Bacteria are classified according to 1) morphology 2) gram stain as well as 3) growth requirements.

(1) Morphology See right hand column

(2) 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. 

(3) Growth Requirements

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.

Methods used to detect bacteria

• 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. 

• Direct detection 

• 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. 

Tools for Bacterial Detection

• genomic analysis of isolates can be down according to the following strategies:

  • 1) the presence of certain plasmids 

  • 2) PCR: Caroll (J. Clin. Microbiol. May 2000, 38(5), 1753-1757 disclsoed 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 amplifcaiton 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.