Biotech 2nd Edition Sample Ch. 4 Biotechnology Sample Ch. 4 - Page 32

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)