Classifcation of Microorganisms

Taxonomy

• Placing organisms into categories to show degrees of similarities

• Domain → Kingdom → Phylum → Class → Order → Family → Genus → Species

Three Domains

• Archaea: prokaryotes; no kingdoms

• Bacteria: prokaryotes; no kingdoms

• Eukaryotic: Animals, Plants, Fungi, Protists

Biochemical Tests

• Using enzymatic activities to differentiate the different bacteria

• Ex: Enteric, G-

• Large heterogenous group of microbes

• Natural habitat = intestinal tract

• Pathogenic & cause diarrhea

• Identify types based on biochem tests

• All: oxidase -

• Ex: Escherichia, Enterobacter, Citrobacter ferment lactose

• Salmonella & Shigella cannot ferment lactose

• Differentiate different types of bacteria with different selective & differential media

Numerical Identification

• Rapid identification methods are developed for medically important bacteria (Enterics)

• Look at photo for guidance

• Color change = positive reaction or usage

• Add up each section → code

Serology

• Science that studies serum & immune responses that are evident in serum

Antigenic

Stimulate formation of AB by host microbial cells

Antiserum

Solution of AB used to identify many of medically importance microbes

Slide Agglutination Test

• Unknown bacteria is placed in a drop saline on several slides

• Different antiserum solutions are added to each sample

• Positive test: clumping of bacteria (antigen) and antiserum (AB)

Enzyme-linked Immunosorbent Assay (ELISA)

• Fast and read by computer scanner

• AB placed in wells of microplate with unknown added to each

• Reaction between the two identifies type of bacteria/virus

• Ex: ID of HIV

Western Blotting

• Based on antigen-AB reaction

• Helps ID HIV & Bornella burgdorferi (Lyme disease)

Phage Typing

• Test for determining which phases a bacterium is susceptible

• Helps in food-associated infections

DNA Base Composition

• % of G + C

• Two organisms that are closely related will have similar amounts of bases in DNA

• Difference of more than 10% = NOT related

• Two organisms have same percentage does NOT mean closely related automatically → need other data

Nucleic Acid Hybridication

1) Heat to separate strands

2) Combine single strands of DNA

3) Cool to allow repatriation of double-stranded DNA

4) Determine degree of hybridization

Complete: organisms IDENTICAL

Partial: RELATED

NO: UNRELATED

Southern Blotting

1) Salmonella DNA fragments is cloned in e.coli

2) Cloned DNA fragments are marked with fluorescent dye & separated into single strands → DNA probes

3) Unknown bacteria are collected on filter

4) Cells are lysed & DNA is released

5) DNA is separated into single strands

6) DNA probes are added to DNA from unknown bacteria

7) DNA probes HYBRIDIZE with Salmonella DNA from sample; excess probes is washed off. Fluorescence = SALMONELLA

DNA Chips

• _ can be manufactured to contain hundreds → thousands of synthetic single-stranded DNA sequences. Assume each DNA sequence was unique to different gene

• Unknown DNA from sample is separated into single strands, enzymatically cut, labeled with fluorescent dye

• Unknown DNA inserted into and allowed to hybridize with DNA on

• Hybridization → color on chip → identify bacteria

Fluorescent In-Situ Hybridization (FISH)

• Cells are treated so that fluorescent dye labeled RNA or DNA probes entered cell & reacts with target DNA in cells

• Use: determine identity, abundance, & relative activity of microorganisms in environment

• Also use: detect bacteria without need to culture (BYPASS incubation)

Dichotomous Keys

Series of successive questions to determine specific species

Cladograms

Maps showing evolutionary relationships based on rRNA sequencing