Laboratory 4l
Characterizing E. coli Using a Light
Microscope and Gram Staining
Background
In Lab 4g and 4h, E. coli cells were cultured and their DNA was extracted. How do you know,
though, if you’ve cultured E. coli or if you have mistakenly cultured some other bacteria?
Scientists study an organism’s characteristics, such as structure (morphology), nutritional
requirements, and behavior. Once these traits are characterized, it is easier to recognize the
organism in the future.
Gram staining provides an opportunity to characterize bacteria based on the cell wall
structure. By using a Gram stain, you can identify six different groups of bacteria. There are three
shapes of bacteria: coccus (spherical), bacillus (rod-shaped), and spirillum (spiral). Gram staining
separates these three groups into two color groups: purple and red. The purple or red color is
determined by the bacterium’s cell wall type. Bacteria that have a thick cell wall retain the crystal
violet stain and are purple in color. Bacteria that have a thin cell wall lose the purple color during
decolorization and must be stained with a red counterstain, called safranin. When properly
Gram-stained, E. coli appear as light red rods (Gram– bacilli) on 1000X.
Purpose
Is there evidence that a bacteria culture contains Gram-negative (Gram–) E. coli cells?
Materials
E. coli broth culture, small
sample saved from Lab 4g
Microscope, compound, with
100X objective lens
Microscope slides
Immersion oil
Microscope slide coverslips,
glass
dH2O
Inoculating loop
Bunsen burner
Crystal violet stain
Ethanol (EtOH)
Safranin stain
Procedure
1. Make a wet mount of 10 µL of the E. coli broth culture. Use the oil immersion lens to observe
these unstained bacteria cells. In your notebook, draw and label the cells showing their cell
wall, cytoplasm, and nucleoid area (darkened by DNA)