Difference between revisions of "Category:Bacteria - Overview"

Introduction

A typical bacterial cell is composed of an outer capsule, a cell wall, a cell membrane, cytoplasm containing nuclear material and ifmotile, appendages such as flagella and fimbrae or pili. Some species of bacteria are more resistant to environmental influences than others, particularly those species of bacteria that are able to produce spores which can remain inactive until the appropriate environmental conditions prevail allowing the bacteria to resist conditions such as freezing, wet, dry or hot conditions.
The structural features of pathogenic bacteria are important in the production of disease and also very useful for the identification and diagnosis of infection in veterinary medicine.
Bacterial Cell Structure

Bacterial Growth and Measurement

Bacterial Genetics

Laboratory Diagnosis of Bacterial Disease

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

Quinn et al., P.J.. (2002) Veterinary Microbiology and Microbial Disease. Oxford: Blackwell Science Limited. pp.Bacterial Counting Techniques, P23.

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 top 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

Quinn et al., P.J.. (2002) Veterinary Microbiology and Microbial Disease. Oxford: Blackwell Science Limited. pp.Bacterial Counting Techniques, P24.

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

Quinn et al., P.J.. (2002) Veterinary Microbiology and Microbial Disease. Oxford: Blackwell Science Limited. pp.Bacterial Counting Techniques, P25.

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.