Bacterial Disease - Laboratory Diagnosis
Laboratory techniques are often required for identifying the aetiological agent and/or the antimicrobial susceptibility of pathogens. It should be noted that any laboratory analysis should be accompanied by a full clinical examination and history.
Identification of Pathogenic Bacteria
Pathogenic bacteria can be identified by the examination of stained smears, cultural and biochemical characteristics and detection by immunological and molecular methods.
Stained Smears
There are a number of different routine methods used to stain bacteria for examination on a microscope slide. Gram stain smears are rapid and able to detect bacteria in large numbers and is often used for a 'rough and ready' analysis of tissue samples. Below is a table showing most of the main stains;
| Staining Method | Comments |
|---|---|
| Gram Stain | Most common in bacterial smears. The stain contains crystal violet which is retained in the cell wall of the bacterium. Gram positive bacteria are blue and gram negative bacteria do not retain the crystal violet and appear red, the colour of the counterstain. |
| Giemsa | Used to identify Dermatophilus congolensis, rickettsiae and Borrelia species which stain blue. |
| Dilute carbol fuchsin | Used for identifying Campylobacter species, Brachyspira species and Fusobacterium species which stain red. |
| Polychrome methylene blue | Used to identify Bacillus anthracis in blood smears which stain blue with distinctive pink capsules |
| Ziehl-Neelsen stain | Red staining bacteria are described as acid-fast or Ziehl-Neelsen positive |
Bacterial Culture
As noted above, the selection of the culture medium, atmospheric conditions and pH are among many variables that need to be considered for the successful culture of bacteria in the lab. A routine culture undertaken involves using a combination of blood agar (see below) and MacConkey agar (see below) together with incubation for between 24-48hours. Blood agar is able to support most pathogenic species of bacteria and is usually appropriate for routine primary isolation. Selective media is then normally used for particular organisms.
Agar plates should be inoculated using a streaking technique facilitating growth of isolated colonies. The aseptic technique of inoculation should also be used to prevent contamination.
Below is a table detailing the main types of medium used in bacterial culture;
| Medium | Comments |
|---|---|
| Nutrient Agar | Most commonly used basic medium. Non-fastidious bacteria (unable to produce their own vitamins) can grow on this medium. This medium is also suitable for demonstrating colonial morphology and pigment production. This type of agar is also commonly used as part of bacterial counting techniques as described above. |
| Blood Agar | This medium contains blood and is able to support the growth of pathogenic bacteria. This medium also allows the recognition of bacterial haemolysin production |
| MacConkey Agar | A selective medium containing bile which is useful for the isolation of enterobacteria and other gram negative bacteria. This medium also allows differentiation of lactose and non-lactose fermenting species. Colonies of lactose fermenters turn the surrounding medium pink as the medium also has a pH indicator. |
| Selenite broth, Rappaport-Vassiliadis Broth | Selective enriched medium used to isolate salmonellae from samples containing other gram negative enteric organisms |
| Edwards Medium | A blood agar based selective medium used for the isolation and recognition of steptococci |
| Chocolate Agar | Heat-treated chocolate agar which provides special growth factors for the isolation of Haemophilus species and for the culture of Taylorella equigenitalis. |
| Brilliant green agar | Indicator medium for the presumptive identification of Salmonella species. Salmonella colonies and surrounding medium have a pink appearence. |
| Buffered peptone water | Non-selective medium used for isolation of pathogens when present in low numbers in samples collected from foods and environmental sources |
Biochemical techniques
Biochemical tests relate to the catabolic activities of bacteria and use this to demonstrate the utilisation of particular substrates. The range of sugars utilised by bacteria is relatively small and therefore catabolism of sugars is often used as a method of identification. Commercial testing kits are available which usually consist of a strip of plastic cupules containing a test to which a suspension of the bacterium is added. The identity of the bacteria is then deduced from the pattern of the various cupules.
Below is a table of the commonly used biochemical tests;
| Test | Pathogens | Comments |
|---|---|---|
| CAMP Reaction | Steptococcus agalactiae, Rhodococcus equi, Actinobacillus pleuropneumoniae, Listeria monocytogenes | Haemolysis caused by Staphylococcus aureus is enhanced by pathogenic bacteria growing close to staphylococcal colonies |
| Pitting of Loeffler's serum slope | Arcanobacterium pyogenes | Proteolytic digestion of the medium around colonies |
| Haem-agglutination | Bordetella bronchiseptica | Agglutination of suspended ovine red blood cells by the bacteria |
| Nagler Test | Clostridium perfringens | Breakdown of lecithin in egg yolk agar by alpha toxin (lecithinase) produced by the organism. Surface application of antitoxin inhibits the alpha toxin activity |
Immunological Techniques
Immunological identification or serotyping uses the surface antigens on bacteria. Fluorescent antibody staining, antigen capture and direct enzyme-linked immunosorbent assays (ELISA) have been developed to identify bacterial pathogens. In all of these techniques the bacteria is bound by a specific antibody which has some form of indicator attached such as colour change enzymes or fluorescence.
Bacteriophage Typing
Some bacteriophages are very species specific and therefore phage typing represents another method that can be used to identify species of bacteria. This method allows bacterial species to be sub-divided into subtypes which are defined by their susceptibility to particular phages. Phage typing is commonly used to differentiate between Staphylococcus aureus and Salmonella enterica sub species.
Molecular Techniques
The most common molecular technique used to identify species of pathogenic bacteria are nucleic acid hybridisation and polymerase chain reactions (PCR). Nucleic acid hybridisation uses synthetic nucleic acid probes (specific for a particular species) that are applied to genetic material extracted from the pathogen. Probes can be used to detect DNA and RNA.
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