Methods of Studying Microorganisms

Methods of Studying Microorganisms

1. specimen collection

   • blood, sputum, urine, stool, tissues, foods, water, oil, etc.
   • isolated by sampling device that holds, maintains and preserves the microbes in samples
     • swab, syringe, inoculation needle/loop

2. five basic techniques

   • 1.inoculation
     • the act of introducing a bacterial sample into a sterile nutrient media
     • inoculum: microbes that are introduced into a culture medium to initiate growth
   • 2.incubation
     • the process of growing microorganisms in conditions that are perfect for their expansion (conditions vary depending upon species)
     • mesophilic: 37 Celsius
       • E.coli
     • thermophilic: 70 Celsius
       • Thermus aquaticus
     • promotes multiplication of microbes over a period of hours, days/weeks
     • produces a culture (the visible growth of the microbes in the medium)
   • 3.isolation
     • the process of separating out an individual bacterial species/strain from a mixed culture
       • separate colonies (discrete mounds of cells) on solid media
       • turbidity in broths
     • streak culture
       • a small droplet of culture or sample is spread thinly over the surface of the medium so that individual colonies can be seen
       • important to isolate clonal bacterial cells
     • lawn culture
       • like spread method, but only concentrated bacteria are used
       • provides a uniform surface growth of the bacterium
       • 1.bacteriophage typing
         • 2.antibiotic sensitivity testing
           • 3.preparation of bacterial antigens and vaccines
     • stroke culture
     • stab culture
       • puncturing a suitable medium with bacterial culture
       • 1.study the oxygen requirements of the bacterium
         • 2.maintenance of stock cultures
     • spread plate
       • bacterial culture is serially diluted, poured over an agar plate and spread with a spreader
       • plate is incubated for a set amount of time to dilute the culture enough to get individual colonies which are then isolated/further studied
     • liquid culture
       • inoculated by touching with a loop containing bacterial culture
       • 1.amplification
         • 2.sterility tests
           • 3.continuous culture methods
       • disadv.: does not provide a pure culture from mixed inoculation
     • anaerobic culture methods
       • production of vacuum
         • displacement of oxygen with other gases
     • pour plate
   • 4.inspection
     • cultures are observed macroscopically for obvious growth characteristics (color, texture, size) useful in analysing specimen contents
       • a single visible colony
         • =pure culture
       • slides are made to assess microscopic details (shapes, size, motility) (light microscopy)
       • staining techniques may be used to gather specific information on microscopic morphology
   • 5.identification
     • determination of the type of microbe
       • physiochemical properties (shape, size, staining, flagella, capsule, spore, etc.)
     • more specialized biochemical tests to determine metabolic activities specific to the microbe
       • API Test

3. culture media: subtance full of nutrients neccesary for microbial growth

   • defined: precise amounts of highly purified chemicals
   • complex (undefined): highly nutritious substances (e.g. FBS)
   • selective: contains compounds that selectively inhibit certain species
     • bile salts and crystal violet
       • inhibits gram+ve bacteria (any bacteria growing is gram-ve)
   • differential: contains an indicator (e.g. color change)
     • pH indicator and lactose
       • gram -ve enteric bacteria can ferment lactose, producing lactic acid
         • lactic acid causes pH indicator to turn red
   • agar
     • jelly-like substance isolated from seaweed
     • remains solid (except being degraded by a few microbes)
     • liquifies at ~100 Celcius and remains liquid until temp. drops to 40-45 Celsius
     • autoclaved to sterilize it
       • kept warm, poured into a plate and solidies when cool

4. microscopy

   • magnification: the amount or degree of visual enlargement of an observed object
     • measured by multiples
     • length of the Image divided by the Actual length
   • resolution: capacity to distinguish or separate two adjacent objects; higher resolution=finer detail of image
   • light
   • eletron
   • fluorescent