{"id":317,"date":"2024-08-13T15:42:43","date_gmt":"2024-08-13T15:42:43","guid":{"rendered":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/?post_type=chapter&p=317"},"modified":"2026-07-30T17:42:32","modified_gmt":"2026-07-30T17:42:32","slug":"exercise-6-gram-stain","status":"publish","type":"chapter","link":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/chapter\/exercise-6-gram-stain\/","title":{"raw":"Exercise 6: Gram Stain","rendered":"Exercise 6: Gram Stain"},"content":{"raw":"

\u00a0Introduction<\/h2>\r\n

Differential staining procedures allow a microbiologist to detect differences between organisms in addition to determining morphology (shape and arrangement). A differential stain is a procedure that includes dyes with contrasting colors. Bacterial cells will retain one or the other dye at the end of the procedure based on differences in their cell structure. A trained eye will be able to recognize the difference in color with a light microscope under 1000X (oil immersion) power.<\/p>\r\n

The Gram stain is the most common and useful differential stain. This Gram staining procedure consists of the following steps in order: a primary stain, a mordant, a decolorizer and a counterstain. Crystal violet stain is the primary stain<\/strong> and is a dark purple color. Adding the mordant<\/strong> (iodine) helps the crystal violet adhere to the cell structures. Decolorization occurs after the mordant is added and is the most critical step in the procedure. Ethanol is the decolorizer <\/strong>used to extract the primary dye from the smear. It must be added for a brief period of time only (usually 10 \u2013 15 seconds). During this time some cell types (Gram-negatives) will lose the crystal violet while other cell types (Gram-positives) retain it. The final step is the addition of the counterstain<\/strong>, safranin. The Gram-negative<\/strong> cells that have lost their purple color will be colorized by the red counterstain. The Gram-positive<\/strong> cells retain their purple color and are unchanged by the addition of safranin.<\/p>\r\n

Differences in Gram-positive and Gram-negative wall composition result in the different responses to decolorization. Keep in mind that all cells can be stained with the crystal violet and safranin as simple stains. So, it is not the stains themselves that make this a differential stain. It is the staining process (steps performed in the correct sequence) that determines the outcome. The more consistently you can perform these steps, the more reliable your results will be. Unfortunately, some Gram-positive organisms (especially Bacillus<\/em> species) break down quickly leading to a Gram-variable<\/strong> result. Variable stains will have a mixture of purple and red cells with identical morphology. Using a young culture (12-18 hours) can usually resolve the issue, so Gram stains with variable results should always be repeated.<\/p>\r\n

In addition to the Gram stain results, students should also note the cellular morphology<\/strong> (shape and arrangement) of the specimens. Each bacterial species that you observe will have a characteristic cellular morphology. Cells may be spheres (cocci<\/strong>, singular coccus<\/strong>), rods (bacilli<\/strong>, singular bacillus<\/strong>), or spiral shaped (spirilla<\/strong>). Additional varieties of these shapes include slightly curved rods (vibrios<\/strong>), short rods (coccobacilli<\/strong>), and flexible spirals (spirochetes<\/strong>). The cell arrangement describes how the cells look in small groups and is determined by planes in which division occurs and whether the cells stay attached after division (see figure 6-1). Common arrangements include strepto<\/strong>-<\/strong> (chains), staphylo<\/strong>-<\/strong> (grape-like clusters), and tetrads<\/strong> (packs of 4).<\/p>\r\n \r\n\r\n[caption id=\"\" align=\"alignnone\" width=\"675\"]\"Chart Figure 6.1: Prokaryotic cell arrangements with names, descriptions, and illustrations of seven different cell arrangements. Common Prokaryotic Cell Arrangements<\/em> (Figure 3.14) by Nina Parker, Mark Schneegurt, Anh-Hue Thi Tu, Philip Lister, and Brian M. Forster, from Microbiology<\/em> by OpenStax, is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License<\/a>. View the original source<\/a>\u00a0[\/caption]\r\n\r\n[pb_glossary id=\"622\"]Fig 6.1 long description [\/pb_glossary]\r\n

Course Intended Outcomes<\/h2>\r\n

Perform and understand the basis of different types of microbiological staining techniques (e.g., simple stain and gram stain) to characterize and identify bacteria.<\/p>\r\n \r\n

Materials Used<\/h2>\r\n

Students will work in small groups. Each group will need the following materials for this exercise.<\/p>\r\n\r\n