{"id":357,"date":"2024-08-14T22:27:17","date_gmt":"2024-08-14T22:27:17","guid":{"rendered":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/?post_type=chapter&p=357"},"modified":"2025-12-19T01:48:59","modified_gmt":"2025-12-19T01:48:59","slug":"oxygen-requirements","status":"publish","type":"chapter","link":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/chapter\/oxygen-requirements\/","title":{"raw":"Exercise 10: Oxygen Requirements","rendered":"Exercise 10: Oxygen Requirements"},"content":{"raw":"

Introduction<\/h2>\r\n

Microorganisms can survive within a range of environmental conditions but will be most productive and grow most densely in areas where conditions are MOST favorable. Oxygen (O2<\/sub>) is one important resource that can influence microbial growth. Some organisms require oxygen for growth. Others don\u2019t use it at all and are not affected by its presence or absence. Some organisms cannot even survive in the presence of oxygen. Aerobic respiration<\/strong> is a metabolic process that requires oxygen to synthesize ATP. Anaerobic respiration<\/strong> and fermentation<\/strong> are metabolic processes that do NOT require oxygen.<\/p>\r\n

Organisms that are obligate aerobes<\/strong> are only capable of aerobic respiration and therefore require oxygen for growth. In thioglycollate medium, they grow only at the top where oxygen is most plentiful. Facultative anaerobes<\/strong> can switch between aerobic or anaerobic processes. These organisms grow best in the presence of oxygen but can grow without it. Growth will be seen throughout the medium but is most dense at the top of the tube. Aerotolerant<\/strong> anaerobes<\/strong> are organisms that don\u2019t utilize oxygen but can tolerate exposure to it without adverse effects. Growth of these organisms is uniform throughout the medium from top to bottom. Obligate<\/strong> anaerobes<\/strong> are organisms for which oxygen is a lethal toxin and, therefore will only grow in the lower portions of the medium where there is no dissolved oxygen. Microaerophiles<\/strong>, as the name suggests, survive only in environments containing very small levels (3-10%) of oxygen. Too much or too little oxygen will kill these organisms making them very difficult with which to work.<\/p>\r\n

Fluid thioglycollate medium is adapted for cultivation of organisms with any oxygen requirements including obligate anaerobes and microaerophiles. It contains a small amount of agar to slow the diffusion of oxygen. It also contains an indicator (reazurin<\/strong>) that turns pink in the presence of oxygen. The anaerobic jar is an airtight container used with a gas generating pouch that allows cultivation of anaerobic bacteria. The large white envelope (gas generator packet) placed in the jar removes all oxygen, making conditions inside the jar anaerobic.<\/p>\r\n \r\n

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

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.<\/p>\r\n

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.<\/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
  • Flint striker<\/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. Groups will inoculate one thioglycollate tube for each organism assigned.<\/li>\r\n \t
    3. Check that thioglycollate tubes have a pink layer at the top.<\/li>\r\n \t
    4. Use a sterile loop to inoculate thioglycollate tubes from the top to the bottom without disturbing the oxygenated (pink) and anaerobic (amber) layers.<\/li>\r\n \t
    5. After inoculations make sure that your caps are loosened one half turn to allow air to enter and exit the tube during incubation.<\/li>\r\n \t
    6. Groups will also inoculate two plates \u2013 one for growth in the presence of oxygen (aerobic) and one for growth in the absence of oxygen (anaerobic).<\/li>\r\n \t
    7. The plates used in this lab exercise are tri-part plates. The media surface is divided for you into three sections to accommodate inoculation of three organisms on the same plate.<\/li>\r\n \t
    8. Groups will inoculate the plates in duplicate. In other words, the plates will be identical with the same organisms in the same order. Remember to label and inoculate carefully.<\/li>\r\n \t
    9. Aerobic incubation will occur in a standard incubator with atmospheric O2<\/sub> levels.<\/li>\r\n \t
    10. The anaerobic jar will be used with a gas generator packet to create an oxygen free environment for the anaerobic plates.<\/li>\r\n<\/ol>\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 your tubes using label tape. Include your group name or initials, section day\/time, and the organism name.<\/li>\r\n \t
        • Clearly label plates (on the bottom side) with your group name or initials, organism name, and section day\/time.<\/li>\r\n \t
        • Label one of your duplicate plates \u201cAerobic\u201d for standard incubation.<\/li>\r\n \t
        • Label the other duplicate plate \u201cAnaerobic\u201d for incubation in the anaerobic jar.<\/li>\r\n \t
        • Continue with inoculation as directed by your instructor.<\/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. Observe your thioglycollate tubes for the pattern of growth.<\/li>\r\n \t
          3. Determine the oxygen requirements of each organism based on the growth pattern.<\/li>\r\n \t
          4. Observe your aerobic and anaerobic plates side by side. Focus on one organism at a time to determine if it had growth on the aerobic plate and\/or aerobic plate.<\/li>\r\n \t
          5. It is sometimes helpful to quantify the bacterial growth using a basic scale as follows:\r\n
              \r\n \t
            • No growth: \u2013<\/li>\r\n \t
            • Minimal growth: +\/-<\/li>\r\n \t
            • Good growth: +<\/li>\r\n \t
            • Heavy growth: +++<\/li>\r\n<\/ul>\r\n<\/li>\r\n \t
            • Determine the oxygen requirements of that organism based on its growth patterns under the both conditions.<\/li>\r\n \t
            • Repeat the procedure for the other two organisms inoculated.<\/li>\r\n<\/ol>\r\n \r\n\r\n[caption id=\"attachment_375\" align=\"alignnone\" width=\"647\"]\"M. Figure 10.1: Plates incubated in aerobic (left) and anaerobic (right) conditions.[\/caption]\r\n\r\n[caption id=\"attachment_376\" align=\"alignnone\" width=\"645\"]\"Thioglycollate Figure 10.2: Thioglycollate Tubes inoculated with M. luteus (L), C. sporogenes (C), and E. coli (R).[\/caption]\r\n\r\n \r\n

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

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

              Remove labels from tubes and place in the tube rack ABOVE the biohazard container. Never throw away tubes. They are always autoclaved, washed and reused.<\/p>\r\n

              Plates are disposable and should be thrown away in the biohazard container.<\/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\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

              Thio Broth<\/p>\r\n<\/th>\r\n

              		\r\n

              Aerobic Plate<\/p>\r\n<\/th>\r\n

              		\r\n

              Anaerobic<\/p>\r\n

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

              		\r\n

              Oxygen Requirement<\/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

              \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

              How can you determine if your anaerobic jar actually created an oxygen free environment during incubation?<\/p>\r\n

              If your thioglycollate tube was pink from top to bottom before you began inoculations, how would this affect your results?<\/p>\r\n

              If the organism inoculated was a microaerophile, on which plates would you expect to see growth after incubation?<\/p>","rendered":"

              Introduction<\/h2>\n

              Microorganisms can survive within a range of environmental conditions but will be most productive and grow most densely in areas where conditions are MOST favorable. Oxygen (O2<\/sub>) is one important resource that can influence microbial growth. Some organisms require oxygen for growth. Others don\u2019t use it at all and are not affected by its presence or absence. Some organisms cannot even survive in the presence of oxygen. Aerobic respiration<\/strong> is a metabolic process that requires oxygen to synthesize ATP. Anaerobic respiration<\/strong> and fermentation<\/strong> are metabolic processes that do NOT require oxygen.<\/p>\n

              Organisms that are obligate aerobes<\/strong> are only capable of aerobic respiration and therefore require oxygen for growth. In thioglycollate medium, they grow only at the top where oxygen is most plentiful. Facultative anaerobes<\/strong> can switch between aerobic or anaerobic processes. These organisms grow best in the presence of oxygen but can grow without it. Growth will be seen throughout the medium but is most dense at the top of the tube. Aerotolerant<\/strong> anaerobes<\/strong> are organisms that don\u2019t utilize oxygen but can tolerate exposure to it without adverse effects. Growth of these organisms is uniform throughout the medium from top to bottom. Obligate<\/strong> anaerobes<\/strong> are organisms for which oxygen is a lethal toxin and, therefore will only grow in the lower portions of the medium where there is no dissolved oxygen. Microaerophiles<\/strong>, as the name suggests, survive only in environments containing very small levels (3-10%) of oxygen. Too much or too little oxygen will kill these organisms making them very difficult with which to work.<\/p>\n

              Fluid thioglycollate medium is adapted for cultivation of organisms with any oxygen requirements including obligate anaerobes and microaerophiles. It contains a small amount of agar to slow the diffusion of oxygen. It also contains an indicator (reazurin<\/strong>) that turns pink in the presence of oxygen. The anaerobic jar is an airtight container used with a gas generating pouch that allows cultivation of anaerobic bacteria. The large white envelope (gas generator packet) placed in the jar removes all oxygen, making conditions inside the jar anaerobic.<\/p>\n

               <\/p>\n

              Course Intended Outcomes<\/h2>\n

              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.<\/p>\n

              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.<\/p>\n

               <\/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
              • Flint striker<\/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. Groups will inoculate one thioglycollate tube for each organism assigned.<\/li>\n
                3. Check that thioglycollate tubes have a pink layer at the top.<\/li>\n
                4. Use a sterile loop to inoculate thioglycollate tubes from the top to the bottom without disturbing the oxygenated (pink) and anaerobic (amber) layers.<\/li>\n
                5. After inoculations make sure that your caps are loosened one half turn to allow air to enter and exit the tube during incubation.<\/li>\n
                6. Groups will also inoculate two plates \u2013 one for growth in the presence of oxygen (aerobic) and one for growth in the absence of oxygen (anaerobic).<\/li>\n
                7. The plates used in this lab exercise are tri-part plates. The media surface is divided for you into three sections to accommodate inoculation of three organisms on the same plate.<\/li>\n
                8. Groups will inoculate the plates in duplicate. In other words, the plates will be identical with the same organisms in the same order. Remember to label and inoculate carefully.<\/li>\n
                9. Aerobic incubation will occur in a standard incubator with atmospheric O2<\/sub> levels.<\/li>\n
                10. The anaerobic jar will be used with a gas generator packet to create an oxygen free environment for the anaerobic plates.<\/li>\n<\/ol>\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 your tubes using label tape. Include your group name or initials, section day\/time, and the organism name.<\/li>\n
                    • Clearly label plates (on the bottom side) with your group name or initials, organism name, and section day\/time.<\/li>\n
                    • Label one of your duplicate plates \u201cAerobic\u201d for standard incubation.<\/li>\n
                    • Label the other duplicate plate \u201cAnaerobic\u201d for incubation in the anaerobic jar.<\/li>\n
                    • Continue with inoculation as directed by your instructor.<\/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. Observe your thioglycollate tubes for the pattern of growth.<\/li>\n
                      3. Determine the oxygen requirements of each organism based on the growth pattern.<\/li>\n
                      4. Observe your aerobic and anaerobic plates side by side. Focus on one organism at a time to determine if it had growth on the aerobic plate and\/or aerobic plate.<\/li>\n
                      5. It is sometimes helpful to quantify the bacterial growth using a basic scale as follows:\n
                          \n
                        • No growth: \u2013<\/li>\n
                        • Minimal growth: +\/-<\/li>\n
                        • Good growth: +<\/li>\n
                        • Heavy growth: +++<\/li>\n<\/ul>\n<\/li>\n
                        • Determine the oxygen requirements of that organism based on its growth patterns under the both conditions.<\/li>\n
                        • Repeat the procedure for the other two organisms inoculated.<\/li>\n<\/ol>\n

                           <\/p>\n

                          \"M.
                          Figure 10.1: Plates incubated in aerobic (left) and anaerobic (right) conditions.<\/figcaption><\/figure>\n
                          \"Thioglycollate
                          Figure 10.2: Thioglycollate Tubes inoculated with M. luteus (L), C. sporogenes (C), and E. coli (R).<\/figcaption><\/figure>\n

                           <\/p>\n

                          Lab Clean-up<\/h2>\n

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

                          Remove labels from tubes and place in the tube rack ABOVE the biohazard container. Never throw away tubes. They are always autoclaved, washed and reused.<\/p>\n

                          Plates are disposable and should be thrown away in the biohazard container.<\/p>\n

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

                           <\/p>\n

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

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

                          		\n

                          Thio Broth<\/p>\n<\/th>\n

                          		\n

                          Aerobic Plate<\/p>\n<\/th>\n

                          		\n

                          Anaerobic<\/p>\n

                          Plate<\/p>\n<\/th>\n

                          		\n

                          Oxygen Requirement<\/p>\n<\/th>\n<\/tr>\n<\/thead>\n

                          \n

                          \n<\/td>\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

                          		\n

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

                          \n

                          \n<\/td>\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

                          		\n

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

                          \n

                          \n<\/td>\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

                          How can you determine if your anaerobic jar actually created an oxygen free environment during incubation?<\/p>\n

                          If your thioglycollate tube was pink from top to bottom before you began inoculations, how would this affect your results?<\/p>\n

                          If the organism inoculated was a microaerophile, on which plates would you expect to see growth after incubation?<\/p>\n","protected":false},"author":24,"menu_order":1,"template":"","meta":{"pb_show_title":"on","pb_short_title":"","pb_subtitle":"","pb_authors":[],"pb_section_license":""},"chapter-type":[],"contributor":[],"license":[],"class_list":["post-357","chapter","type-chapter","status-publish","hentry"],"part":351,"_links":{"self":[{"href":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/wp-json\/pressbooks\/v2\/chapters\/357","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/wp-json\/pressbooks\/v2\/chapters"}],"about":[{"href":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/wp-json\/wp\/v2\/types\/chapter"}],"author":[{"embeddable":true,"href":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/wp-json\/wp\/v2\/users\/24"}],"version-history":[{"count":8,"href":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/wp-json\/pressbooks\/v2\/chapters\/357\/revisions"}],"predecessor-version":[{"id":498,"href":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/wp-json\/pressbooks\/v2\/chapters\/357\/revisions\/498"}],"part":[{"href":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/wp-json\/pressbooks\/v2\/parts\/351"}],"metadata":[{"href":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/wp-json\/pressbooks\/v2\/chapters\/357\/metadata\/"}],"wp:attachment":[{"href":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/wp-json\/wp\/v2\/media?parent=357"}],"wp:term":[{"taxonomy":"chapter-type","embeddable":true,"href":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/wp-json\/pressbooks\/v2\/chapter-type?post=357"},{"taxonomy":"contributor","embeddable":true,"href":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/wp-json\/wp\/v2\/contributor?post=357"},{"taxonomy":"license","embeddable":true,"href":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/wp-json\/wp\/v2\/license?post=357"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}