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:
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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: The clinical condition of the patient: 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.
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After heat-fixing the dry film (by passing through a flame), flood with crystal violet for 15 seconds. Then wash the excess. Flood with Lugol’s iodine for 30 seconds (to fix the stain), wash the excess. Critical step: Decolorize with acetone or alcohol for about 5 seconds. When no blue colour comes off the smear, wash immediately with water. Counterstain will dilute carbolfuchsin for 30 seconds (for neutral red for 2 minutes). 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.
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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) Examination of microscopic morphology and staining characteristics: a stained film of the colony helps identification 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.
