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 Growth

Appropriate environmental conditions are needed for bacterial growth including moisture, pH, temperature, osmotic pressure, atmosphere and nutrients. The time required for the production of daughter cells (generation time) is dependant on genetic and nutritional factors. Generation time can range from 20 mins to 20 hours.

Bacterial Nutrition

Bacterial growth requires nutrients to be available in the immediate environment. Bacteria are mainly chemoheterotrophs (using organic chemicals as energy). Specialised forms of agar plate containing different types of media have been developed to facilitate bacterial growth in controlled specific conditions. Therefore it is possible to determine the species of bacteria based on, amongst other things, the type of media they are able to grow on.
Most pathogenic bacteria can be grown on a nutrient medium at 37C, although they can grow between 20-45C. Most bacteria also grow optimally at a neutral pH and it is standard practice to buffer culture media so maintain it around pH 7. Another key determinant in bacterial growth is their preference for different types of atmosphere. Bacterial preference for oxygen means it is possible to assign all bacteria into four main groups; aerobes, anaerobes, facultative anaerobes and microaerophiles. Therefore anaerobic bacteria are unable to grow in an atmosphere containing oxygen.

Methods for Counting bacteria

Method	Technique	Comments
Microscopic Counting
Direct Smear	Smear on microscope slide from defined volume and dilution. Count performed using 50 microscope fields.	Slow and unreliable method and cannot differentiate viable and non-viable bacteria
Chamber Counting	Count fixed volume of bacterial suspension using a calibrated slide	No differentiation between viable and non-viable bacteria
Colony Counting
Spread Plate	Known vol of bacterial suspension spread onto agar plate and incubated for 24-48 hours	Number of colonies counted and expressed as colony-forming units (CFU)/ml of suspension
Pour Plate	A small vol of a known bactrial dilution is added to a petri dish with 20ml of molton agar at 45-48C and mixed	Colony counting carried out as above.
Miles-Misra	Diluted bacterial solution placed on plate in 5 seperate positions	Number of colonies counted and expressed as colony-forming units (CFU)/ml of suspension
Membrane Filtration	Following filtration of a known vol of bacterial dilution through a 0.22um pore size, filter is placed on an agar plate and incubated for 24-48 hours	The number of viable bacteria are expressed as CFU/ml of fluid
Other Counting Methods
Opacity Tubes	A bacterial suspension is matched visually with Mcfarland's opacity standard tubes	This test indicates the total bacterial cell numbers per ml
Electronic Counting	Counting machines such as the Coulter Counter can give rapid and accurate results	Reliability of results is dependant on quality control and test only gives a total cell count.

Quinn et al., P.J.. (2002) Veterinary Microbiology and Microbial Disease. Oxford: Blackwell Science Limited. pp.Bacterial Counting Techniques, P13.

Bacterial Genetics

Replication of Bacteria

Bacteria are haploid and have one circular chromosome of double stranded DNA. Bacteria replicate through binary fission producing genetically identical daughter cells. Each molecule of DNA in the daughter cells is composed of a strand from the parent and a newly synthesised complementary strand. This process of DNA replication is called semiconservative replication.

Plasmids

Plasmids are small pieces of genetic material found in the cytoplasm and these plasmids are able to replicate independantly of the bacterial chromosome. Most species of bacteria contain plasmids that are composed of double stranded DNA which are circular in shape. In pathogenic bacteria it is often the plasmid that encodes virulence factors and traits such as antibiotic resistance.
Replication of most plasmids is not directly related to the replication of the host bacterium and it has been found that the distribution of plasmids to daughter cells is a random process as plasmids in the cytoplasm may or may not be transferred when the cytoplasm of the cell is seperated prior to forming the daughter cells.
Bacterial plasmids can not only be transferred during bacterial replication but also via processes called conjugation and transformation (although the transformation process rarely occurs in nature).

Bacteriophages

A bacteriophage is a term used to describe a virus that is able to infect a bacterial cell and they can be either virulent or temperate depending on their method of replication. Virulent bacteriophages undergo a lytic cycle within the bacterium which eventually results in the production of bacteriophage progeny from the cell and the lysis of the bacterium.
A bacteriophage can be composed of either DNA or RNA and iehter single or double stranded. The capsid (outer protective layer) of the bacteriophage often remains outside the bacterial cell after the viral nucleic material has entered the cell cytoplasm. The host specificity of bacteriophages is related to the chemical affinity between attachment structures on the surface of the bacteriophage capsid and the receptors on the surface of the bacterium.

Genetic Variation

Genetic variation can occur in a number of ways and the genotype of the bacteria determines its inheritable potential. Below are the main ways that genetic mutation can occur in bacteria;

Mutation
A mutation is a stable inheritable alteration in the bacterial genome. This means that base pairs within a gene are altered. Genes with altered base pairs may, or may not, depending on the mutation be functional or can incorrectly code for an amino acid in a protein resulting in a phenotypic change rather than simply a gene alteration. The type of mutations occurring in bacteria are silent, non-sense, mis-sense, frame shift, deletion of base pairs, insertions, translocations and inversions.

Genetic Recombination
Genetic recombination occurs when sequences of DNA from seperate sources become integrated. This new genetic material can be introduced via conjugation, transduction and transformation.

Conjugation
Conjugation represents the process whereby DNA is transferred from a donor cell to a recipient cell. The donor cell synthesises a modified "pilus" which the donor cell inserts into the recipient cell. This is often called a sex pilus. Genetic material is then transferred through the pilus to the recipient. During conjugation, plasmid genetic material is mostly transferred, although chromosomal DNA can also be transferred via this process. Conjugation is most frequently associated with gram negative bacteria, but can occur in some gram positive bacteria. A sex pilus is not formed in gram positive bacteria and instead plasmid DNA is transferred when the bacteria are in close physical contact.

Tranduction
During transduction, DNA from a donor bacterium is incorporated into the nucleic acid of a bacteriophage and it is the progeny of the bacteriophage infecting another bacterium that allows the transfer of the genetic material.

Transformation
The process of transformation involves the transfer of genes from a segment of chromosomal DNA from a lysed donor bacterium to a fully functional recipient. Natural transformation is uncommon and is usually restricted to propcedures carried out in the lab.

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.