Growth of Biofilms

STUDENT HANDOUT:

• Biofilms Student Handout

LABORATORY REPORT FORM:

• Biofilms Laboratory Report Form

INTRODUCTION:

If you ever rubbed your tongue across your teeth, you may have felt a film.  That’s biofilm!  If you ever looked down a sink pipe that has not been cleaned, you may have seen some slime or sludge in the pipe.  That’s biofilm!  

A biofilm is a microbial community made up of bacteria (Figure 1).  They can consist of a single species or can contain multiple species of bacteria (Figure 1, green dots).  These bacteria are able to attach to a surface due to their ability to form extracellular polymeric substances (Figure 1, EPS Matrix).   These biofilms are able to develop into a three-dimensional shape. 

Figure 1.  Schematic of a biofilm.  Image Wikimedia Commons.

Certain infectious diseases are due to biofilm producing bacteria.  These diseases can affect the skin (Pseudomonas aeruginosa), middle ear (Haemophilus influenzae, Streptococcus pneumoniae or Moraxella catarrhalis), urinary tract infections (Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis ).  These are just to name a few.  The EPS also protects the bacteria against antimicrobial agents.  For this reason, treating and removing biofilms can be difficult.

OBJECTIVES:

1. Learn how to grow a biofilm.

2. Learn how we can visualize a biofilm.

PRECAUTIONS:

1. Note the general safety precautions of this manual, especially in relation to use of Bunsen burners.  

2. Do not attempt this experiment until you have learned proper aseptic technique.  If you need a review, refer to the Aseptic Technique Lab.

3. Remember that even non-pathogenic bacteria can cause infections if not properly handled.  

4. Discard all materials in biohazard containers for proper disposal.

5. Autoclave ALL materials as soon as they are no longer needed.

6. Never discard cultures in the trash can.  

7. Wash your hands with an antibacterial soap such as liquid Dial® and wipe tabletops thoroughly with Lysol®. 

EXPERIMENT #1:  Microscopy

MATERIALS (Work in pairs):

1. beaker or fish tank

2. pond water

3. assorted nutrients (hay, mud)

4. clothespin and string

5. microscope slide

6. coverslip

7. microscope

8. stain (either alcian blue or crystal violet) (0.1% --> 1% concentration)

9. deionized water

10. staining rack

11. biohazard container

PROCEDURE:

1. Set up a beaker or fish tank with some pond water.  Add some assorted nutrients that your teacher has provided to encourage the growth of bacteria in the pond water.  

2. Clean a slide with alcohol.

3. Using string and clothespins, suspend the slide in your beaker or tank (Figure 2).  

4. Put your beaker and slide off to the side of the classroom and let it sit for several weeks.  Do not disturb the beaker.  The biofilm will form at the air-water interface.  

After several weeks have passed…

5. Lift the slide out, wipe off the bottom of the slide with a kimwipe and keep it horizontal.  Keep the slide wet until you can add a stain.  If you let it dry out or hold it vertical, it may disrupt the biofilm that has formed on the slide. 

6. Place the slide on a staining tray and flood the entire slide with alcian blue or crystal violet for 3 minutes. 

7. Rinse gently with deionized water.

8. Gently place a coverslip on the slide.  DO NOT PRESS the coverslip down as you may disrupt the biofilm.

Figure 2.  Experimental set-up.  Image made using ChatGPT (AI).

OBSERVATIONS/CONCLUSIONS:

1. Place your slide on the microscope.  Adjust the iris diaphragm on your microscope so it is open, begin looking at your slide at the lowest magnification and work your way up to 400x magnification.  Try to look for:

(a)  Microcolonies

(b)  Bacteria with EPS

(c)  Channels between microcolonies

2. Record your observations in Table 1 of the Laboratory Report Form. 

3. Walk around and look at another groups’ biofilms.  Record your observations of their biofilms in Table 1 of the Laboratory Report Form.   

4. When you have finished, dispose of your slide in the biohazard container.  

5. What differences did you find between your biofilm and others?

EXPERIMENT #2:  Well-Plate Stain Assay

MATERIALS (Work in pairs):

1. Marker or sharpie

2. 3 tubes of sterile nutrient broth (5 ml each)

3. Test tube rack

4. Cultures of Escherichia coli, Bacillus subtilis and Micrococcus luteus.

5. Bunsen burner

6. Metal inoculating loop or sterile, plastic disposal loops

7. Sterile transfer pipettes (or micropipette if available)

8. Sterile 96-well plate

9. 0.1% crystal violet solution

10. Gloves

11. Paper towels

MATERIALS (Shared by the whole class):

1. 37°C incubator

2. 4 tupperware containers:  three of them filled with deionized water.

3. Biohazard container

PROCEDURE:

1. Take a tube of sterile nutrient broth and label it E. coli, followed by your group’s initials.

2. Take a second tube of sterile nutrient broth and label it B. subtilis, followed by your group’s initials.

3. Take a third tube of sterile nutrient broth and label it M. luteus, followed by your group’s initials.

4. Obtain from your teacher cultures of Escherichia coli, Bacillus subtilis and Micrococcus luteus.

5. Using the Bunsen burner and your inoculating loop, inoculate the tubes with the appropriate culture.  If you do not remember how to do this, look back at the first lab activity of the manual (Tube Transfer - Broth).  If you are using a metal loop, remember to flame your loop before and after using the loop.  If you are using a plastic disposal loop, dispose of your loop in the biohazard bin immediately after using it. 

6. Gently mix your inoculated test tubes.

10. Cover the plate and incubate at 37°C until the next class period.  

• Allow the bacteria to grow for at least two days.

• If it will be more than two days before the next lab period, your instructor will take the plate out and store it in the fridge.  

11. Set up four small tupperware containers in a series and add 1 to 2 inches of deionized water to the last three. The first tray is used to collect waste, while the others are used to wash the assay plates.

12. Wear gloves for the next set of steps in the procedure.

13. Remove planktonic bacteria from each microtiter dish by briskly shaking the dish out over the waste tray. To wash wells, submerge plate in the first water tray and then vigorously shake out the liquid over the waste tray.  

14. Add 0.1% crystal violet solution to each well. Stain 15 min at room temperature.

15. Shake each microtiter dish out over the waste tray to remove the crystal violet solution. Wash dishes successively in each of the next two water trays (i.e., the two not used in step 12), and shake out as much liquid as possible after each wash. This step will remove any crystal violet that is not specifically staining the adherent bacteria. The wash trays can be reused for a number of plates, but the water should be replaced when its color becomes dark or when the efficiency of the washes is observed to decrease. 

16. Invert each microtiter dish and vigorously tap on paper towels to remove any excess liquid. Allow plates to air-dry. At this stage, the staining is stable and the dried plates may be stored at room temperature for at least several weeks.

17. Use clorox wipes to clean out all waste trays.  Dispose of clorox wipes in a biohazard container.

OBSERVATIONS/CONCLUSIONS:

1. Look closely at the stained wells.  The crystal violet stains the residual biomass left on the plates due to the bacteria.  

2. Record your observations in Table 2 of the Laboratory Report Form. 

3. Which strain produced the greatest amount of biofilm?  How did you determine this?

QUESTIONS:

1. What component of EPS allows biofilm to stick to a surface?

2. Why did we need to stain the biofilms?

3. Biofilms contain channels.  What function do you think those channels have?

KEY TERMS:

• antimicrobial agents

• aseptic technique

• bacteria (bacterium)

• biofilm

• extrapolymeric substances (EPS)

• infectious disease

• iris diaphragm

• planktonic bacteria

SUGGESTED READINGS:

The Center for Biofilm Engineering is found at Montana State University.  Learn more about what they do by clicking HERE.