{"id":379,"date":"2025-12-15T17:00:57","date_gmt":"2025-12-15T17:00:57","guid":{"rendered":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/?post_type=chapter&p=379"},"modified":"2026-07-28T18:09:32","modified_gmt":"2026-07-28T18:09:32","slug":"exercise-11-enriched-media","status":"publish","type":"chapter","link":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/chapter\/exercise-11-enriched-media\/","title":{"raw":"Exercise 11: Enriched Media","rendered":"Exercise 11: Enriched Media"},"content":{"raw":"

Introduction<\/h2>\r\n \r\n\r\nAlthough many organisms grow on general purpose media such as TSA and Nutrient Agar some microorganisms require more specific nutritional requirements. Organisms that require these special nutrients are called fastidious<\/strong> organisms. Examples include Neisseria gonorrheae<\/em>, Haemophilus influenza<\/em>, and some Streptococcus<\/em> species. Blood and blood products such as serum are common supplements added to general purpose media to allow growth of the fastidious organisms. With the supplement it is considered an enriched medium<\/strong>. The most common type of enriched media is blood agar plates (BAP)<\/strong> that are made by adding a concentration of 5% sheep blood to TSA medium.\r\n\r\nIn addition to improving growth of some fastidious organisms, BAP also has differential properties. Certain organisms produce special exotoxins called hemolysins<\/strong> that break down red blood cells and hemoglobin. This hemolysis of the blood causes color changes in the bright red BAP medium. The three possible results for hemolysis testing are \u03b2-hemolysis, \u03b1-hemolysis, or \u03b3-hemolysis.\r\n
    \r\n \t
  • beta-hemolysis (\u03b2):<\/strong> The complete destruction of RBCs, results in clearing of the medium around colonies<\/li>\r\n \t
  • alpha-hemolysis (\u03b1)<\/strong>: Partial destruction of RBCs, produces an olive-greenish discoloration of the medium around colonies<\/li>\r\n \t
  • gamma-hemolysis (\u03b3)<\/strong>: No hemolysis occurs; appears as simple growth with no change to the medium<\/li>\r\n<\/ul>\r\nSeveral species of Streptococcus<\/em> produce hemolysins called streptolysins<\/strong>. Streptolysin O is an oxygen labile form that is destroyed by oxygen and only works anaerobically. Some Enterococcus<\/em> species also produce hemolysins making the BAP a useful diagnostic medium.\r\n

    Course Intended Outcomes<\/h2>\r\n
      \r\n \t
    1. Describe different types of media according to their physical state, chemical composition and their function and list some of their applications; interpret microbial growth patterns.<\/li>\r\n \t
    2. Describe and perform at least 3 biochemical tests and describe their roles in microbial identification. Interpret results obtained and draw conclusions from biochemical tests performed.<\/li>\r\n<\/ol>\r\n

      Materials Used<\/h2>\r\nStudents will work in small groups. Each group will need the following materials for this exercise.\r\n
        \r\n \t
      • Bunsen burner<\/li>\r\n \t
      • Inoculation loop<\/li>\r\n \t
      • Test tube rack<\/li>\r\n<\/ul>\r\nAdditional materials and cultures will be assigned by your instructor.\r\n\r\n \r\n

        Procedure, Day 1<\/h2>\r\n

        Technique:<\/h3>\r\n
          \r\n \t
        1. Refer to Exercise 2 for proper use of instruments and aseptic technique.<\/li>\r\n \t
        2. Depending on the directions provided by your instructor you may begin inoculation of the BAP with a sterile loop or a sterile swab.<\/li>\r\n \t
        3. If placing multiple organisms on the same plate, students should use a single line streak for inoculation. Make sure to avoid cross-contamination of the bacterial cultures used.<\/li>\r\n \t
        4. Any time just one organism is used for inoculation (such as during unknown identification) students should perform the isolation streak. This will provide isolated colonies and allow for quick recognition of contamination.<\/li>\r\n \t
        5. After initial inoculation use the tip of the loop or a sterile needle to \u201cstab\u201d some of the culture to the bottom of the plate in a few spots. This will allow recognition of any oxygen labile hemolysins.<\/li>\r\n<\/ol>\r\n \r\n

          Sampling\/Inoculation:<\/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.\r\n
              \r\n \t
            • Place all tubes in a tube rack.<\/li>\r\n \t
            • Never hold a culture tube by its cap.<\/li>\r\n \t
            • Bacterial culture tubes are always to be shared with your table group.<\/li>\r\n<\/ul>\r\n<\/li>\r\n \t
            • Clearly label plate (on the bottom side) with your group name or initials, organism name(s), and section day\/time.<\/li>\r\n \t
            • Place inoculated plates in the rack labeled \u201cto be incubated\u201d on the back bench. Always place the plates with lids down and bottoms up.<\/li>\r\n<\/ol>\r\n \r\n

              Procedure, Day 2<\/h2>\r\n
                \r\n \t
              1. Collect your group\u2019s plates and tubes from the back bench area labeled \u201clast week\u2019s lab\u201d.<\/li>\r\n \t
              2. Examine the plate or plates inoculated during the previous period.<\/li>\r\n \t
              3. Hold the plate up toward the ceiling so that it is backlit by the lights.<\/li>\r\n \t
              4. Focus on the edge of the bacterial growth and look for clearing or discoloration in the media.<\/li>\r\n \t
              5. If you cannot determine the reaction, you can use a sterile loop to scrape away a small section of growth so you can see the media underneath it. If the media has turned green\/brown it is alpha. If there\u2019s no change it is gamma. If the blood has been cleared it is beta.<\/li>\r\n<\/ol>\r\n[caption id=\"attachment_261\" align=\"alignnone\" width=\"589\"]\"Showing Figure 11.1: Image showing gamma, nonhemolytic reaction (A), alpha hemolytic reaction (B) and beta hemolytic reaction (C) on 5% Sheep Blood Agar (BAP).[\/caption]\r\n\r\n \r\n

                Lab Clean-up<\/h2>\r\nReturn bacteria cultures to the front bench after inoculations are complete.\r\n\r\nPlates are disposable and should be thrown away in the biohazard container when everyone in the group has completed the exercise.\r\n\r\nDisinfect your benchtop and wash your hands before leaving the lab.\r\n\r\n \r\n
                \r\n

                Results and Interpretation<\/h2>\r\n1.\u00a0\u00a0\u00a0\u00a0\u00a0 Record your results in the table below.\r\n

                Medium Used: Blood Agar Plate (BAP)<\/p>\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n
                \r\n

                Organism Name<\/p>\r\n<\/th>\r\n

                		\r\n

                Growth\u00a0 (G,NG,i)<\/p>\r\n<\/th>\r\n

                		\r\n

                Result (\u03b1,\u03b2,\u03b3)<\/p>\r\n<\/th>\r\n

                		\r\n

                Comments<\/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<\/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<\/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<\/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<\/tr>\r\n<\/tbody>\r\n<\/table>\r\n \r\n

                Critical Thinking Questions<\/h2>\r\n1.\u00a0\u00a0\u00a0\u00a0\u00a0 Streaking throat cultures on BAP was a common practice for many years. What diagnostic purpose did it serve?\r\n\r\n<\/div>\r\n \r\n\r\n \r\n
                \r\n\r\n2.\u00a0\u00a0\u00a0\u00a0\u00a0 The presence of some hemolysins may be missed if you do NOT stab the media during inoculation. Why?\r\n\r\n \r\n\r\n<\/div>\r\n ","rendered":"

                Introduction<\/h2>\n

                 <\/p>\n

                Although many organisms grow on general purpose media such as TSA and Nutrient Agar some microorganisms require more specific nutritional requirements. Organisms that require these special nutrients are called fastidious<\/strong> organisms. Examples include Neisseria gonorrheae<\/em>, Haemophilus influenza<\/em>, and some Streptococcus<\/em> species. Blood and blood products such as serum are common supplements added to general purpose media to allow growth of the fastidious organisms. With the supplement it is considered an enriched medium<\/strong>. The most common type of enriched media is blood agar plates (BAP)<\/strong> that are made by adding a concentration of 5% sheep blood to TSA medium.<\/p>\n

                In addition to improving growth of some fastidious organisms, BAP also has differential properties. Certain organisms produce special exotoxins called hemolysins<\/strong> that break down red blood cells and hemoglobin. This hemolysis of the blood causes color changes in the bright red BAP medium. The three possible results for hemolysis testing are \u03b2-hemolysis, \u03b1-hemolysis, or \u03b3-hemolysis.<\/p>\n

                  \n
                • beta-hemolysis (\u03b2):<\/strong> The complete destruction of RBCs, results in clearing of the medium around colonies<\/li>\n
                • alpha-hemolysis (\u03b1)<\/strong>: Partial destruction of RBCs, produces an olive-greenish discoloration of the medium around colonies<\/li>\n
                • gamma-hemolysis (\u03b3)<\/strong>: No hemolysis occurs; appears as simple growth with no change to the medium<\/li>\n<\/ul>\n

                  Several species of Streptococcus<\/em> produce hemolysins called streptolysins<\/strong>. Streptolysin O is an oxygen labile form that is destroyed by oxygen and only works anaerobically. Some Enterococcus<\/em> species also produce hemolysins making the BAP a useful diagnostic medium.<\/p>\n

                  Course Intended Outcomes<\/h2>\n
                    \n
                  1. Describe different types of media according to their physical state, chemical composition and their function and list some of their applications; interpret microbial growth patterns.<\/li>\n
                  2. Describe and perform at least 3 biochemical tests and describe their roles in microbial identification. Interpret results obtained and draw conclusions from biochemical tests performed.<\/li>\n<\/ol>\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<\/ul>\n

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

                       <\/p>\n

                      Procedure, Day 1<\/h2>\n

                      Technique:<\/h3>\n
                        \n
                      1. Refer to Exercise 2 for proper use of instruments and aseptic technique.<\/li>\n
                      2. Depending on the directions provided by your instructor you may begin inoculation of the BAP with a sterile loop or a sterile swab.<\/li>\n
                      3. If placing multiple organisms on the same plate, students should use a single line streak for inoculation. Make sure to avoid cross-contamination of the bacterial cultures used.<\/li>\n
                      4. Any time just one organism is used for inoculation (such as during unknown identification) students should perform the isolation streak. This will provide isolated colonies and allow for quick recognition of contamination.<\/li>\n
                      5. After initial inoculation use the tip of the loop or a sterile needle to \u201cstab\u201d some of the culture to the bottom of the plate in a few spots. This will allow recognition of any oxygen labile hemolysins.<\/li>\n<\/ol>\n

                         <\/p>\n

                        Sampling\/Inoculation:<\/h3>\n
                          \n
                        1. Your instructor will assign the cultures to be used for inoculation by your group.<\/li>\n
                        2. Collect the required materials for the lab.\n
                            \n
                          • Place all tubes in a tube rack.<\/li>\n
                          • Never hold a culture tube by its cap.<\/li>\n
                          • Bacterial culture tubes are always to be shared with your table group.<\/li>\n<\/ul>\n<\/li>\n
                          • Clearly label plate (on the bottom side) with your group name or initials, organism name(s), and section day\/time.<\/li>\n
                          • Place inoculated plates in the rack labeled \u201cto be incubated\u201d on the back bench. Always place the plates with lids down and bottoms up.<\/li>\n<\/ol>\n

                             <\/p>\n

                            Procedure, Day 2<\/h2>\n
                              \n
                            1. Collect your group\u2019s plates and tubes from the back bench area labeled \u201clast week\u2019s lab\u201d.<\/li>\n
                            2. Examine the plate or plates inoculated during the previous period.<\/li>\n
                            3. Hold the plate up toward the ceiling so that it is backlit by the lights.<\/li>\n
                            4. Focus on the edge of the bacterial growth and look for clearing or discoloration in the media.<\/li>\n
                            5. If you cannot determine the reaction, you can use a sterile loop to scrape away a small section of growth so you can see the media underneath it. If the media has turned green\/brown it is alpha. If there\u2019s no change it is gamma. If the blood has been cleared it is beta.<\/li>\n<\/ol>\n
                              \"Showing
                              Figure 11.1: Image showing gamma, nonhemolytic reaction (A), alpha hemolytic reaction (B) and beta hemolytic reaction (C) on 5% Sheep Blood Agar (BAP).<\/figcaption><\/figure>\n

                               <\/p>\n

                              Lab Clean-up<\/h2>\n

                              Return bacteria cultures to the front bench after inoculations are complete.<\/p>\n

                              Plates are disposable and should be thrown away in the biohazard container when everyone in the group has completed the exercise.<\/p>\n

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

                               <\/p>\n

                              \n

                              Results and Interpretation<\/h2>\n

                              1.\u00a0\u00a0\u00a0\u00a0\u00a0 Record your results in the table below.<\/p>\n

                              Medium Used: Blood Agar Plate (BAP)<\/p>\n\n\n\n\n\n\n\n\n
                              \n

                              Organism Name<\/p>\n<\/th>\n

                              		\n

                              Growth\u00a0 (G,NG,i)<\/p>\n<\/th>\n

                              		\n

                              Result (\u03b1,\u03b2,\u03b3)<\/p>\n<\/th>\n

                              		\n

                              Comments<\/p>\n<\/th>\n<\/tr>\n<\/thead>\n

                              \n

                              \n<\/td>\n

                              		\n

                              \n<\/td>\n

                              		\n

                              \n<\/td>\n

                              		\n

                              \n<\/td>\n<\/tr>\n

                              \n

                              \n<\/td>\n

                              		\n

                              \n<\/td>\n

                              		\n

                              \n<\/td>\n

                              		\n

                              \n<\/td>\n<\/tr>\n

                              \n

                              \n<\/td>\n

                              		\n

                              \n<\/td>\n

                              		\n

                              \n<\/td>\n

                              		\n

                              \n<\/td>\n<\/tr>\n

                              \n

                              \n<\/td>\n

                              		\n

                              \n<\/td>\n

                              		\n

                              \n<\/td>\n

                              		\n

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

                               <\/p>\n

                              Critical Thinking Questions<\/h2>\n

                              1.\u00a0\u00a0\u00a0\u00a0\u00a0 Streaking throat cultures on BAP was a common practice for many years. What diagnostic purpose did it serve?<\/p>\n<\/div>\n

                               <\/p>\n

                               <\/p>\n

                              \n

                              2.\u00a0\u00a0\u00a0\u00a0\u00a0 The presence of some hemolysins may be missed if you do NOT stab the media during inoculation. Why?<\/p>\n

                               <\/p>\n<\/div>\n

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