This document discusses various methods used to identify and characterize prokaryotes, including phenotypic, genotypic, and strain-level characterization techniques. Phenotypic methods include microscopy, staining techniques like Gram stain, culture characteristics, and biochemical tests. Genotypic identification uses nucleic acid-based methods like PCR and sequencing. Strain differentiation analyzes biochemical profiles, serological reactions, genomic fingerprinting using pulsed-field gel electrophoresis, ribotyping, and phage typing susceptibility patterns.

1. SEROTYPING, GENOTYPING & PHAGETYPING LECTURE HOZA, A.S BLS 206

6. Metabolic differences Metabolic differences include – culture characteristics – selective media and – biochemical tests.

14. Biochemical tests • The basic strategy for identifying bacteria based on biochemical test relies on the use of a dichotomous key, which is a flow chart of tests that give either a positive or negative result. • The biochemical tests are usually initiated simultaneously to speed identification.

16. Serology • In some cases, proteins and polysaccharides present on the surface of the bacterium are considered as identifying markers. • The most useful of these are the molecules that make up surface structures including the cell wall, glycocalyx, flagella and pili. • Antibodies directed against surface proteins and polysaccharides are frequently used to identify various bacteria. • Methods which use antibodies for the detection of antigens are called serology. • Some serological tests such as used to identify Streptococcus pyogenes are quite specific, simple and rapid.

22. Polymerase chain reaction (PCR) • PCR can be used to amplify specific nucleotide sequences of microorganisms from samples such as body fluids, soil, food, and water. • This technique can be used to detect microorganisms that are present in extremely low numbers as well as those can not be grown in culture. • In order to use PCR to detect microorganism of interest, a sample should be first treated to release and denature DNA. • All ingredients needed for PCR along with specific primers known and designed for particular microbe are then added.

23. • After ~30 cycles of PCR, sufficiently amplified DNA fragment is visualized as discrete band on an ethidium bromide stained agarose gel. • Alternatively, a DNA probe can be used to detect the amplified DNA.

24. Sequencing ribosomal RNA genes • Ribosomal RNA genes (DNA sequences) are highly conserved, and can be used to identify organisms. • This method is particularly useful for identification of those prokaryotes which are difficult or currently impossible to grow in culture. • Three different rRNAs: 5S, 16S, and 23S. • Some regions of 16S rRNA are virtually same in all prokaryotes whereas others have quite variable sequence and this variable region is used to identify an organism. • In certain cases, 16S rDNA is used to identify uncultivable organisms.

28. Biochemical and serological typing 1. Biochemical typing • Biochemical tests are mainly used to identify various species of bacteria but they can also be used to distinguish strains. • A strain that has characteristic biochemical pattern is called a biovar or biotype.

33. • One method of genomic typing is to compare the pattern of fragment sizes produced when the same restriction enzyme is used to digest DNA from each organism. • When the lengths of restriction fragments vary among organisms, it is termed ‘restriction fragment length polymorphisms’ (RPLFs). 3. Genomic typing

35. Phage typing

37. • Strains of a given species sometimes differ in their susceptibility to various types of bacteriophages. • The susceptibility of an organism to a particular type of phage can be readily demonstrated in the laboratory. • The patterns of clearing around the bacteriophage spot indicate the susceptibility of the test organism to different phages. • Different patterns are compared to determine strain differences. • Bacteriophage typing is largely replaced by molecular methods. Phage typing

38. plaque assay

40. Antibiograms

41. • Antibiotics susceptibility patterns or antibiograms, are also used to distinguish among different strains. • Again this method has largely been replaced by molecular techniques. • Different strains will have different patterns of clearing around antibiotics disks.