{"id":70,"date":"2024-05-29T14:12:00","date_gmt":"2024-05-29T14:12:00","guid":{"rendered":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/chapter\/__unknown__\/"},"modified":"2025-12-17T16:18:53","modified_gmt":"2025-12-17T16:18:53","slug":"__unknown__","status":"publish","type":"chapter","link":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/chapter\/__unknown__\/","title":{"raw":"Exercise 2: Aseptic Technique","rendered":"Exercise 2: Aseptic Technique"},"content":{"raw":"

Introduction<\/h2>\r\n

Clinical microbiologists are required to transfer live microorganisms from one place to another in the lab without contamination while identifying the agent of a disease. The methods used to prevent contamination are called aseptic technique<\/strong>. Students must also learn these techniques to get good results and accurately identify their unknown cultures. To prevent contamination of working cultures, inoculating<\/strong> instruments should always be sterilized prior to use. To inoculate means to \u201cintroduce\u201d microorganisms into a growth medium. Wire inoculating loops and needles are sterilized immediately before use in a Bunsen burner flame. The mouths of tubes containing cultures or media may also be flamed at the time of transfer by passing their openings over the burner.<\/p>\r\n

Any growth medium containing live microorganisms is called a culture<\/strong>. If that culture contains only organisms of the same species that are genetically identical, it is considered to be a pure culture<\/strong>. It is essential to transfer pure cultures to a sterile medium aseptically<\/strong>. The addition of any other organisms will result in contamination and the culture will be considered mixed. Mixed cultures<\/strong> cannot be used for testing and identification as results will be unreliable.<\/p>\r\n

There are three common forms of growth media, each with specific applications. Broth<\/strong> media<\/strong> are fluid and can be used to grow microbes when fresh cultures or large numbers of cells are required. Agar slants<\/strong> contain 1.5% agar to make the medium semisolid \u2013 like gelatin. Slants can be used for differential testing or to grow stock cultures that can be refrigerated after incubation. Plate<\/strong> media<\/strong> are also semisolid and are typically used for isolating pure cultures, differential testing or quantifying bacterial densities. Aseptic techniques should be used regardless of the type of growth medium being used.<\/p>\r\n \r\n

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

Demonstrate and apply basic aseptic techniques, standard lab safety procedures, and maintain aseptic environment in a microbiology laboratory.<\/p>\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 \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
  • Rubber tubing<\/li>\r\n \t
  • Striker<\/li>\r\n \t
  • Inoculating loop<\/li>\r\n \t
  • 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

    Broth Inoculation Technique:<\/h3>\r\n
      \r\n \t
    1. Light your Bunsen burner with the striker and adjust your burner so it produces a double blue flame.<\/li>\r\n \t
    2. Hold the inoculating loop or needle like a pencil in your dominant hand and relax.<\/li>\r\n \t
    3. Flame entire loop and wire until it glows orange before use and after use. Allow to cool for 10 seconds after flaming.<\/li>\r\n \t
    4. While holding your loop remove the cap from the tube of prepared culture using your ring and pinky fingers. Do NOT put down the cap as this will invite contamination. Practice holding both the loop and the cap. You will get used to it.<\/li>\r\n \t
    5. Use the sterile loop to remove a small amount of culture from the surface of the slant. Remember that bacteria only grow on the surface of the agar. Don\u2019t gouge the agar.<\/li>\r\n \t
    6. Return the cap to the culture tube then pick up the nutrient broth tube. Remove the cap as before without putting it down.<\/li>\r\n \t
    7. Transfer the small amount of culture from the loop to the liquid broth in the bottom of the tube. Briefly shake or spin the loop to release the culture into the broth.<\/li>\r\n \t
    8. Sterilize the loop again before placing on the bench top.\r\n
        \r\n \t
      • It\u2019s also good practice to quickly flame the tube after opening and before closing.<\/em><\/li>\r\n \t
      • Take your time and do NOT rush. It is not a race.<\/em><\/li>\r\n<\/ul>\r\n<\/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.<\/em><\/li>\r\n \t
          • Never hold a culture tube by its cap.<\/em><\/li>\r\n \t
          • Bacterial culture tubes are always to be shared with your table group.<\/em><\/li>\r\n<\/ul>\r\n<\/li>\r\n \t
          • Clearly label and arrange all of your media before you begin.\r\n
              \r\n \t
            • Use label tape for tubes<\/em><\/li>\r\n \t
            • Label plates directly on the agar side without tape. The sharpie will work on the plastic.<\/em><\/li>\r\n \t
            • Always include your initials, the organism or specimen ID, and the section day\/time at a minimum.<\/em><\/li>\r\n<\/ul>\r\n<\/li>\r\n \t
            • Place newly inoculated tubes in the rack to be incubated at 35 C. Return bacterial cultures to the front desk.<\/li>\r\n<\/ol>\r\n \r\n

              Procedure, Day 2<\/h2>\r\n
                \r\n \t
              1. Collect your tubes inoculated in the previous lab from the back bench.\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<\/ul>\r\n<\/li>\r\n \t
                • Examine tubes for bacterial growth. Growth in liquid broth cultures is noted by turbidity in the medium. Greater turbidity in the broth indicates more growth. Sometimes new growth will sink to the bottom forming a pellicle<\/strong>.<\/li>\r\n \t
                • Inoculated tubes should show growth\/turbidity.<\/li>\r\n \t
                • Uninoculated control tubes should be sterile and remain clear.<\/li>\r\n \t
                • Record your results in your lab notebook.<\/li>\r\n<\/ol>\r\n \r\n

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

                  Remove labels from tubes and place in the tube rack for sterilization and cleaning.<\/p>\r\n

                  Return other materials to their proper bins on the back bench.<\/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 the name of the culture\/organism used and the result as growth (G) or no growth (NG)<\/p>\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n
                  \r\n

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

                  		\r\n

                  Result<\/strong><\/p>\r\n<\/th>\r\n

                  		\r\n

                  Comments<\/strong><\/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<\/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<\/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<\/tr>\r\n

                  \r\n

                  Positive Control<\/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

                  Negative Control<\/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 would you determine if the growth (turbidity) in your in inoculated broth is a pure culture?<\/p>\r\n

                  Based on your experience, what are the most likely mistakes that can lead to contamination during inoculation?<\/p>","rendered":"

                  Introduction<\/h2>\n

                  Clinical microbiologists are required to transfer live microorganisms from one place to another in the lab without contamination while identifying the agent of a disease. The methods used to prevent contamination are called aseptic technique<\/strong>. Students must also learn these techniques to get good results and accurately identify their unknown cultures. To prevent contamination of working cultures, inoculating<\/strong> instruments should always be sterilized prior to use. To inoculate means to \u201cintroduce\u201d microorganisms into a growth medium. Wire inoculating loops and needles are sterilized immediately before use in a Bunsen burner flame. The mouths of tubes containing cultures or media may also be flamed at the time of transfer by passing their openings over the burner.<\/p>\n

                  Any growth medium containing live microorganisms is called a culture<\/strong>. If that culture contains only organisms of the same species that are genetically identical, it is considered to be a pure culture<\/strong>. It is essential to transfer pure cultures to a sterile medium aseptically<\/strong>. The addition of any other organisms will result in contamination and the culture will be considered mixed. Mixed cultures<\/strong> cannot be used for testing and identification as results will be unreliable.<\/p>\n

                  There are three common forms of growth media, each with specific applications. Broth<\/strong> media<\/strong> are fluid and can be used to grow microbes when fresh cultures or large numbers of cells are required. Agar slants<\/strong> contain 1.5% agar to make the medium semisolid \u2013 like gelatin. Slants can be used for differential testing or to grow stock cultures that can be refrigerated after incubation. Plate<\/strong> media<\/strong> are also semisolid and are typically used for isolating pure cultures, differential testing or quantifying bacterial densities. Aseptic techniques should be used regardless of the type of growth medium being used.<\/p>\n

                   <\/p>\n

                  Course Intended Outcomes<\/h2>\n

                  Demonstrate and apply basic aseptic techniques, standard lab safety procedures, and maintain aseptic environment in a microbiology laboratory.<\/p>\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

                   <\/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
                  • Rubber tubing<\/li>\n
                  • Striker<\/li>\n
                  • Inoculating loop<\/li>\n
                  • 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

                    Broth Inoculation Technique:<\/h3>\n
                      \n
                    1. Light your Bunsen burner with the striker and adjust your burner so it produces a double blue flame.<\/li>\n
                    2. Hold the inoculating loop or needle like a pencil in your dominant hand and relax.<\/li>\n
                    3. Flame entire loop and wire until it glows orange before use and after use. Allow to cool for 10 seconds after flaming.<\/li>\n
                    4. While holding your loop remove the cap from the tube of prepared culture using your ring and pinky fingers. Do NOT put down the cap as this will invite contamination. Practice holding both the loop and the cap. You will get used to it.<\/li>\n
                    5. Use the sterile loop to remove a small amount of culture from the surface of the slant. Remember that bacteria only grow on the surface of the agar. Don\u2019t gouge the agar.<\/li>\n
                    6. Return the cap to the culture tube then pick up the nutrient broth tube. Remove the cap as before without putting it down.<\/li>\n
                    7. Transfer the small amount of culture from the loop to the liquid broth in the bottom of the tube. Briefly shake or spin the loop to release the culture into the broth.<\/li>\n
                    8. Sterilize the loop again before placing on the bench top.\n
                        \n
                      • It\u2019s also good practice to quickly flame the tube after opening and before closing.<\/em><\/li>\n
                      • Take your time and do NOT rush. It is not a race.<\/em><\/li>\n<\/ul>\n<\/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.<\/em><\/li>\n
                          • Never hold a culture tube by its cap.<\/em><\/li>\n
                          • Bacterial culture tubes are always to be shared with your table group.<\/em><\/li>\n<\/ul>\n<\/li>\n
                          • Clearly label and arrange all of your media before you begin.\n
                              \n
                            • Use label tape for tubes<\/em><\/li>\n
                            • Label plates directly on the agar side without tape. The sharpie will work on the plastic.<\/em><\/li>\n
                            • Always include your initials, the organism or specimen ID, and the section day\/time at a minimum.<\/em><\/li>\n<\/ul>\n<\/li>\n
                            • Place newly inoculated tubes in the rack to be incubated at 35 C. Return bacterial cultures to the front desk.<\/li>\n<\/ol>\n

                               <\/p>\n

                              Procedure, Day 2<\/h2>\n
                                \n
                              1. Collect your tubes inoculated in the previous lab from the back bench.\n
                                  \n
                                • Place all tubes in a tube rack.<\/li>\n
                                • Never hold a culture tube by its cap.<\/li>\n<\/ul>\n<\/li>\n
                                • Examine tubes for bacterial growth. Growth in liquid broth cultures is noted by turbidity in the medium. Greater turbidity in the broth indicates more growth. Sometimes new growth will sink to the bottom forming a pellicle<\/strong>.<\/li>\n
                                • Inoculated tubes should show growth\/turbidity.<\/li>\n
                                • Uninoculated control tubes should be sterile and remain clear.<\/li>\n
                                • Record your results in your lab notebook.<\/li>\n<\/ol>\n

                                   <\/p>\n

                                  Lab Clean-up<\/h2>\n

                                  Remove labels from tubes and place in the tube rack for sterilization and cleaning.<\/p>\n

                                  Return other materials to their proper bins on the back bench.<\/p>\n

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

                                   <\/p>\n

                                  Results and Interpretation<\/h2>\n

                                  Record the name of the culture\/organism used and the result as growth (G) or no growth (NG)<\/p>\n\n\n\n\n\n\n\n\n\n
                                  \n

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

                                  		\n

                                  Result<\/strong><\/p>\n<\/th>\n

                                  		\n

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

                                  \n

                                  \n<\/td>\n

                                  		\n

                                  \n<\/td>\n

                                  		\n

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

                                  \n

                                  Positive Control<\/p>\n<\/td>\n

                                  		\n

                                  \n<\/td>\n

                                  		\n

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

                                  \n

                                  Negative Control<\/p>\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 would you determine if the growth (turbidity) in your in inoculated broth is a pure culture?<\/p>\n

                                  Based on your experience, what are the most likely mistakes that can lead to contamination during inoculation?<\/p>\n","protected":false},"author":24,"menu_order":2,"template":"","meta":{"pb_show_title":"on","pb_short_title":"","pb_subtitle":"","pb_authors":[],"pb_section_license":""},"chapter-type":[],"contributor":[],"license":[],"class_list":["post-70","chapter","type-chapter","status-publish","hentry"],"part":74,"_links":{"self":[{"href":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/wp-json\/pressbooks\/v2\/chapters\/70","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":9,"href":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/wp-json\/pressbooks\/v2\/chapters\/70\/revisions"}],"predecessor-version":[{"id":516,"href":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/wp-json\/pressbooks\/v2\/chapters\/70\/revisions\/516"}],"part":[{"href":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/wp-json\/pressbooks\/v2\/parts\/74"}],"metadata":[{"href":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/wp-json\/pressbooks\/v2\/chapters\/70\/metadata\/"}],"wp:attachment":[{"href":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/wp-json\/wp\/v2\/media?parent=70"}],"wp:term":[{"taxonomy":"chapter-type","embeddable":true,"href":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/wp-json\/pressbooks\/v2\/chapter-type?post=70"},{"taxonomy":"contributor","embeddable":true,"href":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/wp-json\/wp\/v2\/contributor?post=70"},{"taxonomy":"license","embeddable":true,"href":"https:\/\/pressbooks.hcfl.edu\/microlabmanual\/wp-json\/wp\/v2\/license?post=70"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}