1. Microbial Ecology – From Isolation to Identification
Ribit Summer 2011
Microbial Ecology – From Isolation to Identification
Dr. Melanie Griffin

2. Growth media
Sterile source of nutrients and water for bacteria to grow
Semisolid media contain dissolved agar which will provide a solid surface for the organisms to grow
Broth media contains no agar
Both types of media are incubated for hours at 25-45˚C.

3. Distinguishing by Colony Morphologies
What to do:
Examine the stock culture plates
Record appropriate descriptive terms for the colony morphology

4. Streak Plate Method What to do:
Initials; Grp#, date, title
What to do:
Sterilize a loop and capture a loopful of broth
Streak 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

5. Streak Plating Technique

6. Distinguishing Cellular Morphology
Observed with 100X obj

7. Prepare the bacteria Prepare a bacterial smear
Suspend one colony in one drop of water on a clean microscope slide
Spread the suspension very thin
Allow the smear to air-dry
Do NOT try to rush this step!
Fix the bacteria to the slide by passing through a flame 2 or 3 times

8. Gram Staining Primary stain – flood smear with crystal violet
Rinse with water
Cover smear with iodine - Mordant
Decolorize – gently rinse slide with acetone-alcohol, 3X
Counterstain with safranin
Dry slide with blotting paper

9. Spore Stain Trim a piece of blotting paper to the size of the smear
Flood with malachite green dye
Steam over open flame for 5 min
Keep paper wet with malachite dye
Rinse slide with a stream of water
Counterstain with safranin
Blot-dry

10. Microscope Objectives
Start with low power (4X) and work your way up to oil immersion (100X)
4X X X

11. 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

12. Visualize your smears
Start with low power and work your way up to oil immersion
Observe shape, arrangements, Gram reaction

13. 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 /call/text me for the ID