Classification and identification (L5)

• Microorganisms include


  • Bacteria
  • Algae
  • Yeast
  • Moulds
  • Protozoa
• Viruses are not generally included as they are 'not-living'
• Most microorganisms are unicellular, but some are multicellular
• Cell walls


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

  • Bacteria
  • Algae
  • Yeasts
  • Moulds
• Culturing microorganisms


  1. Sample called an inoculum is introduced into a collection of nutrients, a medium

  2. Microorganisms that grow from an inoculum is called a culture

  3. Cultures can be grown in liquid - aka broths

  4. Cultures can be grown in solid - aka agar
• Culture


  The act of cultivating microorganisms
• Microorganism identification


  • Observation
  • Looking at individual colonies
  • Characteristics
  • Use of microscopes aids in this identification
• Obtaining pure cultures


  1. Clinical specimens are collected to identify a suspected pathogen

  2. Normal microbiota could also be collected

  3. Several techniques are therefore applied to obtain pure cultures

  4. Cultures composed of cells arising from a single progenitor

  5. A progenitor may be either a single cell or a group of related cells

  6. The progenitor is termed a colony forming unit (CFU)
• Culture Media


  • Should contain source of protein or protein hydrolysate
  • pH control
  • Defined salt concentration
• Agar


  • Complex polysaccharide, derived from the cell walls of certain red algae
  • Most microbes cannot 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


  • General
  • Selective
  • Differential
• Bacterial counting determined by

  • Plate count
  • Direct microscopic count
  • Dry weight
  • Turbidity
  • Coulter counter
• Plate count


  • Agar plates
  • Membrane filtration - if populations are large, but population density is small (e.g. faecal bacteria in drinking water)
• Coulter counter


  • Device that directly counts cells as they interrupt electrical current flowing across a narrow tube in front of an electronic detector
  • Can determine the size of the specimen that you wish to count
  • Debris in media and presence of clumps can give inaccurate results
• Wavelength of radiation


  • Beams of radiation may be also referred to as waves
  • Various forms of radiation differ in wavelength
  • Electrons (e-) are negatively charged particles
  • Moving e- act as waves, with wavelengths dependent on the voltage of the electricity
• Magnification


  • Apparent increase in size of the object
  • Magnification results when beam of radiation refracts as it passes through a lens
  • Lenses focuses light rays on a focal point
• Resolution


  • Ability to distinguish objects that are close together
  • Resolution distance dependent on wavelength of electromagnetic radiation
  • Resolution distance dependent on numerical aperture of the lens (ability of the lens to gather light)
• Contrast


  • Refers to differences in intensity between two objects or between an object and its background
  • Important in determining resolution
• Electron microscopy


  • Electrons travelling as waves have wavelengths 0.01 nm – 0.001 nm
  • Resolving and magnification power of electron microscopes in therefore greater
  • They provide detailed views of small bacteria, viruses, internal cellular structures etc.
  • There are two types - Transmission electron microscopes (TEM) and Scanning electron microscopes
  • Electron microscopes must work in a vacuum
  • Specimens must be thin in TEM
• Staining


  • Most microorganisms are colourless
  • Staining increases contrast and resolution
  • Necessary for both light microscopy and electron microscopy
• Simple stains


  • Composed of simple basic dye such as crystal violet
  • 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
• Gram Stain


  1. Primary stain

  2. Mordant

  3. Decolourizing agent

  4. Counterstain
• Gram stain results


  • Gram negative
  • Gram positive
• Acid-fast stain (Zeihl-Neelsen stain)


  • Stains Mycobacteria and Nocardia
  • Flood slide with carbol fuchsin and heat. the heat drives the stain through the waxy wall and into the cell
  • Decolourize with HCl and alcohol
  • Counterstain with methylene blue
• Endospore stain


  • Conventional stains will not work, as the spore coat is practically impermeable to al chemicals
  • Schaeffer-Fulton endospore stain uses heat to drive the primary stain, malachite green, into endospore
  • After cooling, slide decolourized with water and counterstained with safranin
• Growth characteristics


  • Oxygen requirements
  • Other requirements - temp., pH, etc.
• Biochemical characteristics


  • Specific tests for presence of enzymes
  • Enzymes used in the utilization of O2
  • Catalase test
  • Oxidase test
  • Urease test - Proteus spp.
  • Carbohydrate fermentation
  • Gelatinase test
• Analytical Profile Index strips

  • API 20E is used to identify gram negative bacilli
  • Substrates are stored dehydrated
  • Rehydrated using a bacterial suspension
  • Contains miniature biochemical tests

  
• Genetic characterization of bacteria

  • Genomics
  • Individual genes detected
  • Serology
  • Non-genetic profiling
• Genomics


  Whole genomes of bacteria have been sequenced
• Individual genes detected


  16s ribosomal RNA molecule sequencing has become the 'gold standard'
• Serology


  e.g. Enzyme-linked immunosorbent assay (ELISA)
• Non-genetic profiling


  Small molecule or proteins - e.g. fatty acids
• Bacterial identification
• Why is agar a good material to hold nutrients?

  As most microbes can't digest agar
• Selective: contains substances that favour growth of a particular microorganism or inhibit the growth of unwanted microorganisms