Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. Cut the slide holder bars in half using heavy-duty scissors before student use.
2. Obtain or have students collect dirt or mud samples one day prior to beginning this experiment.

Safety Precautions

Wear gloves when working with the microscope slides and the infusion solution. Wipe down the lab area with a dilute bleach solution before and after the experiment. Allow to air dry. Sodium hypochlorite (bleach) provided for disinfection is corrosive and toxic. Avoid contact with acids, which can generate toxic chlorine gas. Wash hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Biofilm slides should be soaked in a 10% bleach solution for several hours and then washed thoroughly with soap and water if they are to be reused according to Flinn Biological Suggested Disposal Method Type I.

Teacher Tips

• Enough materials are provided in this kit for 30 students working in pairs.
• The washers and paper clips are used to weigh down the slides in the infusion solution.
• Set up the number of slides as is appropriate for your teaching plan and student group sizes. Many racks of slides can be placed in one container of infusion solution.
• The dirt and/or mud should be as free of chemicals as possible. Do not use dirt that has been recently treated with fertilizers or pesticides.
• A few examples of substances that may be used as the slide treatments are as follows: petroleum jelly, paint, motor oil, detergent solution, suntan lotion, vegetable oil, Chap stick^®, toothpaste, corn syrup, honey, milk, shaving cream and olive oil.
• Gloves should be worn when removing the slides from the infusion solutions.
• This lab procedure lends itself nicely for student inquiry lab designs. Many variables can be identified and students can easily design “controlled” experiments to test each variable. With relatively simple equipment, many experiments can be run simultaneously.
• Have students perform the procedure with different types of infusion media (e.g., hay, dried leaves).

Sample Data

Answers to Questions

1. Compare and contrast the results obtained with the various slide treatments. Describe conditions that seemed to accelerate or slow down the biofilm growth and explain any differences.

   Some treatments, such as petroleum jelly and motor oil, provided larger colonies or thick layers of organisms. Others, such as detergent and paint, seemed to prohibit the growth of biofilms, as only a few sporadic organisms appeared on those slides.

2. Why did biofilm appear on some of the slides? Where did the biofilm come from?

   Biofilms appeared when bacteria and other microorganisms present in the infusion media clung to the microscope slide and formed colonies.

3. What potential problems could biofilms cause? What about beneficial uses?

   Biofilms could be problematic in terms of sterilization of instruments or formation on objects, such as contact lenses or water pipes, as they could cause contamination.

4. How could items be treated to prevent biofilms from forming on them?

   Items could be treated using solutions or materials that coat the slides and form impermeable protective coatings that do not allow air and water to penetrate.

Introduction

Grow biofilms, experiment with their growing conditions, and examine them under a microscope.

Concepts

• Biofilms
• Colonies
• Infusions

Background

“Slime cities” thrive wherever there is water—in laundry rooms, on contact lenses, on teeth and in flower vases to name a few. These slimy surfaces are referred to as biofilms. Simply put, biofilms are a collection of microorganisms surrounded by the slime they secrete. The study of biofilms has emerged as a major research field in recent years. The reason for the interest is related to the destructive properties associated with certain biofilm types. Understanding biofilms and developing ways to control them have enormous economic and health implications.

Biofilms are composed of populations or communities of microorganisms adhering to environmental surfaces and are usually encased in an extracellular polysaccharide that the organisms themselves synthesize. They may be found on nearly any environmental surface containing sufficient moisture for microbial growth. The development of biofilms is most rapid in flowing water systems where adequate nutrients are available.

A biofilm forms in a fairly predictable, almost stepwise fashion going through several different stages of development. The first step usually consists of a surface conditioning step that does not involve microorganisms. Organic molecules on the surface form a conditioning layer, which tends to neutralize surface change which may prevent a charged microbe cell wall from approaching the surface. Next, a “pioneer” bacteria approaches the surface and if the flow of the water is sufficiently slow, it will cling to the surface. In a rather complex process, the microbe eventually becomes affixed to the surface by the extracellular substances it secretes. Attachment is mediated by these extracellular polymers that extend outward from the bacterial cell wall much like a spider’s web. This web of polymeric material not only helps attachment but also starts to act as an ion exchange system for trapping and concentrating trace nutrients from the overlying water.

As nutrients accumulate, the pioneer cells start to reproduce—each producing its own extracellular web and adhering to its neighboring microbes. Pretty soon a thriving colony of bacteria is established. In a mature biofilm, more of the volume is occupied by the extracellular matrix than by the bacterial cells themselves.

As the matrix builds, it ensnares or traps other types of microbes. Some utilize the waste products of others and eventually a “microbial cooperative” is functioning. The biofilm thus grows and flourishes as a colony and develops its own immunity and protection from outside forces. At this stage, the biofilm can be very difficult to get rid of. If biofilms form in areas such as water pipes, heart pump lines, kidney dialysis tubes or jet fuel lines, they can be a big problem. On the other hand, biofilms may also be used effectively in a water filtration systems to remove bacteria. Learning how biofilms grow and how to get rid of very persistant ones has become a huge research area in recent years. The economic consequences of the destructive properties of biofilms are just starting to be realized.

Experiment Overview

In this activity, microscope slides treated with different substances will be used to observe the growth (or lack thereof) of biofilms.

Materials

Safety Precautions

Wear gloves when working with the microscope slides and the infusion solution. Wipe down the lab area with a dilute bleach solution before and after the experiment. Allow to air dry. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

1. Start by creating an infusion solution. The infusion solution will be an ideal environment for biofilms to form. Fill a large container or bucket with tap water. Place some organic material (dirt or mud) into the container. Be sure the organic matter sinks to the bottom of the water, if possible. Let this infusion solution sit for 24 hours.
2. Insert five plastic microscope slides into the slide holders as shown in Figure 1.
   {10851_Procedure_Figure_1_Slide holder}
3. Attach a washer to a paper clip as shown in Figure 2. Repeat with another paper clip.
   {10851_Procedure_Figure_2}
4. Clip the paper clips to the bottom of the slide holders (see Figure 3). The washers will be used to weigh down the rack of slides.
   {10851_Procedure_Figure_3}
5. Using cotton swabs, treat the top of each slide surface with a different substance (oils, petroleum jelly, ointments, paints and other substances of your choice). Leave one slide untreated to serve as a control. Predict which substances will stimulate or inhibit biofilm growth. Use a marker to label all slides and record the type of treatments for each slide.
6. Place the rack of slides in the infusion solution (paper clip and washer end first). Be sure that the rack of slides is completely submerged in the infusion solution. Place the bucket or container containing the rack of slides in a place where it will not be disturbed for two weeks. Add additional tap water, if needed, to keep the slides completely submerged.
7. After two weeks, carefully remove the rack of slides from the infusion solution. Perform an initial inspection of the slides and record any observations on the Biofilm Worksheet.
8. Carefully remove the slides from the holder and dry the underside of each slide.
9. Examine the slides under a microscope. Use various powers of the microscope and focus so that the bacteria living at different levels can be observed. Record all observations and draw sketches of the biofilms.
10. Using a pipet, place a couple drops of methylene blue stain on each of the slides to make any biofilms more visible. Record all observations and draw sketches of the biofilms.
11. Consult the instructor about disposal.

Student Worksheet PDF

10851_Student1.pdf