DIAGNOSTIC MICROBIOLOGYDIAGNOSTIC MICROBIOLOGY AND LABORATORY METHODS AND LABORATORY METHODS

BY BY ::

KARLINA HARDJAWINATAKARLINA HARDJAWINATA

DIAGNOSTIC MICROBIOLOGYDIAGNOSTIC MICROBIOLOGY

The study of specimens taken from The study of specimens taken from patients suspected of having patients suspected of having infections.infections.

The end result is a report, which The end result is a report, which should assist the clinician in reaching should assist the clinician in reaching a definitive diagnosisa definitive diagnosis and a and a decision on decision on antimicrobial therapyantimicrobial therapy..

The clinicians should be acquainted The clinicians should be acquainted with the techniques behind with the techniques behind laboratory analysis.laboratory analysis.

THE DIAGNOSIS OF AN THE DIAGNOSIS OF AN INFECTIOUS DISEASE:INFECTIOUS DISEASE:

Entails a number of decisions and Entails a number of decisions and actions by many people.actions by many people.

The diagnostic cycle begins when the The diagnostic cycle begins when the clinician takes a microbiological clinician takes a microbiological sample and ends when the clinician sample and ends when the clinician receives the laboratory report and receives the laboratory report and uses the information to manage the uses the information to manage the condition.condition.

The steps in the diagnostic cycle area:The steps in the diagnostic cycle area:

Clinical request and provision of Clinical request and provision of clinical informationclinical information

Collection and transport of Collection and transport of appropriate specimen(s)appropriate specimen(s)

Laboratory analysisLaboratory analysis Interpretation of the microbiology Interpretation of the microbiology

report and use of the informationreport and use of the information

Clinical request:Clinical request:

The first stage in the diagnosis cycle The first stage in the diagnosis cycle comprises the specimen and the comprises the specimen and the accompanying request form.accompanying request form.

The following, which influence the The following, which influence the quality of the specimen, should be quality of the specimen, should be noted:noted:

1.1. The clinical condition of the patient:The clinical condition of the patient:2.2. Antibiotic therapy will alter the Antibiotic therapy will alter the

quality and quantity of the quality and quantity of the organisms.organisms.

Provision of clinical information:Provision of clinical information:

The appropriate tests for each specimen The appropriate tests for each specimen have to be selected by the microbiologist have to be selected by the microbiologist according to clinical information given in according to clinical information given in the accompanying request form.the accompanying request form.

Informations are important for the Informations are important for the rationalization of investigation and should rationalization of investigation and should be supplied with the specimen, such as: be supplied with the specimen, such as: age, main clinical condition, date of onset age, main clinical condition, date of onset of illness, recent / current antibiotic of illness, recent / current antibiotic therapy, antibiotic allergies, and history of therapy, antibiotic allergies, and history of previous specimens.previous specimens.

SPECIMENS COLLECTIONSPECIMENS COLLECTION Always collect appropriate specimens.Always collect appropriate specimens. Specimen should be as fresh as possible: Specimen should be as fresh as possible:

many organisms (e.g anaerobes) do not many organisms (e.g anaerobes) do not survive for long in specimens at room survive for long in specimens at room temperature. Others, such as coliforms temperature. Others, such as coliforms and staphylococci, may multiply at room and staphylococci, may multiply at room temperature and subsequent analysis of temperature and subsequent analysis of such specimens will give misleading such specimens will give misleading results.results.

TRANSPORT SPECIMENSTRANSPORT SPECIMENS

Transport specimens in an appropriate Transport specimens in an appropriate medium, otherwise dehydration and/or medium, otherwise dehydration and/or exposure of organisms to aerobic conditions exposure of organisms to aerobic conditions occurs, with the resultant death and occurs, with the resultant death and reduction in their numbers.reduction in their numbers.

The transport medium should be compatible The transport medium should be compatible with the organisms that are believed to be with the organisms that are believed to be present in the clinical sample.present in the clinical sample.

Transport specimens in safe, robust Transport specimens in safe, robust containers to avoid contamination.containers to avoid contamination.

LABORATORY ANALYSIS OF A PUS LABORATORY ANALYSIS OF A PUS SPECIMEN FROM THE DENTAL ABSCESS:SPECIMEN FROM THE DENTAL ABSCESS:

Make a smear of the specimen, Gram stain and examine by Make a smear of the specimen, Gram stain and examine by microscopy.microscopy.

Inoculate the specimen on two blood agar plates for culture Inoculate the specimen on two blood agar plates for culture under aerobic and anaerobic conditions (the primary plates).under aerobic and anaerobic conditions (the primary plates).

Incubate the blood agar plates for 2-3 days at 37Incubate the blood agar plates for 2-3 days at 37ooC (for isolating C (for isolating aerobes an 18-hour incubation period is adequate)aerobes an 18-hour incubation period is adequate)

Inspect plates for growth. Note the shape, and size of different Inspect plates for growth. Note the shape, and size of different colony types for subculture.colony types for subculture.

Isolate the putative pathogen(s) by subculturing on to fresh Isolate the putative pathogen(s) by subculturing on to fresh blood agar plate(s) and incubating at 37blood agar plate(s) and incubating at 37ooC for 24-48 hours.C for 24-48 hours.

Harvest a pure culture of the pathogen and identify using Harvest a pure culture of the pathogen and identify using biochemical reactions, selective media or specific antibody biochemical reactions, selective media or specific antibody reactions.reactions.

Antibiotic sensitivity tests can be performed on the mixed growth Antibiotic sensitivity tests can be performed on the mixed growth obtained from pus (primary antibiotic tests) or on the pure obtained from pus (primary antibiotic tests) or on the pure organism(s) obtained (secondary antibiotic tests).organism(s) obtained (secondary antibiotic tests).

INTERPRETATION OF THE INTERPRETATION OF THE MICROBIOLOGY REPORT AND MICROBIOLOGY REPORT AND

USE OF INFORMATIONUSE OF INFORMATION

Interpretation of most microbiology Interpretation of most microbiology reports may be straightforwardreports may be straightforward

The clinician should contact the The clinician should contact the microbiologist, e.g. for guidance in microbiologist, e.g. for guidance in relation to antibiotic therapy and the relation to antibiotic therapy and the necessity for further sampling.necessity for further sampling.

Good collaboration between the clinician Good collaboration between the clinician and the microbiologist is essential to and the microbiologist is essential to achieve optimal therapyachieve optimal therapy

LABORATORY METHODSLABORATORY METHODS

Non-cultural methods.Non-cultural methods. Cultural methodsCultural methods Immunological methodsImmunological methods

NON-CULTURAL METHODSNON-CULTURAL METHODS

These are many and varied, and These are many and varied, and include:include:

- microscopic methods (light - microscopic methods (light microscopy, electron microscopy)microscopy, electron microscopy)

- detection of microbes by probing - detection of microbes by probing into for their genesinto for their genes

CULTURAL METHODSCULTURAL METHODS

Classic methods of diagnosis, in which:Classic methods of diagnosis, in which:- solid or liquid media are used for solid or liquid media are used for

bacterial and fungal growthbacterial and fungal growth- Cultural cells derived from animals Cultural cells derived from animals

and humans are used for viral and humans are used for viral growth.growth.

IMMUNOLOGICAL METHODSIMMUNOLOGICAL METHODS

These are used to:These are used to:- identify organismsidentify organisms- detect antibodies in a patient’s detect antibodies in a patient’s

body fluids (e.g. serum, saliva), body fluids (e.g. serum, saliva), especially when the organism especially when the organism cannot be cultured in laboratory cannot be cultured in laboratory media.media.

MICROSCOPIC METHODSMICROSCOPIC METHODS

Light microscopyLight microscopy Bright-field or standard microscopyBright-field or standard microscopy Dark-ground microscopyDark-ground microscopy Phase-contrast microscopyPhase-contrast microscopy Fluorescence microscopyFluorescence microscopy Electron microscopyElectron microscopy

Bright-field (standard) microscopy.Bright-field (standard) microscopy.

Routinely used in diagnostic Routinely used in diagnostic microbiology, stained smears from microbiology, stained smears from lesions are examined with the oil lesions are examined with the oil immersion objective (x100) using the immersion objective (x100) using the x10 eye-piece, yielding a x10 eye-piece, yielding a magnification of x1000. magnification of x1000.

Wet films are examined with a dry Wet films are examined with a dry objective (x40) e.g. to demonstrate objective (x40) e.g. to demonstrate motility of bacteriamotility of bacteria

Dark-ground microscopy:Dark-ground microscopy:

The specimen is illuminated obliquely The specimen is illuminated obliquely by a special condenser so that the by a special condenser so that the light rays do not enter the objective light rays do not enter the objective directly.directly.

Instead the organisms appear bright, Instead the organisms appear bright, as the light rays hit them, against the as the light rays hit them, against the dark background.dark background.

Phase-contrast microscopy:Phase-contrast microscopy:

Although rarely employed in Although rarely employed in diagnostic microbiology, this diagnostic microbiology, this technique may be used to define the technique may be used to define the detailed structure of unstained detailed structure of unstained microbes.microbes.

Fluorescence microscopy:Fluorescence microscopy: Fluorescence techniques are widely used, Fluorescence techniques are widely used,

especially in immunology.especially in immunology. This method employs the principle of emission This method employs the principle of emission

of a different wavelength of light when light of of a different wavelength of light when light of one wavelength strikes a fluorescence object.one wavelength strikes a fluorescence object.

Ultraviolet light normally used, and the Ultraviolet light normally used, and the bacteria or cells are stained with fluorescence bacteria or cells are stained with fluorescence dyes such as auramine; for example, to detect dyes such as auramine; for example, to detect microbial antigen in a specimen the latter is microbial antigen in a specimen the latter is ‘stained’ with specific antibodies tagged with ‘stained’ with specific antibodies tagged with fluorescent dyes (immunofluorescence).fluorescent dyes (immunofluorescence).

Electron microscopy:Electron microscopy:

In electron microscopy light waves In electron microscopy light waves are replaced by a beam of electron, are replaced by a beam of electron, which allows resolution of extremely which allows resolution of extremely small organisms such as virion, e.g. small organisms such as virion, e.g. 0,001 0,001 μm.μm.

Electron microscopy can be used in Electron microscopy can be used in diagnosis virology, for instance for diagnosis virology, for instance for direct examination of specimens.direct examination of specimens.

LIGHT MICROSCOPY LIGHT MICROSCOPY AND STAINS AND STAINS

In light microscopy bacterial stains are In light microscopy bacterial stains are used:used:

- to visualize bacteria clearlyto visualize bacteria clearly- to categorize them according to to categorize them according to

staining propertiesstaining properties- The most commonly used stain in The most commonly used stain in

diagnostic microbiology is the Gram diagnostic microbiology is the Gram stain.stain.

GRAM STAIN TECHNIQUEGRAM STAIN TECHNIQUE

1.1. After heat-fixing the dry film (by passing After heat-fixing the dry film (by passing through a flame), flood with crystal violet for through a flame), flood with crystal violet for 15 seconds. Then wash the excess.15 seconds. Then wash the excess.

2.2. Flood with Lugol’s iodine for 30 seconds (to Flood with Lugol’s iodine for 30 seconds (to fix the stain), wash the excess.fix the stain), wash the excess.

3.3. Critical step: Decolorize with acetone or Critical step: Decolorize with acetone or alcohol for about 5 seconds. When no blue alcohol for about 5 seconds. When no blue colour comes off the smear, wash colour comes off the smear, wash immediately with water.immediately with water.

4.4. Counterstain will dilute carbolfuchsin for 30 Counterstain will dilute carbolfuchsin for 30 seconds (for neutral red for 2 minutes).seconds (for neutral red for 2 minutes).

5.5. Wash with water, blot dry.Wash with water, blot dry.

ZIEHL-NEELSEN TECHNIQUEZIEHL-NEELSEN TECHNIQUE

Some bacteria, such as tubercle bacilli, are Some bacteria, such as tubercle bacilli, are difficult to stain by the Gram method because difficult to stain by the Gram method because they possess a thick, waxy outer cell wall.they possess a thick, waxy outer cell wall.

Instead, the Ziehl-Neelsen technique is used.Instead, the Ziehl-Neelsen technique is used. The organisms are exposed to hot, The organisms are exposed to hot,

concentrated carbolfuchsin for about 5 concentrated carbolfuchsin for about 5 minutes, decolorized with acid and alcohol minutes, decolorized with acid and alcohol (hence the term acid- and alcohol-fast bacilli), (hence the term acid- and alcohol-fast bacilli), and finally counterstained with methylenblue and finally counterstained with methylenblue or malachite green.or malachite green.

The bacilli will stain red against a blue The bacilli will stain red against a blue background.background.

OTHER STAINSOTHER STAINS

A number of other stains are used in A number of other stains are used in microbiology to demonstrate flagella, microbiology to demonstrate flagella, capsules and granules, and for capsules and granules, and for staining bacteria in tissue sections.staining bacteria in tissue sections.

CULTURAL METHODSCULTURAL METHODS Bacteria grow well on artificial media, unlike Bacteria grow well on artificial media, unlike

viruses which require live cells for growth.viruses which require live cells for growth. Blood agar is the most widely used bacterial Blood agar is the most widely used bacterial

culture medium. It is an example of a non-culture medium. It is an example of a non-selective medium as many organisme can grow on selective medium as many organisme can grow on it.it.

When chemicals are incorporated into media to When chemicals are incorporated into media to prevent the growth of certain bacterial species prevent the growth of certain bacterial species and to promote the growth of others, selective and to promote the growth of others, selective media can be developed (e.g. the addition of bile media can be developed (e.g. the addition of bile salts help isolation of enterobacteria from a stool salts help isolation of enterobacteria from a stool sample by suppressing the growth of most gut sample by suppressing the growth of most gut commensals).commensals).

Some selective media used in microbiologySome selective media used in microbiology

BACTERIOLOGICAL MEDIABACTERIOLOGICAL MEDIA

The main constituents of bacteriological media The main constituents of bacteriological media are:are:

1.1. WaterWater2.2. Agar: a carbohydrate obtained from seaweed Agar: a carbohydrate obtained from seaweed

(as agar melts at 90(as agar melts at 90ooC) and solidifies at 40C) and solidifies at 40ooC, C, heat-sensitive nutrients can be added to the heat-sensitive nutrients can be added to the agar base before the medium solidifies)agar base before the medium solidifies)

3.3. Growth-enriching constituents: e.g. yeast Growth-enriching constituents: e.g. yeast extract, meat extract (these contain extract, meat extract (these contain carbohydrates, proteins, inorganic salts and carbohydrates, proteins, inorganic salts and growth factors for bacterial growth)growth factors for bacterial growth)

4.4. Blood: defibrinated horse blood or sheep blood.Blood: defibrinated horse blood or sheep blood.

PREPARATION OF SOLID MEDIA AND PREPARATION OF SOLID MEDIA AND INOCULATION PROCEDUREINOCULATION PROCEDURE

When all the necessary ingredients When all the necessary ingredients have been added to the molten agar, have been added to the molten agar, it is dispensed, while still warm, into it is dispensed, while still warm, into plastic or glass petri dishes.plastic or glass petri dishes.

The agar will gradually cool and set at The agar will gradually cool and set at room temperature, yielding a plate room temperature, yielding a plate ready for inoculation of the ready for inoculation of the specimen.specimen.

The objective of inoculating the The objective of inoculating the specimen or a culture of specimen or a culture of bacteria on to a solid medium is bacteria on to a solid medium is to obtain discrete colonies of to obtain discrete colonies of organisms after appropriate organisms after appropriate incubation.incubation.Hence a standard technique Hence a standard technique should be used.should be used.

Solid media are more useful than Solid media are more useful than liquid media as they facilitateliquid media as they facilitate::

Discrete colony formation, allowing single, pure Discrete colony formation, allowing single, pure colonies to be picked from the primary plate for colonies to be picked from the primary plate for subculture on a secondary plate. The pure growth from subculture on a secondary plate. The pure growth from the secondary culture can then be used for the secondary culture can then be used for identification of the organism using biochemical tests, identification of the organism using biochemical tests, etc.etc.

Observation of colonial characteristics helpful in Observation of colonial characteristics helpful in identification of organismsidentification of organisms

Quantitation of organisms as colony forming units Quantitation of organisms as colony forming units (CFU). This is valuable both in research and in (CFU). This is valuable both in research and in diagnostic microbiology (e.g. if a urine specimen yields diagnostic microbiology (e.g. if a urine specimen yields more than 10more than 105 5 CFU/ml the patient is deemed to have a CFU/ml the patient is deemed to have a urinary tract infection, a mixed saliva sample with urinary tract infection, a mixed saliva sample with more than 10more than 106 6 CFU/ml of CFU/ml of Streptococcus mutansStreptococcus mutans indicates high cariogenic activity).indicates high cariogenic activity).

Liquid mediaLiquid media

Liqud media are used in microbiology to:Liqud media are used in microbiology to: Promote growth of small numbers of bacteria Promote growth of small numbers of bacteria

present in specimens contaminated with present in specimens contaminated with antibiotics. The antibiotic is diluted in the fluid antibiotics. The antibiotic is diluted in the fluid medium, thereby promoting growth of the medium, thereby promoting growth of the organism.organism.

Preferentially promote the growth of a specific Preferentially promote the growth of a specific bacterium while suppressing other bacterial bacterium while suppressing other bacterial commensals present in the sample. These are commensals present in the sample. These are called enrichment media (e.g. selenite F broth).called enrichment media (e.g. selenite F broth).

Test the biochemical activities of bacteria or Test the biochemical activities of bacteria or identification purposesidentification purposes

Transport media: Specimens are Transport media: Specimens are transported from the clinic to the transported from the clinic to the laboratory in a transport medium, which laboratory in a transport medium, which helps to maintain the viability of the helps to maintain the viability of the organisms in transit.organisms in transit.

Bacteriological transport media: A Bacteriological transport media: A semisolid , non-nutrient agar, such as semisolid , non-nutrient agar, such as Stuart transport medium is widely used. Stuart transport medium is widely used. It also contains thioglycolic acid as It also contains thioglycolic acid as agent, and electrolytes.agent, and electrolytes.

ATMOSPHERIC REQUIREMENTS ATMOSPHERIC REQUIREMENTS AND INCUBATIONAND INCUBATION

Once inoculated the agar plates may be incubated:Once inoculated the agar plates may be incubated: Aerobically: but addition of 10% carbon dioxide Aerobically: but addition of 10% carbon dioxide

enhances the growth of most human pathogens.enhances the growth of most human pathogens. Anaerobically: most bacteria, especially the oral Anaerobically: most bacteria, especially the oral

pathogens are strict anaerobes and grow only in pathogens are strict anaerobes and grow only in the absence of oxygen. Anaerobic condition can the absence of oxygen. Anaerobic condition can be produced in a sealed jar or in large anaerobic be produced in a sealed jar or in large anaerobic incubators. In either case the environmental incubators. In either case the environmental oxygen is replaced by nitrogen, hydrogen and oxygen is replaced by nitrogen, hydrogen and carbon dioxide.carbon dioxide.

At body temperature: 37At body temperature: 37ooC ( a few bacteria grow C ( a few bacteria grow well at a higher or a lower temperature, fungi well at a higher or a lower temperature, fungi usually grow at ambient temperature).usually grow at ambient temperature).

BACTERIAL IDENTIFICATIONBACTERIAL IDENTIFICATION

When the putatives pathogen from the When the putatives pathogen from the clinical specimen is isolated as a clinical specimen is isolated as a pure culture it is important to identify pure culture it is important to identify the organism(s).the organism(s).

BACTERIAL IDENTIFICATION BACTERIAL IDENTIFICATION INITIALLY ENTAILS:INITIALLY ENTAILS:

1.1. Inspection of the colonial characteristics: Inspection of the colonial characteristics: size, shape, elevation (flat, size, shape, elevation (flat, conves,umbonate) margin (entire, undulate, conves,umbonate) margin (entire, undulate, filamentous), colour, smell and texture, filamentous), colour, smell and texture, effect on blood ( effect on blood ( α-, β-α-, β-, or non-haemolytic), or non-haemolytic)

2.2. Examination of microscopic morphology and Examination of microscopic morphology and staining characteristics: a stained film of the staining characteristics: a stained film of the colony helps identificationcolony helps identification

3.3. Identification a growth condition: aerobic Identification a growth condition: aerobic conditions: aerobic, anaerobic, capnophilic conditions: aerobic, anaerobic, capnophilic (I.e. grows well in carbon-dioxide excess), (I.e. grows well in carbon-dioxide excess), growth on selective and enrichment media.growth on selective and enrichment media.

BIOCHEMICAL TESTSBIOCHEMICAL TESTS

Each bacterial species has a Each bacterial species has a characteristic biochemical profile characteristic biochemical profile valuable for its identification. These valuable for its identification. These include:include:

Sugar fermentation and assimilation Sugar fermentation and assimilation profile. The pure culture is incubated profile. The pure culture is incubated with specific sugars and checked for with specific sugars and checked for the production of acid and gas or the production of acid and gas or both.both.

Enzyme profileEnzyme profile

Enzyme profile:Enzyme profile:

The organism is incubated with an appropriate The organism is incubated with an appropriate enzyme substrate. enzyme substrate.

If the enzyme is secreted by the organism this If the enzyme is secreted by the organism this will react with the substrate and cause a will react with the substrate and cause a colour change. In addition, some bacteria can colour change. In addition, some bacteria can be identified primarily by production of a be identified primarily by production of a characteristic enzyme characteristic enzyme

Thus, coagulase produced by Thus, coagulase produced by Staphylococcus Staphylococcus aureusaureus clots (or coagulates) plasma and is a clots (or coagulates) plasma and is a specific enzyme for this organisms.specific enzyme for this organisms.

Commercial identification kitsCommercial identification kits

Definitive identification of an organism Definitive identification of an organism requires testing for a spectrum of enzymes requires testing for a spectrum of enzymes as well as its ability to ferment (anaerobic as well as its ability to ferment (anaerobic breakdown) or assimilate (aerobic breakdown) or assimilate (aerobic breakdown) a number of carbohydrates.breakdown) a number of carbohydrates.

This is facilitated by commercially This is facilitated by commercially available kits, such as the API and AnIdent available kits, such as the API and AnIdent systems, which incorporate a wide range systems, which incorporate a wide range of the foregoing tests (usually 20) in a of the foregoing tests (usually 20) in a single kit system.single kit system.

Method of identification:Method of identification: A pure culture of the test organism is A pure culture of the test organism is

inoculated into each small well (cupule) inoculated into each small well (cupule) containing the appropriate carbohydrate containing the appropriate carbohydrate or the chemical and incubated overnight. or the chemical and incubated overnight. The resultant colour or turbidity change The resultant colour or turbidity change for each test is then compared with a for each test is then compared with a standard colour chart (provided by the standard colour chart (provided by the manufacturers) and scored.manufacturers) and scored.

The numerical profile thus obtained for The numerical profile thus obtained for the organism is compared with a profile the organism is compared with a profile compiled from the type cultures, and the compiled from the type cultures, and the degree of concordance between the degree of concordance between the profiles of the two organisms enables profiles of the two organisms enables identification of the test bacterium.identification of the test bacterium.

Bacterial typingBacterial typing

Sometimes the process of identifying Sometimes the process of identifying an organism has to be extended an organism has to be extended further than speciation (I.e. further than speciation (I.e. identifying the bacteria beyond the identifying the bacteria beyond the species level) decribed above, this is species level) decribed above, this is called bacterial typing.called bacterial typing.

Typing of bacteriaTyping of bacteriaIt is important to realize that bacteria of the It is important to realize that bacteria of the

same species may have different same species may have different characteristics (just as individual members of characteristics (just as individual members of the species the species Homo sapiens Homo sapiens vary in vary in characteristics, such as skin colour, stature, characteristics, such as skin colour, stature, etc). etc).

This is especially important when tracing the This is especially important when tracing the epidemic spread of an organism either in the epidemic spread of an organism either in the community or in a hospital ward (like tracing a community or in a hospital ward (like tracing a criminal in a vast population)criminal in a vast population)

Tracing such an organism can be performed by Tracing such an organism can be performed by strain differentiation.strain differentiation.

Strain differentiation using the Strain differentiation using the following typing procedures:following typing procedures:

Serotyping: differentiates bacteria Serotyping: differentiates bacteria according to antigenic structureaccording to antigenic structure

Biotyping: differentiates bacteria Biotyping: differentiates bacteria according to the biochemical reactivityaccording to the biochemical reactivity

Phagetyping: differentiates bacteria on the Phagetyping: differentiates bacteria on the basis of susceptibility to a panel of known basis of susceptibility to a panel of known bacteriophages (viruses that kill bacteria)bacteriophages (viruses that kill bacteria)

Bacteriocin typingBacteriocin typing Genetic typingGenetic typing

Bacteriocin typing:Bacteriocin typing: Bacteriocins are potent proteins of bacteria which Bacteriocins are potent proteins of bacteria which

inhibit the growth of other members of the same class inhibit the growth of other members of the same class species.species.

A panel of bacteriocins can be used to test the A panel of bacteriocins can be used to test the susceptibility of a test organism, and the profile thus susceptibility of a test organism, and the profile thus obtained used for typing.obtained used for typing.

Genetic typing: Genetic typing: A number of novel genetic typing methods are now A number of novel genetic typing methods are now

available and these produced very accurate ‘fingerprint’ available and these produced very accurate ‘fingerprint’ of bacteria.of bacteria.

LABORATORY INVESTIGATIONS RELATED TO LABORATORY INVESTIGATIONS RELATED TO ANTIMICROBIAL THERAPYANTIMICROBIAL THERAPY

Once the putative pathogen has been Once the putative pathogen has been identified from a specimen its antimicrobial identified from a specimen its antimicrobial sensitivity can be predicted with some sensitivity can be predicted with some degree of accuracy, based on previous degree of accuracy, based on previous experience and available data.experience and available data.

Prescribing in this manner is called empirical Prescribing in this manner is called empirical therapy (e.g. based on the sensitivity of therapy (e.g. based on the sensitivity of staphylococcal to flucloxacillin).staphylococcal to flucloxacillin).

However, it is essential to base rational However, it is essential to base rational therapy on the results of laboratory therapy on the results of laboratory antibiotic tests performed on the isolated antibiotic tests performed on the isolated pathogen.pathogen.

Susceptibility of organisms to Susceptibility of organisms to antimicrobial agentsantimicrobial agents

In clinical microbiology a microbe is In clinical microbiology a microbe is considered sensitive (or susceptible) to an considered sensitive (or susceptible) to an antimicrobial agent if it is inhibited by a antimicrobial agent if it is inhibited by a concentration of the drug normally concentration of the drug normally obtained in human tissues after a standard obtained in human tissues after a standard therapeutic dose.therapeutic dose.

The reserve is true for a resistant organism.The reserve is true for a resistant organism. Organisms are considered intermediate in Organisms are considered intermediate in

susceptibility if the inhibiting concentration susceptibility if the inhibiting concentration of the antimicrobial agent is slightly higher of the antimicrobial agent is slightly higher than that obtained with a therapeutic dose.than that obtained with a therapeutic dose.

Laboratory testing for Laboratory testing for antimicrobial sensitivityantimicrobial sensitivity

The action of an antimicrobial drug The action of an antimicrobial drug against an organism can be against an organism can be measured:measured:

Qualitatively (disc diffusion tests)Qualitatively (disc diffusion tests) Quantitatively (minimum inhibitory Quantitatively (minimum inhibitory

concentration or minimum concentration or minimum bactericidal concentration tests).bactericidal concentration tests).

A semiquantitative technique:A semiquantitative technique:

This technique called the break-point This technique called the break-point test.test.

These in vitro tests indicate whether These in vitro tests indicate whether the expected therapeutic the expected therapeutic concentration of the drug given in concentration of the drug given in standard dosage inhibits the growth standard dosage inhibits the growth of a given organisms in vivo.of a given organisms in vivo.

Laboratory results can only give an Laboratory results can only give an indication of the activity of the drug indication of the activity of the drug in vitro.in vitro.

The activity of the drug in vivo The activity of the drug in vivo depend on factors:depend on factors:

The ability of the drug to reach the The ability of the drug to reach the site of infectionsite of infection

The immune status of the host. A The immune status of the host. A strong host defence response may strong host defence response may give the impression of ‘successful’ give the impression of ‘successful’ drug therapy, even though the drug therapy, even though the infecting organism was ‘resistant’ to infecting organism was ‘resistant’ to a specific drug when laboratory tests a specific drug when laboratory tests were used.were used.

DISC DIFFUSION TESTDISC DIFFUSION TEST

Is the most commonly used method of Is the most commonly used method of testing the sensitivity of a microorganism testing the sensitivity of a microorganism to an antimicrobial agent.to an antimicrobial agent.

The isolate to be tested is seeded over the The isolate to be tested is seeded over the entire surface of an agar plate.entire surface of an agar plate.

Drug-impregnated filter paper discs are Drug-impregnated filter paper discs are applied.applied.

After overnight incubation at 37After overnight incubation at 37ooC, zones C, zones of growth inhibition are observed around of growth inhibition are observed around each disc, depending on the sensitivity of each disc, depending on the sensitivity of a particular organism to a given agent.a particular organism to a given agent.

Antimicrobial sensitivity tests Antimicrobial sensitivity tests can be divided into:can be divided into:

Primary sensitivity (direct) testPrimary sensitivity (direct) test Secondary sensitivity (indirect) testSecondary sensitivity (indirect) test

Primary sensitivity (direct) test:Primary sensitivity (direct) test:

Primary sensitivity (direct) test is carried out Primary sensitivity (direct) test is carried out by inoculating the clinical sample, say pus, by inoculating the clinical sample, say pus, directly on to the test zone of the plate. directly on to the test zone of the plate.

The advantage of this is that the overall The advantage of this is that the overall sensitivity results for the organisms present sensitivity results for the organisms present in pus will available after 24-48 hours’ in pus will available after 24-48 hours’ incubation.incubation.

This is particularly useful when treating This is particularly useful when treating debilitated patients with acute infections debilitated patients with acute infections such as dentoalveolar abscesses.such as dentoalveolar abscesses.

However this is a rough estimate.However this is a rough estimate.

Secondary sensitivity test:Secondary sensitivity test:

Secondary sensitivity test performed Secondary sensitivity test performed on a pure culture of the organism on a pure culture of the organism isolated.isolated.

The results are not available for at The results are not available for at least 2-4 days after sampling.least 2-4 days after sampling.

Assessment of MIC and MBC:Assessment of MIC and MBC:

Determining the minimum inhibitory Determining the minimum inhibitory concentration (MIC) and the concentration (MIC) and the minimum bactericidal concentration minimum bactericidal concentration (MBC) gives a quantitative (MBC) gives a quantitative assessment of the potency of an assessment of the potency of an antibiotic.antibiotic.

Method of determining MIC and Method of determining MIC and MBC:MBC:

A range of twofold dilutions of an antimicrobial agent A range of twofold dilutions of an antimicrobial agent can be incorporated into a suitable broth in a series of can be incorporated into a suitable broth in a series of tubes (tube dilution technique).tubes (tube dilution technique).

The broth is inoculated with a standardized suspension The broth is inoculated with a standardized suspension of the test organism and incubated for 18 hours.of the test organism and incubated for 18 hours.

The minimum concentration of the drug that inhibits The minimum concentration of the drug that inhibits the growth of the test organism in the tube is recorded the growth of the test organism in the tube is recorded as the MIC, I.e. the lowest concentration that will as the MIC, I.e. the lowest concentration that will inhibit the visible growth in vitro.inhibit the visible growth in vitro.

Subsequently, a standard inoculum from each of the Subsequently, a standard inoculum from each of the tubes in which no growth occurred may be subcultured tubes in which no growth occurred may be subcultured on blood agar to determine the minimum on blood agar to determine the minimum concentration of the drug required to kill the organism concentration of the drug required to kill the organism (MBC).(MBC).

The MBC is defined as the minimum The MBC is defined as the minimum concentration of the drug that kills 99,9% of concentration of the drug that kills 99,9% of the test organisms in the original inoculum.the test organisms in the original inoculum.

These MIC / MBC tests are not routinely These MIC / MBC tests are not routinely performed but are useful in patients with performed but are useful in patients with serious infections where optimal serious infections where optimal antimicrobial therapy is essential, i.e. to antimicrobial therapy is essential, i.e. to establish sensitivity of streptococci isolated establish sensitivity of streptococci isolated from blood cultures from patients with from blood cultures from patients with infective endocarditis, and of bacteria infective endocarditis, and of bacteria causing septicaemia in immunosuppressed causing septicaemia in immunosuppressed patients.patients.

APPROPRIATE SPECIMENS FOR ORAL APPROPRIATE SPECIMENS FOR ORAL INFECTIONS:INFECTIONS:

Sampling for pathogens within the oral environment Sampling for pathogens within the oral environment poses many problems due to the multitude of poses many problems due to the multitude of indigenous commensal flora that thrive in the oral indigenous commensal flora that thrive in the oral cavity.cavity.

Many of the patogens are endogenous in origin and Many of the patogens are endogenous in origin and cause disease when an opportunity arises cause disease when an opportunity arises (opportunistic pathogens)(opportunistic pathogens)

Obtaining an uncontaminated sample from sites such Obtaining an uncontaminated sample from sites such as the depths of periodontal pockets where the disease as the depths of periodontal pockets where the disease activity and hence the numbers of periodontopathogens activity and hence the numbers of periodontopathogens are likely to be high is extremely difficult.are likely to be high is extremely difficult.

For these reasons, judicial and appropriate sampling For these reasons, judicial and appropriate sampling techniques should be used when diagnosing oral techniques should be used when diagnosing oral infections.infections.

The specimens submitted to an oral The specimens submitted to an oral microbiology laboratory can be microbiology laboratory can be catagorized as those useful for the catagorized as those useful for the management of:management of:

Purulent infectionsPurulent infections Mucosal infectionsMucosal infections Periodontal infectionsPeriodontal infections Caries.Caries.

Purulent infections:Purulent infections:

The appropriate specimen is an The appropriate specimen is an aspirated sample of pus, if possible.aspirated sample of pus, if possible.

Take care to avoid needlestick Take care to avoid needlestick injuries when re-sheathing the injuries when re-sheathing the needlecap, drainage of residual pus needlecap, drainage of residual pus by incision, after aspiration sampling, by incision, after aspiration sampling, is obligatory.is obligatory.

The laboratory steps in the The laboratory steps in the diagnosis of a purulent infection:diagnosis of a purulent infection:

Mucosal infections:Mucosal infections: A common oral mucosal infection is oral A common oral mucosal infection is oral

candidiasis.candidiasis. Here, the lesion is sampled with a dry swab, and a Here, the lesion is sampled with a dry swab, and a

smear taken immediately thereafter.smear taken immediately thereafter. When evaluating the oral carriage of yeasts (or When evaluating the oral carriage of yeasts (or

other organisms such as Enterobacteriaceae) then other organisms such as Enterobacteriaceae) then the oral rinse should be collected.the oral rinse should be collected.

This entails requesting the patient to rinse the This entails requesting the patient to rinse the mouth for 60 seconds with 10 ml of phosphate-mouth for 60 seconds with 10 ml of phosphate-buffered saline and then expectorating the rinse buffered saline and then expectorating the rinse into a container, which is transported to the into a container, which is transported to the laboratory for quantification of yeast growth (in laboratory for quantification of yeast growth (in term of CFUs).term of CFUs).

Periodontal infections and Periodontal infections and caries:caries:

The value of microbiological sampling for the The value of microbiological sampling for the diagnosis of caries and periodontal diseases is diagnosis of caries and periodontal diseases is limited.limited.

In the case of dental caries salivary counts of In the case of dental caries salivary counts of lactobacilli and lactobacilli and Streptococcus mutansStreptococcus mutans could be could be used, and for this purpose saliva samples should used, and for this purpose saliva samples should be collected.be collected.

The diagnosis of periodontal disease by The diagnosis of periodontal disease by microbiologic means is problematic. A deep microbiologic means is problematic. A deep gingival smear is useful for the diagnosis of acute gingival smear is useful for the diagnosis of acute necrotizing ulcerative gingivitis, while paper point necrotizing ulcerative gingivitis, while paper point sample appear useful for DNA analysis of sample appear useful for DNA analysis of periodontopathic bacteria.periodontopathic bacteria.