see also Morphology
Introduction:
Bacteria are classified according to 1) morphology 2) gram stain as well as 3) growth requirements. Isolation and identification in Culture is the “gold standard for identification of bacteria. Types of culture media used include 1) agar plates, 2) liquid media (broth) which is used in blood culture bottles, 3) selective and differential media: See right hand panel.
The Gram stain is the most important tool for bacterial identification. It is a differential stain in which a decolorization step occurs between the application of two basic stains. The primary stain is crystal violet. Iodine is added as a mordant to enhance crystal violet staining by forming a crysal violet-iodine complex. Declolorization follows and is the most critical step in the procedure. Gram negative cells are declorozed by the solution whereas Gram positive cells are not. Gram negative cells can thereafter be colorized by the counterstain safranin. At the end, Gram positive cells appear purpose and gram negative cells appear reddish pink.
Although all bacteria have minimum nutrient requirements for growth which are 1) a carbon and nitrogen sources, 2) energy source, 3) water and 4) various ions, a great deal of diversity exists within the prokaryotic kingdom in specific growth requirements. For example, some bacteria are unable to grow in the presence of oxygen and are are referred to as obligate anaerobes whereas others require the presence of oxygen and are called obligate aerobes. The vast majority of bacterial grow in either the presence or the absence of oxygen and are called facultative. One may also classify bacteria based on their sources of energy and carbon. Those that derive energy from the oxidation of metals and get carbon from CO2 are referred to as chemotrophs. Those that derive energy from light and carbon from CO2 are referred to as phototrophs. Most pathogenic bacteria derive their energy and carbon from organic sources and are referred to as organotrophs.
Genomic Analysis:
16S rRNA sequencing:
The human microbiome has a large impact on oral health and can inform our understanding of disease mechanisms. Researchers in South Korea used 16S rRNA sequencing to characterize the microbiome in subjects with dental implant infections, which increases the risk for additional bone loss. In comparison to the microbiome of subjects with healthy gums or subjects with severe gum disease, bacterial communities associated with dental implant infections showed higher diversity. Data analysis identified both well-known pathogens associated with gum disease and several bacteria previously unrecognized in gum tissue. Results indicated that dental implant infections cause a more complex inflammatory response that involves more bacteria compared to severe gum disease. See Illumina
Caroll (J. Clin. Microbiol. May 2000, 38(5), 1753-1757 disclosed a nested PCR protocol for the detection of and discrimination between 14 species of gram positive and negative bacterial. First round PCR was with pan bacterial oligonucleotide primers based on conserved sequences of the 16S ribosomal gene, followed by a gram negative specific PCR which resulted in a 985 bp amplifcation product and a multiplex PCR which resulted in two PCR products, a 1,025 bp amplicon (all bacteria) and a 355 bp amplicon (gram positive bacteria only). 3) nucleic acid probes.
Next Generation Sequencing (NGS):
NGS is a key tool for food testing in public health. Two outbreaks of Salmonella food poisoning occurred concurrently in one district of South Africa, one at a daycare center and one at a restaurant. Researchers used small whole-genome sequencing of stool samples and food samples from both outbreaks to find epidemiological links between the two cases. The strains found in the samples from the daycare center and the samples from the restaurant were highly related with fewer than five allele differences between them. The close relationship of the Salmonella strains from this investigation indicated a common contaminated food source, likely eggs. see Illumina
Serology:
Serology which is evidence of a specific immune response to the bacterium. The problem with this approach is that it is retrospective. IgM is the first Ig to appear usually about 7-8 days after infection and indicates a recent infection. IgG appears later.