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

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 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: 2261-01 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

Streak Plating Technique

Distinguishing Cellular Morphology Observed with 100X obj

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

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

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

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