Exercise 5: Preparing a Smear

Introduction

To enhance visibility with the microscope most microbiological specimens are stained prior to viewing. Stains are solutions containing a charged dye of a particular color called a chromogen. Basic stains are positively charged and attracted to the surface of most bacterial cells. Common basic stains include methylene blue, crystal violet, and safranin. Before the specimen can be stained it is applied to a glass slide as a thin smear, allowed to dry completely, and then heat-fixed. The smears must be thin to allow observation of individual and small groups of cells. Heat fixing kills the bacteria, makes them adhere to the slide, and coagulates proteins to increase absorption of the stain.

Course Intended Outcomes

Perform and understand the basis of different types of microbiological staining techniques (e.g., simple stain and gram stain) to characterize and identify bacteria.

Materials Used

Students will work in small groups. Each group will need the following materials for this exercise.

• Bunsen burner
• Inoculation loop
• Glass slides, cleaned
• Test tube rack

Additional materials and cultures will be assigned by your instructor.

Procedure, Day 1

Technique:

1. Place one loop-full of DI water on a clean slide using a sterile inoculating loop.
2. Aseptically add a small amount of bacteria to the water using inoculating loop.
3. Mix in the bacteria and spread the drop out into a thin smear. Avoid splattering the emulsion. Sterilize the loop.
4. Allow the smear to air dry completely. If prepared correctly, the smear should be slightly cloudy.
5. Using a wooden clothes-pin to hold the slide, pass the slide (smear side up!) through the upper part of a flame 2 or 3 times. Avoid overheating. DO NOT hold the slide still directly over the flame.
6. Let the slide cool.

 (Fig. 5.1 Long description)

Sampling/Inoculation:

1. Your instructor will assign the cultures to be used for inoculation by your group.
2. Collect the required materials for the lab.
   • Place all tubes in a tube rack.
   • Never hold a culture tube by its cap.
   • Bacterial culture tubes are always to be shared with your table group.
3. Label your slides using a wax pencil with the organism initials (e.g., EC = Escherichia coli). This will prevent mixing up your slides and help with slide orientation.

Lab Clean-up

Return bacteria cultures to the front bench.

Wash slides with detergent, rinse and dry. Return slides to your slide box and store in the bin labeled for your section.

Disinfect your benchtop and wash your hands before leaving the lab.

Results and Interpretation

Preparing a smear is an initial step in specimen preparation for staining and evaluation. Interpretation of results will be completed after staining procedures.

Critical Thinking Questions

What potential problems occurs if the smear on the slide is too thick?

What effect does over-heating have on the smear? What if you forget to heat fix?