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Bacteria Identification Techniques

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Streak Plate Technique

Materials required

  • Petri dish
  • Cotton swab (inoculation loop)
  • Inoculums (broth)
  • Agar

Procedure

  • Spread the sample across one quadrant of petri dish containing a growth medium of agar plate sterilized with autoclave.
  • Re-sterilize the loop and then drag it severally on previously inoculated quardrant.
  • Incubate the plate for between 26 to 36 hours, this gives bacteria the chance to reproduce.

Results

  1. Visible colonies form in areas touched by the inoculation loop.
  2. Based on morphological differences, single bacterial species can be clearly identified from the mixed colonies. 

Description of colonies

The colonies vary in size, shape and colour

Gram Staining Technique

Materials:

a) Supplies

  • Glass slides (25 by 75 mm)
  • Immersion oil
  • Bunsen burner
  • Sterile Pasteur pipettes and wood applicator sticks

b) Reagents

Hucker’s modifications: crystal violet, grams iodine and de-colourizers (ethanol, 95%)

Acetone-alcohol mixture with ethanol at, 95% (100 ml) and acetone (reagent grade,  100 ml)

c) Equipments

  • Centrifuge
  • Electric slide warmer, 60C
  • Cytospin centrifuge
  • Vortex mixer
  • Tissue grinder
  • Scalpels, sterile scissors and forceps
  • Sterile tubes, screw cap

Procedure

  • Wear latex gloves for protection when handling clinical specimens
  • Put a slide containing a bacterial smear on a staining rack
  • For between 1-2 minutes, stain the slide with crystal violet
  • Pour out the stain
  • For between 1 and 2 minutes, flood the slide with Gram’s iodine after which pour off the iodine
  • With the help of acetone, decolorize the slide by washing for 2-3 seconds
  • Immediately wash with water to get rid of the acetone
  • Use safranin couterstain to flood the slide
  • Wash with water
  • Blot excess water and dry over Bunsen flame

Results

i) Gram reaction

Given that gram reaction is based on the structure of the bacterial cell wall, the dark purple violet stain is on the thick layer of peptidoglycan which is visible on the outer cell layer for Gram positive bacteria.

ii) Cell morphology

Observe the different cell shapes formed

Microscopy (as part of gram staining)

Use of microscope (handling and care)

Use two hands at all times when moving the microscope with one hand around the arm and the other under the base of scope for support, always be gentle when setting the microscope to avoid setting the lenses loose. Make sure you have clean hands when handling microscope

Procedure (proper use of objectives and focusing,)

  • Start by observing the specimen using the lowest power object first.
  • With the help of coarse adjustment knob, bring the object into focus
  • Use fine adjustment knob to bring the object into sharp focus
  • Keep both eyes open to minimize eyestrain, if necessary, focus and then move to higher power objective.

Performed differential biochemical or other differential tests

Differential biochemical tests are used to characterize bacteria species. For every biochemical process performed, there is need to have the right specie-specific enzymes. An identification of organisms can therefore be done by looking at the pattern of biochemical activity.

Materials required to carry out differential tests

  • Micropipettors
  • Vortexer
  • Thermocycler
  • Microcentrifuge tube
  • Sodium chloride
  • Yeast extract
  • Broth

The tests include:

a)    Nitrate reduction test.

This test determines the ability of an organism to reduce nitrate. Some organisms reduce nitrate to nitrite and others produce nitrite that end up in nitrogen gas. In the event that nitrite is produced, the broth will turn red, if the broth remains clear, there is need to conduct further tests to establish if no reduction has taken place.

Add zinc dust to reduce any left nitrate this will turn the broth pink, an indication of negative teat for reduction in nitrate.

If after the addition of zinc dust, the broth stay clear, then the organism reduced nitrate to some other nitrogenous compound, something that is called “positive complete”

The identified organisms are therefore Eschericha coli for red, Pseudomonas aeruginosa for clear and Acinetobacer calcoaceticus for pink broth respectively.

b)   Phenol red broth.

This is a differential test that helps in determining the ability of an organism to ferment sugars. To conduct this test, add sugar and phenol to peptone medium using a small inverted tube to capture any gas produced.

If an organism has the ability to metabolize sugar, an acid is subsequently produced and the indicator turns yellow. Any gas by product produced creates a bubble in the small inverted tube. If the results are yellow and there is no gas then the organism present is Staphylococcus aureus.

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