Microbial Ecology – From Isolation to Identification

Dr. Melanie Griffin

Ribit Summer 2011

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

Streak Plating Technique

Distinguishing Cellular Morphology

Observed with

100X obj

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

• mgriff40@kennesaw.edu