STUDENT HANDOUT:

• Size, Shape and Arrangements Student Handout

LABORATORY REPORT FORM:

• Size, Shape and Arrangements Laboratory Report Form

INTRODUCTION:

The term morphology refers to the size and shape of an organism regardless of its function.  Colony morphology refers to the appearance of bacteria when they grow on a solid surface, and this growth can usually be seen with the naked eye.  The terms used to describe the variety of appearance on a solid surface are described in the Aseptic Technique lab.  

Cellular morphology refers to the size, shape, and arrangement of individual bacterial cells.  These characteristics can only be determined using a microscope.  Most bacterial cells fall into one of the following categories (Table 1). 

Table 1.  Cellular morphologies.

Some bacteria group together in a particular arrangement as a result of how many planes the cell divides in (Table 2).  This grouping can help to identify the bacteria. 

Table 2.  Cellular arrangements.

In nature, bacteria are usually very small and colorless, making them difficult to see, even with the light microscope.  To view these microorganisms, we stain them with a dye that is absorbed by the cell.  Bacterial cells differ chemically from their surroundings and thus can be stained to contrast with their environment.  Simple staining uses one dye to stain the cells which allows the viewer to see the relative size, shape and arrangement of the microorganisms (Figure 1).

Microorganisms differ from one another chemically and physically and therefore may react differently to the same staining procedure. This is the basic principle of differential staining, a method of distinguishing between types of bacteria (Figure 1).  

Figure 1.  Comparison of simple and differential stains.

The most important differential staining procedure is the Gram stain, first developed by Hans Christian Gram in the late 1880s.  It is used to distinguish between types of bacteria based on the chemical composition of their cell walls.  The molecule that comprises the cell wall of bacteria is known as peptidoglycan. 

The Gram differentiation is based on the application of four chemical reagents:  primary dye,  mordant, decolorizer and counterstain.  The primary dye, crystal violet, imparts a purple color to all organisms.  Gram’s [Lugol’s] iodine acts as a mordant, enhancing the bond between the crystal violet dye and the cell to form a complex.  The decolorizer solution of alcohol extracts the purple complex from certain cells more readily than others.  A safranin counterstain colors those organisms that lost the purple complex.  

Those organisms that retain the purple complex are gram-positive [purple] while those losing the purple complex are gram-negative [pink or red] (Figure 2).  Gram-positive bacteria have thick cell walls of peptidoglycan.  Gram-negative bacteria have thin cell walls of peptidoglycan.  

Figure 2.  Mixed culture of gram-positive and gram-negative bacilli (rods).  Image from Wikimedia Commons.

PRECAUTIONS:

1. Handle the bacterial cultures with care. Wash your hands with antimicrobial soap before and after handling cultures, and wash work surfaces with disinfectant.

2. Clean up spills using disposable plastic gloves, paper towels, and disinfectant. Dispose of all cleanup materials in the biohazard bag.

3. Be extremely careful when using the Bunsen burner.

4. Be sure to wear gloves, especially when working with stains.

5. Check with your teacher as to where stain waste should be collected before disposal.

EXPERIMENT #1:  Simple Staining

OBJECTIVES:

1. Understand the difference between colony and cellular morphology.

2. Prepare a smear of bacteria.

3. Perform a simple stain procedure.

MATERIALS (Work individually):

1. Plate cultures of :  Escherichia coli, Micrococcus luteus, Rhodospirillum rubrum

2. Gram’s crystal violet

3. Gram’s safranin

4. Methylene blue

5. 3 glass microscope slides

6. Permanent glass marker (wax pencil)

7. Metal inoculating loop or sterile, plastic disposable loops

8. Bunsen burner

9. Bibulous paper to blot slides

10. Dish detergent

11. Paper towels

12. Staining rack

13. Paper towels

14. Biohazard bag

PROCEDURE: Preparing the Bacterial Smear

You can watch a video of how to make a bacterial smear and heat fix using the Bunsen Burner.

1. Clean the microscope slides with dish detergent [such as Dawn®], rinse them thoroughly with tap water and dry them with a paper towel.

2. Using a glass marker, mark a slide with the name of the organism [e.g. E. coli], the dates and circle to indicate where the bacteria will be placed.

3. Turn the slide over so that the markings are on the underside.

4. Apply one loopful of sterile water on the slide over the circle.

5. Using aseptic technique, apply a small amount of  E. coli  to the water drop and mix with the loopful of water.

6. Spread into a thin film.

7. Allow the slide to air dry.

8. With the bacteria on the top side of the slide, heat-fix the slide by passing it through the Bunsen burner flame three times.

9. Repeat steps 5 through 8 with M. luteus and R. spirillum. 

PROCEDURE: Staining Process

You can watch a video of how to perform a simple stain.

1. Allow the slides to cool before placing them on a staining rack.

2. Pour the Gram’s crystal violet over the E. coli slide [smear side up] and allow it to stay on the slide for 1 minute.

3. Holding the slide at an angle, rinse it thoroughly with water until no more dye comes off the slide.

4. Using the bibulous paper, gently blot the slide until it is dry.

5. Pour the Gram’s safranin over the M. luteus slide for 2 minutes.

6. Repeat steps 3 to 4.

7. Pour the methylene blue over the R. rubrum for 2 minutes.

8. Repeat steps 3 to 4.

9. Using the procedure described in the Microscopy Lab, observe the slides. 

OBSERVATIONS/CONCLUSIONS:

1. Record results in Table 1 of the Laboratory Report Form

2. Were all the cells the same size?

3. Were some cells easier to see than others?

QUESTIONS:

1. How many different shapes of bacteria did you see?
   
   

2. Name the different arrangements of bacteria that you saw.
   
   

3. Why did you need to stain the bacteria?

EXPERIMENT #2:  Differential Staining - The Gram Stain

OBJECTIVES:

1. Understand the concept of differential staining.

2. Learn a differential staining technique that divides bacteria into two different groups and aids in their identification.

3. Successfully perform a Gram stain.

MATERIALS (Work individually):

1. Plate cultures of: Escherichia coli, Micrococcus luteus, Rhodospirillum rubrum

2. Gram's crystal violet

3. Gram’s iodine

4. Ethyl alcohol

5. Gram's safranin

6. 3 glass microscope slides

7. Permanent glass marker (wax pencil)

8. Metal inoculating loop or sterile, plastic disposable loops

9. Bunsen burner

10. Bibulous paper to blot slides

11. Dish detergent

12. Paper towels

13. Immersion oil

14. Staining rack

15. Biohazard bag

PROCEDURE:

You can watch a video of how to perform the Gram stain.

1. Prepare bacterial smears of the three different bacteria as described in Experiment 1 above.

2. Place the first slide on the staining rack.

3. Apply Gram's crystal violet and allow it to react for 1 minute.

4. Holding the slide at an angle, rinse it thoroughly with water until no more dye comes off the slide.

5. Flood the slide with Gram's iodine and allow it to react for 1 minute.

6. Rinse the slide as in step 4.

7. Holding the slide at an angle, carefully add the ethyl alcohol one drop at a time. As soon as color stops coming off the slide (after about 8 to 10 seconds), rinse with water to stop the decolorizing action.

8. Flood the slide with Gram's safranin and allow it to react for 2 minutes.

9. Drain the excess stain from the slide and rinse it.

10. Gently blot the stained slide using bibulous paper.

11. Repeat steps 3 to 10 for the other three slides.

12. Using the procedure described in the Microscopy Lab, observe the slides. 

OBSERVATIONS/CONCLUSIONS:

1. Record results in Table 2 on the Laboratory Report Form.

2. Were all the cells the same color?

3. Were some cells easier to see than others?

QUESTIONS:

1. What is the purpose of a differential staining procedure?
   
   
   

2.  What is the purpose of Gram’s iodine?
   
   
   

3. Why are the smears heat-fixed?
   
   
   

4. How can the Gram stain help you identify bacteria?

KEY TERMS:

• bacillus

• cellular morphology 

• coccus

• colony morphology 

• counterstain 

• decolorizer 

• differential staining 

• diplobacilli 

• diplococci

• Gram stain 

• mordant 

• morphology 

• primary dye 

• spirillum 

• staphylococci 

• streptobacilli 

• streptococci

SUGGESTED READINGS:

Anekwe, Lilian.  2019.  Hans Christian Gram: The biologist who helped investigate bacteria. NewScientist.