Microbiology Lab

Exercise 6:  The Ubiquity of Bacteria

Exercise 8:  Aseptic Technique

 

Exercise 6:  The Ubiquity of Bacteria

  1. Each student will receive a sterile TSA plate.
    1. Students will label the bottom of the plate (the side with the agar) with their initials, the date, and exposure method using a Sharpie pen
  2. Students will expose their plates to the following environmental contaminations…
    1. Student 1 – To the air in the laboratory for 30 minutes
    2. Student 2 – To the air in a room other than the laboratory for 30 minutes (the bathrooms are off limits)
    3. Student 3 – To the air outside the building for 30 minutes – exposed to sunlight
    4. Student 4 – To the air outside the building for 30 minutes – in the shade
    5. Student 5 – Blow dust onto exposed medium
    6. Student 6 – Moist lips pressed against medium
    7. Student 7 – Fingertips pressed lightly on medium
    8. Student 8 – Several coins pressed temporarily on medium
    9. Student 9 – Hair combed over exposed medium (10 strokes)
    10. Student 10 – Optional – Any method not listed above
  3. Students will return TSA plates to a designated area for incubation at 37°C for 48 hours.

 

 

Ex 8:  Aseptic Technique

 

 

broth media turbidity.JPG

 

agar slant bacterial growth.JPG

 

  1. Each student will aseptically transfer an assigned bacterial broth culture to a new broth tube and an agar slant tube.
  2. Assignments…
    1. Students 1 & 2 – Bacillus megaterium to nutrient broth & nutrient agar slant
    2. Students 3 & 4 – Branhamella catarrhalis to BHI broth & TSA slant
    3. Students 5 & 6 – Escherichia coli to nutrient broth & nutrient agar slant
    4. Students 7 & 8 –  Pseudomonas aeruginosa to nutrient broth & nutrient agar slant
    5. Students 9 & 10 – Staphylococcus aureus to BHI broth & TSA slant
  3. Procedure
    1. Preparation

                                          i.    Put your lab coat on

                                        ii.    Wash your hands

                                       iii.    Disinfect your work area

    1. Gather supplies

                                          i.    Inoculating loop

                                        ii.    Your assigned culture

                                       iii.    A sterile broth & a sterile agar slant

                                       iv.    Sharpie marker

                                        v.    Bunsen burner & striker

    1. Inoculate sterile broth with your assigned bacteria

                                          i.    Label sterile tube with the bacteria’s name, your initials, & date

                                        ii.    Agitate culture tube

                                       iii.    Light Bunsen burner

                                       iv.    Flame loop until it is red-hot

1.    Then let the loop cool

                                        v.    Uncap and flame the mouth of the broth culture tube

                                       vi.    Collect sample of bacteria from broth with the inoculating loop

                                     vii.    Reflame & recap the culture tube

                                    viii.    Uncap & flame the mouth of the sterile tube

                                       ix.    Insert the loop with your bacteria into the media of the sterile tube, gently agitate the broth, then remove the loop

                                        x.    Reflame and recap your tube

                                       xi.    Reflame inoculating loop

    1. Inoculate sterile agar slant with your assigned bacteria

                                          i.    Repeat steps i – viii of c

                                        ii.    Streak the surface of the agar slant in a straight line with the bacteria on your inoculating loop, then remove the loop

                                       iii.    Repeat steps x & xi of c

  1. Clean-up
    1. Extinguish the Bunsen burner
    2. Return assigned culture tubes to instructor’s desk
    3. Put newly inoculated cultures into the incubator
    4. Disinfect your work area
    5. Wash hands