin typing
 Genetic typing
Bacteriocin typing:
 Bacteriocins are potent proteins of bacteria which inhibit

the growth of other members of the same class species.

 A panel of bacteriocins can be used to test the

susceptibility of a test organism, and the profile thus

obtained used for typing.

Genetic typing:
A number of novel genetic typing methods are now
available and these produced very accurate ‘fingerprint’
of bacteria.
LABORATORY INVESTIGATIONS RELATED TO
ANTIMICROBIAL THERAPY
 Once the putative pathogen has been identified
from a specimen its antimicrobial sensitivity can
be predicted with some degree of accuracy, based
on previous experience and available data.
 Prescribing in this manner is called empirical
therapy (e.g. based on the sensitivity of
staphylococcal to flucloxacillin).
 However, it is essential to base rational therapy on
the results of laboratory antibiotic tests performed
on the isolated pathogen.
 Susceptibility of organisms to
antimicrobial agents
 In clinical microbiology a microbe is considered
sensitive (or susceptible) to an antimicrobial agent
if it is inhibited by a concentration of the drug
normally obtained in human tissues after a
standard therapeutic dose.
 The reserve is true for a resistant organism.
 Organisms are considered intermediate in
susceptibility if the inhibiting concentration of the
antimicrobial agent is slightly higher than that
obtained with a therapeutic dose.
 Laboratory testing for
antimicrobial sensitivity
The action of an antimicrobial drug
against an organism can be
measured:
 Qualitatively (disc diffusion tests)

 Quantitatively (minimum inhibitory

concentration or minimum
bactericidal concentration tests).
 A semiquantitative technique:
 This technique called the break-point
test.
 These in vitro tests indicate whether
the expected therapeutic concentration
of the drug given in standard dosage
inhibits the growth of a given
organisms in vivo.
 Laboratory results can only give an
indication of the activity of the drug in
vitro.
 The activity of the drug in vivo
depend on factors:
 The ability of the drug to reach the
site of infection
 The immune status of the host. A
strong host defence response may
give the impression of ‘successful’
drug therapy, even though the
infecting organism was ‘resistant’ to a
specific drug when laboratory tests
were used.
 DISC DIFFUSION TEST
 Is the most commonly used method of
testing the sensitivity of a microorganism
to an antimicrobial agent.
 The isolate to be tested is seeded over the
entire surface of an agar plate.
 Drug-impregnated filter paper discs are
applied.
 After overnight incubation at 37oC, zones of
growth inhibition are observed around each
disc, depending on the sensitivity of a
particular organism to a given agent.
 Antimicrobial sensitivity tests
can be divided into:
 Primary sensitivity (direct) test
 Secondary sensitivity (indirect) test
 Primary sensitivity (direct) test:
 Primary sensitivity (direct) test is carried out
by inoculating the clinical sample, say pus,
directly on to the test zone of the plate.
 The advantage of this is that the overall
sensitivity results for the organisms present in
pus will available after 24-48 hours’
incubation.
 This is particularly useful when treating
debilitated patients with acute infections such
as dentoalveolar abscesses.
 However this is a rough estimate.
 Secondary sensitivity test:
 Secondary sensitivity test performed
on a pure culture of the organism
isolated.
 The results are not available for at
least 2-4 days after sampling.
 Assessment of MIC and MBC:
Determining the minimum inhibitory
concentration (MIC) and the
minimum bactericidal concentration
(MBC) gives a quantitative
assessment of the potency of an
antibiotic.
 Method of determining MIC and

MBC:
A range of twofold dilutions of an antimicrobial agent can be
incorporated into a suitable broth in a series of tubes (tube dilution
technique).
 The broth is inoculated with a standardized suspension of the test
organism and incubated for 18 hours.
 The minimum concentration of the drug that inhibits the growth of the
test organism in the tube is recorded as the MIC, I.e. the lowest
concentration that will inhibit the visible growth in vitro.
 Subsequently, a standard inoculum from each of the tubes in which no
growth occurred may be subcultured on blood agar to determine the
minimum concentration of the drug required to kill the organism (MBC).
 The MBC is defined as the minimum
concentration of the drug that kills 99,9% of
the test organisms in the original inoculum.
 These MIC / MBC tests are not routinely
performed but are useful in patients with
serious infections where optimal antimicrobial
therapy is essential, i.e. to establish sensitivity
of streptococci isolated from blood cultures
from patients with infective endocarditis, and
of bacteria causing septicaemia in
immunosuppressed patients.
 APPROPRIATE SPECIMENS FOR ORAL
INFECTIONS:
 Sampling for pathogens within the oral environment poses many
problems due to the multitude of indigenous commensal flora that thrive
in the oral cavity.
 Many of the patogens are endogenous in origin and cause disease when
an opportunity arises (opportunistic pathogens)
 Obtaining an uncontaminated sample from sites such as the depths of
periodontal pockets where the disease activity and hence the numbers of
periodontopathogens are likely to be high is extremely difficult.
 For these reasons, judicial and appropriate sampling techniques should
be used when diagnosing oral infections.
The specimens submitted to an oral
microbiology laboratory can be
catagorized as those useful for the
management of:
 Purulent infections

 Mucosal infections

 Periodontal infections

 Caries.
 Purulent infections:
 The appropriate specimen is an
aspirated sample of pus, if possible.
 Take care to avoid needlestick
injuries when re-sheathing the
needlecap, drainage of residual pus
by incision, after aspiration sampling,
is obligatory.
 The laboratory steps in the
diagnosis of a purulent infection:
 Mucosal infections:
 A common oral mucosal infection is oral candidiasis.
 Here, the lesion is sampled with a dry swab, and a smear
taken immediately thereafter.
 When evaluating the oral carriage of yeasts (or other
organisms such as Enterobacteriaceae) then the oral rinse
should be collected.
 This entails requesting the patient to rinse the mouth for 60
seconds with 10 ml of phosphate-buffered saline and then
expectorating the rinse into a container, which is transported
to the laboratory for quantification of yeast growth (in term of
CFUs).
 Periodontal infections and
caries:
 The value of microbiological sampling for the diagnosis
of caries and periodontal diseases is limited.
 In the case of dental caries salivary counts of
lactobacilli and Streptococcus mutans could be
used, and for this purpose saliva samples should be
collected.
 The diagnosis of periodontal disease by microbiologic
means is problematic. A deep gingival smear is useful
for the diagnosis of acute necrotizing ulcerative
gingivitis, while paper point sample appear useful for
DNA analysis of periodontopathic bacteria.