Contamination Exercise

 

Objective

·        Observe ubiquitous presence of contaminants (bacteria, fungi, viruses, etc.).

 

Materials

Tryptic soy agar

Petri dishes (95x15 mm) polystyrene, non-TC treated

Agar plate containing S. marceans (from D.Caprette)

Yogurt, apple juice, standing water

Pasteur pipets

 

Safety

1.      Wear disposable latex gloves at all times

2.      Wear safety glasses at all times.

 

Agar plates (prepared by Instructor)

Need to prepare 10 plates per group + 12 plates per section.  Yield is 18-20 plates for 20 g of agar.

1.      Mix 20 g powdered agar with 0.5 L DI water in 2 L Erlenmeyer flask.  Do not use more than 40 g in 1.0 L DI water.

2.      Heat on hot plate until boiling for one minute.

3.      Loosely place foil on top of flask.  Autoclave on liquids setting (#4).  Use autoclave gloves to remove and handle flask when hot.

The following steps need to be done in a laminar flow hood.  Sterile technique should be followed.

4.      Cut open sleeve of Petri dishes at end so that it can be easily resealed/reused for storage.  Set out Petri dishes (one deep) to cover surface of hood.

5.      Using a sterile pipet, aliquot 25 mL of agar into each petri dish.  This operation must be accomplished while agar is still hot/warm.  Minimize the number of bubbles in the agar by touching bubbles with end of pipette.  Set lid on base but leave lid cracked.

6.      Cool for several hours or overnight.

7.      Stack dishes and reload in plastic sleeves.  Store in refrigerator.

 

 

Contamination Study

EACH GROUP NEEDS TO MAKE ONLY ONE SET OF PLATES.

1.  Conditions to test using agar plates:

·        Press fingers onto agar

·        Lick fingers and press onto agar (wash your hands before doing this…)

·        6 drops of tap water (drop in different locations on plate)

·        6 drops of standing water from outside (drop in different locations on plate)

·        6 drops of apple juice (drop in different locations on plate)

·        Smear of yogurt

·        Hair debris/dandruff

·        Leave dish open on lab bench for 2 hrs

·        Leave dish open in hood for 2 hrs

·        Control

      NOTE:  Remember to label each condition and put your initials on the plate.

2.   Store plates on counter (NOT in laminar flow hood).

3.   Check plates in 2 days.  Record observations.

 

 

Serratia marceans Study

1.      Label plates 1 - 12. 

2.      All students put on one glove.  Wet gloves with DI water.  Line up students 1 - 12.

3.      Transfer S. marceans from initial culture plate to glove by gently rubbing dish.

4.      Transfer S. marceans glove to glove through line of students (student 1, 2, 3, …).

5.      Transfer S. marceans from glove to plate (student 1 to plate 1, etc.) by lightly pressing and rubbing gloves onto agar surface.  Be careful not to push too hard and disrupt agar surface.

6.      Record observations.  In which dishes is the S. marceans visible?

7.      Leave plates on counter for 2 days.  Do NOT put plates in laminar flow hood.

8.      Check plates in 2 days.  Record observations.

 

Data and observations should be recorded in your laboratory notebook.  The observations recorded for the Contamination Study (step 3) and the Serratia marceans Study (steps 6 and 8) are each worth 5 pts.