Classification and Identification

• Microorganisms


  • Bacteria
  • Algae
  • Yeast
  • Moulds
  • Protozoa
• Viruses aren't generally included as they're 'not-living', but they are considered under microbiology as they are infective MO
• There are also protein species that can cause deadly infections. Like viruses, they aren't living, but they do need a host to survive
• Most microorganisms are unicellular, but some are multicellular
• Cell walls


  Can be identified using the Gram stain
• Microorganisms that have a cell wall

  • Bacteria
  • Algae
  • Yeasts
  • Moulds
• Nomenclature


  When naming MO, the first part is the genus name (written with a capital letter) and the second part is the species name (written with a lower case letter), written in Latin
• Write MO name in italics: once it has been written in full, then it can be abbreviated by shortening the name of the genus, provided that the shortened form is unambiguous
• Culturing Microorganisms


  • Growing MO is called 'culturing'
  • To cultivate MO, a sample/group of MO called an inoculum is introduced into a collection of nutrients, known as a medium
  • MO that grow from an inoculum is called a culture, and culture can refer to the act of cultivating MO
  • Cultures can be grown in a liquid such as broths or a solid such as agar
• Pure Cultures


  • A pure culture is just one individual species of MO
  • If a sample is taken from a patient, multiple species may be obtained, and as a result, they need to be separated out and grown into pure cultures
  • Clinical specimens are collected to identify a suspected pathogen, however, normal microbiota could also be collected
  • Several techniques are applied to obtain pure cultures, which are composed of cells arising from a single progenitor: a progenitor may be either a single cell or a group of related cells, therefore, the progenitor is termed a colony forming unit CFU
• Culture Media


  • Culture media should contain a source of protein or protein hydrolysate, pH control and defined salt concentration
  • Agar is a complex polysaccharide, derived from the cell walls of certain red algae. Most microbes can't digest agar
  • Nutrient agar contains peptone, beef extract, NaCl and agar
  • Tryptone soya agar contains casein enzymic hydrolysate, papaic digest of soyabean meal, NaCl, methylumbelliferyl-β-D-glucuronide and agar
• Types of Media


  • Defined media
  • Complex media
• Defined media


  Prepared by adding precise amounts of pure inorganic or organic chemicals to distilled water, meaning the exact composition of a defined medium is known
• Complex media


  • Made from digests of microbial, animal or plant products, such as milk protein (casein), beef (beef extract), soybeans (tryptic soy broth), yeast cells (yeast extract) or any of a number of highly nutritious substances
  • The exact nutritional composition is unknown and to form a culture medium, the digests need to be hydrated with distilled water as they're commercially available in dehydrated form
• Other types of media


  • General-purpose media
  • Selective media
  • Differential media
• General-purpose media


  Include nutrient and tryptone soya agar
• Selective media


  Often incorporates compounds that inhibit growth of contaminants/certain MO but not others
• Differential media


  Employ indicator systems: an indicator (typically a dye) is added to colour MO differently depending on the nutrients they utilise and usually hasten bacterial characterisation by yielding appropriate positive or negative results
• Many media are both selective and differential
• Microscopy


  • Beams of radiation may also be referred to as waves and various forms of radiation differ in wavelength
  • Magnification is the apparent increase in size of the object and occurs when beam of radiation refracts as it passes through a lens, which focus light rays on a focal point
  • Resolution, aka resolving power, is the ability to distinguish objects that are close together. Resolution distance is dependent on: wavelength of electromagnetic radiation and numerical aperture of the lens (ability of lens to gather light)
  • Contrast refers to differences in intensity between two objects or between an object and its background and is important in determining resolution
• Light Microscopes


  • Use light which is refracted and is limited on the amount of magnification is available
  • Can't look inside MO and they have to be stained
• Electron Microscopy


  • Uses electrons instead of light, the moving electrons acting as waves with wavelengths dependent on the voltage of the electricity: they have wavelengths 0.01 nm - 0.001 nm
  • Resolving and magnification power is therefore greater due to the shorter wavelengths
  • Provide detailed views of small bacteria, viruses, internal cellular structures etc
  • The two types are transmission electron microscopes TEM and scanning electron microscopes
  • Transmission means the electron beams goes right through the object and it bounces back when it hits something, e.g. a ribosome, cell wall etc. How much it bounces back depends on the density of the object
  • SEM put the surface of the specimen, often with some kind of precious metal, and electron beams bounce off the surface
  • They must work in a vacuum and specimens must be this in TEM
• Staining


  • Most MO are colourless, so they have to be stained, which increases contrast and resolution, which is necessary for both light microscopy and electron microscopy
  • Simple stains are composed of simple basic dye such as crystal violet and involve no more than soaking the specimen, then washing off with water
  • Differential stain use more than one dye to different cells, chemicals or structures; it'll identify differences between different MO, e.g. gram stain, which identifies differences between gram positive and negative bacteria
• Gram Stain


  • Crystal violet is first put onto the bacterial cell and it goes inside the cell wall
  • When iodine is added, it complexes/reacts with CV, making a macromolecular structure (a large structure), which becomes stuck in the cell wall and colours it
  • Gram negative cells have very thin cell walls so don't retain a lot of the gram stain. When decolourising by adding alcohol, it lyses the outer membrane of the gram negative cell, so the gram stain is washed away
  • In gram positive cells, as it has a thick cell wall and has no outer membrane, the decolourising agent has no effect
  • When gram negative cells have been decolourised, a counterstain — carbol fuchsin —is added; it's a general stain
• Acid-fast Stain


  • Stains Mycobacteria and Nocardia and identifies mycolic acid within the cell wall
  • If bacteria have mycolic acid within the cell wall, the mycolic acid is very resistant to substances like gram stains and its role is to act as a form of protection against other toxins
  • The slide is flooded with carbol fuchsin and heat, which melts the mycolic acid. The heat drives the stain through the waxy wall and into the cell
  • The stain is decolourised with HCl and alcohol and the counterstain methylene blue
• Endospore stain


  • Conventional stains won't work, as the spore coast is practically impermeable to all chemicals, so there isn't a specific endospore stain
  • Gram stains can be used to identify any endospores as it slightly coats the endospores and therefore could be potentially detected using a microscope, but it would still be a little bit hard to see them
  • Schaeffer-Fulton endospore uses heat to drive the primary stain, malachite green, into endospore, which stains it
  • After cooling, slide is decolourised with water and counterstained with safranin
• Other Identification Procedures


  • Growth characteristics, such as oxygen requirements and other requirements, e.g. temperature, pH etc
  • Biochemical characteristics such as specific tests for presence of enzymes
  • Enzymes used in the utilisation of oxygen: catalase and oxidase test
  • Urase test — Proteus spp.
  • Carbohydrate fermentation
  • Gelatinase test
  • Analytical Profile Index Strips
  • API 20E is used to identify gram negative bacilli. Substrates are stored as dehydrated strips and rehydrated using a bacterial suspension. It contains miniature biochemical tests
• Genetic Characterisation of Bacteria

  • Genomics is whole genomes of bacteria that have been sequenced
  • Individual genes detected: 16 ribosomal RNA molecule sequencing has become the 'gold standard'
  • Serology, e.g. enzyme-linked immunosorbent assay ELISA
  • Non-genetic profiling, e.g. small molecule or proteins such as fatty acids