8.2: Exercises

Exercise A: Colony Characteristics

Work in groups of 2-4 students. Safety: wear gloves, keep petri plates closed at all times, and wipe work benches with disinfectant when finished. Discard gloves in biohazard (red) waste bags. Examine each of the petri plates provided. You may use the stereoscope or your microscope (2x filter with no lens, or BF filter with 4x lens). Record results in Table 8.1 in the lab report (use technical terms).

Data Table for Microbe Identification by Morphology

Name of Bacterium:	Typically found where?	Average colony size (mm):	Colony Shape:	Margin:	Surface:	Pigmentation (or none)
Micrococcus luteus
Serratia marcescens
Bacillus
Escherichia coli

Table 8.1 Colony Characteristics

Exercise B: Investigating Environmental Microbes

Bacteria, fungi, and viruses can be found just about everywhere on earth! Today, you will collect samples of bacteria and fungi that are either on you or around you. Work with one other student and follow instructions from your instructor. Label your plastic petri dish along the edges, on the agar side, not the lid, using a permanent marker. Include your last name, the surface you will swab, the date, your section number and the incubation temperature.

Create a hypothesis and a prediction for your experiment BEFORE you swab, record them in the lab report section. Dip you sterile swab into sterile water and then rub the surface you want to test, several times, back and forth. Rotate your swab and rub your surface again. Now you are ready to rub the swab gently on the surface of your agar plate. Minimize the agar's exposure to air as much as possible. After 5 minutes, flip petri plates upside down to incubate. Incubate at 37 C if your test surface was your skin, otherwise, incubate at room temperature (25 C). Use Parafilm to seal the lid to the bottom before incubation.

B.1. What surfaces will you swab? Examples: you can compare before and after handwashing (don’t dry your hands), or compare two body areas, or environmental samples such as commonly touched areas (phone vs your pencil).

B.2. What is your hypothesis? Get approval before you swab.

B.3. What is your prediction (what will your results look like if your hypothesis is true)?

B.4. After incubation: Sketch and describe your results below.

B.5. Do your results support your hypothesis? Explain:

Exercise C: Viewing Prepared Slide of Gram-Stained "Three Bacterial Types"

Obtain a prepared gram-stained slide, labeled “Three Bacterial Types,” from the side counter. Follow the instructions on using oil immersion from our Microscopy Lab I. Each student should focus their own slide for all three bacterial samples in this exercise. Then, fill in Table 8.2 in the lab report. Remember to use color for your sketches.

Data Table for Three Bacterial Types

Type:	Describe the shape and arrangement of cells:	Sketch at 1000x (oil)	Gram +/-:
bacillus (bacilli)
coccus (cocci)
spirillum (spirilla)

Table 8.2 Prepared Slide of Gram-Stained "Three Bacterial Types"

Exercise D: Making a Gram-Stained Slide

Materials:

How to make a heat-fixed bacterial smear

1. Label a clean glass slide as demonstrated by your instructor

2. Add a small drop of water to the center of the slide

3. With an inoculation loop, aseptically pick up a small amount of bacteria

4. Mix it well with into your drop of water and then spread the mixture over a wider area of the slide. Make sure you don’t have any clumps and your smear is not thick

5. Air dry (completely) (slide warmers may also be used if available).

6. When slides are completely dry, heat fix the bacterial specimen by passing the slide slowly over the flame twice (your instructor will demonstrate this) for total of about 3 seconds.

Heat fixing kills cells and adheres them to the slide. Cells will be rinsed off of the slide if they are not heat fixed properly. Be careful not to overheat the slides in this procedure, you can distort the shape of the cells and/or crack your glass slide. After heat-fixing is complete, you are ready to stain your slide.

Gram Stain

For all steps in the gram staining procedure, add enough of the solution to cover the entire smear (usually 2-3 drops). All staining should be done over a staining tray. Be sure to put paper towels under the tray in case of spillage. Gloves should be worn while staining and removed before working with the microscope. Each student will be assigned one bacterial culture to stain, you will look at your group member’s stains to sketch the remaining three types of bacteria. Use the instructions for oil immersion from our microscopy lab when you view your Gram-stain. Leave your slide, in-focus on 1000x, on your microscope until everyone is finished with it. Then you may place it into the 10% bleach container in the sink.

Staining Steps

1. Add a few drops of crystal violet (primary stain) to the smear and let it sit for 60 seconds.

2. Rinse the slide with water. All cells are purple after this step. Stopping here would be a simple stain. Do not dry the slide.

3. Add a few drops of Gram's iodine (mordant) to the smear and let it sit for 60 seconds. Gram’s iodine forms a complex with the crystal violet that is inside the cell wall.

4. Rinse the slide with water. Do not dry the slide. All cells are still purple after this step.

5. Decolorize with 95% ethanol: let the alcohol run over surface of slide until no more crystal violet color comes out of the smear (time varies, 3-10 seconds). Gram-positive cells retain crystal violet and remain purple after this step. Gram-negative cells lose crystal violet and are now colorless after this step.

6. IMMEDIATELY rinse with water. Water rinse stops the decolorization process. If you leave the alcohol on longer than 10 seconds you will damage the cells and they will all look gram-negative, this is called over-decolorization. Do not dry your slide.

7. Add a few drops of safranin (counterstain) and let it sit for 60 seconds. Safranin is a pink/red dye.

8. Rinse with water.

9. Blot dry on bibulous paper. Do not rub, press down gently. Gram-positive cells remain purple; gram-negative cells are now pink/red.

10. Dispose of the staining waste in your tray into the special waste container inside the hood. Rinse and dry staining tray. Disinfect your workspace and remove gloves. (biohazardous waste).

11. Observe your slide under the microscope, view all the way to 1000x (oil). Remember not to use a microscope near a flame.

11. For each organism, determine morphology, arrangement, and Gram reaction. Then, draw (in color) a representative set of cells and include the calculation for total magnification below the table.

Data Table for Gram-positive and Gram-negative Bacteria

Bacterium:	Color of cells (100x lens)	Gram result (+/-)	Shape (morphology)	Sketch in color, 100x lens
Micrococcus luteus
Serratia marcescens
Bacillus
Escherichia coli

Table 8.3 Gram Stain Results