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Growth media
Sterile source of nutrients and water for bacteria to growSemisolid media contain dissolved agar which will
provide a solid surface for the organisms to growBroth media contains no agar
Both types of media are incubated for 24-48 hours at 25-45˚C.
Distinguishing by Colony Morphologies
What to do:
• Examine the stock culture plates
• Record appropriate descriptive terms for the colony morphology
Streak Plate Method
What to do:Sterilize a loop and capture
a loopful of brothStreak the plate in 3 or 4
sections, taking care to sterilize the loop between and after each streak
Always label the bottom of the plate
Prepare the bacteria
• Prepare a bacterial smear1. Suspend one colony in one drop of water on
a clean microscope slide
2. Spread the suspension very thin
3. Allow the smear to air-dry• Do NOT try to rush this step!
4. Fix the bacteria to the slide by passing through a flame 2 or 3 times
Gram Staining
1. Primary stain – flood smear with crystal violet• Rinse with water
2. Cover smear with iodine - Mordant• Rinse with water
3. Decolorize – gently rinse slide with acetone-alcohol, 3X
• Rinse with water
4. Counterstain with safranin• Rinse with water
5. Dry slide with blotting paper
Spore Stain
1. Trim a piece of blotting paper to the size of the smear
2. Flood with malachite green dye
3. Steam over open flame for 5 min Keep paper wet with malachite dye
4. Rinse slide with a stream of water
5. Counterstain with safranin
6. Blot-dry
Microscope Objectives
• Start with low power (4X) and work your way up to oil immersion (100X)
4X 10X 100X
The 100X oil immersion lens
• Add ONE drop of oil to the slide
• Click 100X objective into place
• Use course focus (turn very slowly) then fine focus – scan field slowly
Image sharpest in the center
Visualize your smears
• Start with low power and work your way up to oil immersion
• Observe shape, arrangements, Gram reaction
Rapid Biochemical ID
• Inoculate test system and incubate overnight
• Read results using a light background and a UV lamp
• Get your 10-digit code and email/call/text me for the ID