{"id":326,"date":"2024-08-13T18:17:10","date_gmt":"2024-08-13T18:17:10","guid":{"rendered":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/?post_type=chapter&p=326"},"modified":"2026-07-30T14:55:57","modified_gmt":"2026-07-30T14:55:57","slug":"exercise-7","status":"publish","type":"chapter","link":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/chapter\/exercise-7\/","title":{"raw":"Exercise 7: Negative Stain","rendered":"Exercise 7: Negative Stain"},"content":{"raw":"

Introduction<\/h2>\r\n

Certain bacteria are unable to maintain their natural shape and arrangement after heating. The negative stain<\/strong> is used to view cellular morphology without the influence of heat-fixing. The specimen is mixed into a suspension in a negative stain. An acidic dye<\/strong> is used as the chromogen. The negatively charged, acidic chromogen is repelled by the cell\u2019s proteins and other structures leaving the cell unstained against a dark background. One of the most common acidic dyes used for a negative stain is nigrosin<\/strong> that provides a purple background. It may be used to stain delicate cells such as spirochetes so that their shape and size can be properly measured.<\/p>\r\n\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

    \r\n \t
  • Bunsen burner<\/li>\r\n \t
  • Inoculation loop<\/li>\r\n \t
  • Test tube rack<\/li>\r\n \t
  • Bottle of nigrosin stain<\/li>\r\n<\/ul>\r\nAdditional materials and cultures will be assigned by your instructor\r\n\r\n \r\n

    Procedure<\/h2>\r\n

    Technique:<\/strong><\/h3>\r\n
      \r\n \t
    1. Apply 1 drop of nigrosin stain toward one end of a clean slide.<\/li>\r\n \t
    2. Use a sterile inoculating loop to add a small amount of culture to the drop of nigrosin. Emulsify the culture into the stain but do not spread out the suspension.<\/li>\r\n \t
    3. Using the short edge of a second clean slide press it against the slide at a 40 degree angle. Next, draw it backward into the suspension.<\/li>\r\n \t
    4. When the suspension spreads along the edge quickly push the suspension across the surface of the bottom slide.<\/li>\r\n \t
    5. Allow the bottom slide to dry completely then view with the microscope.<\/li>\r\n \t
    6. Disinfect the top\/spreader slide with bleach then wash with detergent and return to your slide box.<\/li>\r\n \t
    7. Do NOT heat fix.<\/li>\r\n<\/ol>\r\n[caption id=\"\" align=\"alignnone\" width=\"755\"]\"Step-by-step Figure 7.1: Step-by-step diagram illustrating the Negative staining procedure.[\/caption]\r\n\r\n[pb_glossary id=\"627\"]Fig 7.1 long description[\/pb_glossary]\r\n\r\n \r\n\r\n[caption id=\"attachment_331\" align=\"alignnone\" width=\"514\"]\"Microscopic Figure 7.2: Negative stain of M. luteus tetrads[\/caption]\r\n

      Sampling\/Inoculation:<\/strong><\/h3>\r\n
        \r\n \t
      1. Your instructor will assign the cultures to be used for inoculation by your group.<\/li>\r\n \t
      2. Collect the required materials for the lab.<\/li>\r\n \t
      3. Label your slides using a wax pencil with the organism initials (e.g., EC = Escherichia coli). This will prevent mixing up your slides and help with slide orientation.<\/li>\r\n<\/ol>\r\n \r\n

        Lab Clean-up<\/h2>\r\n

        Wash slides with detergent, rinse and dry. Return slides to your slide box and store in the bin labeled for your section.<\/p>\r\n

        Return bacteria cultures to the front bench.<\/p>\r\n

        Thoroughly clean your microscope and return it to the lab prep room.<\/p>\r\n

        Disinfect your benchtop and wash your hands before leaving the lab.<\/p>\r\n \r\n

        Results and Interpretation<\/h2>\r\n

        Record your results in your lab notebook creating a table as shown.<\/p>\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n
        \r\n

        Specimen Name \/ Source<\/p>\r\n<\/th>\r\n

        		\r\n

        Background<\/p>\r\n

        Color<\/p>\r\n<\/th>\r\n

        		\r\n

        Cell Color<\/p>\r\n<\/th>\r\n

        		\r\n

        Shape<\/p>\r\n<\/th>\r\n

        		\r\n

        Arrangement<\/p>\r\n<\/th>\r\n<\/tr>\r\n<\/thead>\r\n

        \r\n

        <\/p>\r\n<\/td>\r\n

        		\r\n

        <\/p>\r\n<\/td>\r\n

        		\r\n

        <\/p>\r\n<\/td>\r\n

        		\r\n

        <\/p>\r\n<\/td>\r\n

        		\r\n

        <\/p>\r\n<\/td>\r\n<\/tr>\r\n

        \r\n

        <\/p>\r\n<\/td>\r\n

        		\r\n

        <\/p>\r\n<\/td>\r\n

        		\r\n

        <\/p>\r\n<\/td>\r\n

        		\r\n

        <\/p>\r\n<\/td>\r\n

        		\r\n

        <\/p>\r\n<\/td>\r\n<\/tr>\r\n

        \r\n

        <\/p>\r\n<\/td>\r\n

        		\r\n

        <\/p>\r\n<\/td>\r\n

        		\r\n

        <\/p>\r\n<\/td>\r\n

        		\r\n

        <\/p>\r\n<\/td>\r\n

        		\r\n

        <\/p>\r\n<\/td>\r\n<\/tr>\r\n<\/tbody>\r\n<\/table>\r\n \r\n

        Critical Thinking Questions<\/h2>\r\n

        Why are the cells spread in a suspension instead of applying the stain to an air-dried smear?<\/p>\r\n

        What is the reason that the cells remain colorless after applying the stain?<\/p>","rendered":"

        Introduction<\/h2>\n

        Certain bacteria are unable to maintain their natural shape and arrangement after heating. The negative stain<\/strong> is used to view cellular morphology without the influence of heat-fixing. The specimen is mixed into a suspension in a negative stain. An acidic dye<\/strong> is used as the chromogen. The negatively charged, acidic chromogen is repelled by the cell\u2019s proteins and other structures leaving the cell unstained against a dark background. One of the most common acidic dyes used for a negative stain is nigrosin<\/strong> that provides a purple background. It may be used to stain delicate cells such as spirochetes so that their shape and size can be properly measured.<\/p>\n

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

        Materials Used<\/h2>\n

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

          \n
        • Bunsen burner<\/li>\n
        • Inoculation loop<\/li>\n
        • Test tube rack<\/li>\n
        • Bottle of nigrosin stain<\/li>\n<\/ul>\n

          Additional materials and cultures will be assigned by your instructor<\/p>\n

           <\/p>\n

          Procedure<\/h2>\n

          Technique:<\/strong><\/h3>\n
            \n
          1. Apply 1 drop of nigrosin stain toward one end of a clean slide.<\/li>\n
          2. Use a sterile inoculating loop to add a small amount of culture to the drop of nigrosin. Emulsify the culture into the stain but do not spread out the suspension.<\/li>\n
          3. Using the short edge of a second clean slide press it against the slide at a 40 degree angle. Next, draw it backward into the suspension.<\/li>\n
          4. When the suspension spreads along the edge quickly push the suspension across the surface of the bottom slide.<\/li>\n
          5. Allow the bottom slide to dry completely then view with the microscope.<\/li>\n
          6. Disinfect the top\/spreader slide with bleach then wash with detergent and return to your slide box.<\/li>\n
          7. Do NOT heat fix.<\/li>\n<\/ol>\n
            \"Step-by-step
            Figure 7.1: Step-by-step diagram illustrating the Negative staining procedure.<\/figcaption><\/figure>\n