Methods of Identifying Microorganism

• Filtration is used to sterilize heat-labile substances like serum, plasma, and carbohydrate solutions
• Lyophilization is the process of freeze-drying
• Ultracentrifugation involves the use of a centrifuge
• Chemical methods of destruction include:

  • Disinfection: the process of removing or killing microorganisms using a chemical agent
  • Bactericidal: killing bacteria
  • Bacteriostatic: inhibiting only the growth of bacteria
• Biochemical Test demonstrates the enzyme system within a microbial cell
• CHOentation Test involves the anaerobic breakdown of substances where bacteria gain more energy by using the simplest sugar before other sugars, which must be converted to glucose to enter the Embden-Mayerhoff pathways
• Thru the fermentation process, simple sugars serve as the main source of energy for microorganisms
• Media used in biochemical tests:

  • TSI (triple sugar ion) contains glucose (0.1%), lactose (1%), sucrose (1%), and iron
  • KIA (Klingler iron agar) contains glucose, lactose, and iron
  • RDA (Russel’s double agar) contains glucose and lactose
• TSI (triple sugar ion) results:

  • K/K: no sugar was fermented (red/red)
  • K/A: only glucose was fermented (red/yellow)
  • A/A: all three sugars are fermented (yellow/yellow)
• IMViC reaction (Indole Methyl Red Voges-Proskauer Citrate Test) uses media like Tryptophan broth for the indole test, MRVP medium, or Clark & Lubs Dextrose Broth medium
• Indole Test:

  • To observe for a positive result, add ether or xylene to extract indole, then add Ehrlich’s or Kovac’s reagents
  • Positive result: formation of deep red color
  • Principle: some organisms can split tryptophan into alanine and indole; the liberated indole combines with p-dimethylaminobenzaldehyde in Ehrlich’s or Kovac’s reagent to produce deep red colors
• Methyl Red Test:

  • After incubating the inoculated MRVP medium, add 5 drops of Methyl Red indicator
  • Positive result: formation of deep red color
  • Principle: Organisms fermenting dextrose produce large amounts of acids; the test is based on the final hydrogen ion concentration (pH) reached by the culture
• Voges-Proskauer Test:

  • After incubating the inoculated MRVP medium for 48 hours, add:
  • 15 drops of 5% alpha naphthol in ethyl alcohol
  • 10 drops of 0.3% of creatine in 40% KOH
  • Principle: when glucose is fermented, some organisms produce not only acid but also a compound known as acetyl methyl carbinol or acetoin, which in the presence of KOH will be oxidized to dimethyl carbinol, reacting with guanidine compounds in the broth to produce a deep red color
• Simmon Citrate Test:

  • pH indicator: Bromthymol blue
  • Positive result: Prussian blue color
  • Principle: some organisms can utilize citrate as the sole source of carbon, releasing ammonia gas which makes the medium alkaline, turning the pH indicator from green to blue
• Catalase Test:

  • Negative result due to catalase also being present in red blood cells
  • Principle: some organisms elaborate the enzyme catalase, liberating hydrogen peroxide to form oxygen