Middle School Science and Engineering:
Isolation, Observation, and Identification of Bacteria

Deborah Goldberg
Treasure Mountain Middle School
Park City, Utah
Summer 1994

Introduction

Engineering and Science

One of the goals of the Washington State University and the National Science Foundation Summer Teachers' Institute, is to have teachers prepare a module which will bring the summers engineering experience back to the classroom. The purpose of the summer engineering experience was to strengthen the science and mathematics background of the teacher by showing the teacher how these subjects can be applied to solve real engineering problems. Another outcome of the summer was to acquaint the teacher with engineering, how engineers approach problems and how engineering differs from science. Eventually the teacher should be able to advise students about careers in engineering and demonstrate how engineers solve problems. The following modules were prepared under the guidance of Dr. David Yonge at Washington State University. They focus on the isolation, observation, and identification of bacteria and the removal of sediment from water.

Engineering

There are many different disciplines of engineering. Environmental engineering is just one of the several engineering disciplines. According to Wanilesta in Engineering and the Environment an environmental engineer is "an engineer who applies engineering principles to the problems arising in the interaction of humans and their environment so as to ensure that ecological balances can be preserved". Because of changing environmental challenges, environmental engineering is constantly shifting directions. In recent years, environmental engineering has begun to tackle more difficult problems because the problems are more global, response times are longer, and the problems are less visible. Today environmental engineers depend more on scientific measurements and computer models. Some typical environmental engineering problems might involve water pollution, hazardous substances and risk analysis, air pollution, the greenhouse effect, energy supplies, stratospheric ozone depletion, solid wastes and residues, and treatment of water and wastes.

For example one current project at Washington State University deals with assimilable organic carbon or AOC. Organic carbon compounds are used by heterotrophic bacteria for the production of new cellular material (assimilation) and as an energy source (dissimilation). Most organic carbon in water supplies come from living and decaying vegetation. The goal of this particular project is to find out how the AOC concentration changes with the depth of the soil. Predictions have been made that as you go deeper into the soil there will be less bacteria present. The number of bacteria present may also be related to the type of soil or the type of carbon present.

Not only are AOC concentrations important in subsurface environments, but they have been found to be especially important in water distribution systems. High AOC levels in water distribution systems are related to the growth of bacteria. Regrowth of bacteria within the distribution system is unwanted because of the problems in those systems that arise such as the potential for pathogenic bacterial growth, taste, color, and odor problems, coliform density is increased, and pipe materials are corroded. The AOC concentration must be less than 10 ug / liter to inhibit the growth of HPC ( heterotrophic plate count ) bacteria. One goal is to reduce the AOC concentrations without using higher disinfectant doses. More disinfectant is not necessarily better because chlorine disappears from the water during distribution, chlorine has a limited effect on bacteria, and chlorine resistant species are still able to survive. It is most beneficial for water distribution utilities to design treatment processes that reduce AOC concentrations prior to entering the distribution systems and, if possible, in the groundwater or surface water source.

This teaching module concentrates on isolating, observing, and identifying bacteria that might be common in soil or water. Local pond water will be used so that the students will be able to observe the condition of their water before it reaches the treatment plant.

Additional information about bacteria would be presented to facilitate completion of the accompanying experiments. There are four basic shapes of bacteria: rod shaped, cocci (round), coccobacillary (oval), and spirillum (cork screw shaped). Bacteria can be classified by shape and by staining. Classification by shape is recommended because it easier and less expensive. However one may classify bacteria by a procedure called gram staining. A bacterial cell has a cell wall and a cell membrane. The gram stain will attach to the sugar molecule that is built into certain types of bacterial cell walls. The stain indicates if that particular sugar is present and classifies the bacteria accordingly.

Bacteria can also be grouped by their dependence on temperature. Most bacteria prefer to grow at room temperature or between 10 - 30 C. These bacteria are called mesophiles. Some bacteria are cold tolerant and will grow at 5 C and below. The cold tolerant bacteria are called psychrophiles. Thermophiles are heat tolerant bacteria that grow at 30 C and above. Remember that most bacteria grow at room temperature. Therefore, the teaching module includes incubation at room temperature to insure maximum growth for the broadest range of bacterial species.

Upon opening a plate with bacteria one must be careful because it may give off an odor. The bacteria that give off this odor are called anaerobes. Anaerobes means that these bacteria are growing without oxygen. The smell is a result of anaerobically produced metabolic end products. Sulfur containing compounds called mercaptans are often the cause of the strongest odors. Aerobes, on the other hand, do not typically yield odor causing compounds. Their final breakdown products of AOC are water and carbon dioxide.

Bacteriology and engineering are not always easily distinguishable. This teaching module will try to show the connection between science and engineering in a cooperative learning environment. It will also show the connection between the community, engineering, and science by utilizing a local water source for identification of bacteria.

Goals

Module Goals

The goal of this module is to familiarize the student with the process of isolating, observing, and identifying bacteria by shape and gram staining. In addition, it is designed to promote the skills of observing, communicating, analyzing, and inferring in a "hands on" classroom environment.

Student Learning Objectives

By the end of the module the student will be able to:

  1. Isolate bacteria on a petri dish.
  2. Predict places bacteria might be found in the classroom.
  3. Determine where certain bacteria are found.
  4. Separate bacterial colonies and determine the purity of the colonies.
  5. Observe how pH effects the growth of bacteria.
  6. Observe how temperature effects the growth of bacteria.
  7. Stain bacteria for identification.
  8. Identify bacteria and place into one of the four groups of bacteria based on shape of individual bacterium.

Procedures

The following procedures will contain several different student experiments and teacher demonstrations. They are easily interchangeable to fit the needs of the classroom. One caution: It would work best to begin the experiments on a Friday in order to allow the bacteria sufficient time to be incubated. The preparation of bacteria for incubation could take up to 1 class period. To complete the laboratory work and corresponding test one should allow from 3 - 4 days.

Safety Precautions

  1. Insure proper disposal. All discarded cultures should be placed in a special container and sterilized in an autoclave. If an autoclave is unavailable, scrap out the agar into a throw away container like an old round cake pan. Bake the agar at 450 degrees for 15 minutes. Place in a plastic bag and seal tightly. Now the cake pan and agar may be disposed of in an outside garbage dumpster. To resterilize petri dishes soak in Chlorox.
  2. Wash hands thoroughly with soap and water at the end of the experiment and wipe down the desks with disinfectant.
  3. Be careful of bright yellow, whitish amoeba shaped and pinkish red bacteria, they could be pathogenic.
  4. Keep paper, pencils, and fingers; and other objects out of your mouth during all experimental procedures. Leave the cover on pathogenic bacteria.
  5. In case a living culture is spilled, immediately notify the teacher. The spill should be covered with disinfectant like Lysol or a dilute solution of liquid Chlorox ( 1 part Chlorox to 5 parts water ). Then wash your hands with disinfectant soap and water.
  6. Never pour a living culture in the sink.
  7. All cultures should be grown at room temperature, unless otherwise directed.

Bacterial Diversity Test

Objectives

  1. Predict the different places bacteria can be found.
  2. Classify the different types of bacteria by shape of individual bacterium.

Materials (per group)

petri dish with agar ( can be purchased ready for use or prepared by students )
permanent marking pen
various objects ( lunch room fork, ear wax, hair, eyelash, pen, pencil )
tupperware container ( 12" x 4" )
paper towels
water
microscope slides
loop
lighter
gram - staining kit and wash bottle
hot plate
beaker
Bacto - Nutrient Broth ( dehydrated )

 

Procedure

  1. Use pre-made petri dishes with agar or make agar and pour into the petri dishes the night before the experiment to allow sufficient time to harden.
  2. To make agar, dissolve 8 g. of Bacto-Nutrient Broth (dehydrated) in 1 L. of water in a large beaker. Put the beaker on a hot plate for 15 minutes or until the agar is dissolved. Add an additional 15 g. of Bacto-Agar to the beaker. While the agar is hot, pour it into the petri dishes using just enough to cover the bottom of the plate. Be careful not to use too much, it is a waste of agar. Keep pouring until all the agar is gone. Rinse the beaker immediately when finished with hot water so the agar will not harden inside the beaker. Cover the plates and allow to harden overnight.
  3. On the underside of the petri dish divide the plate into 4 sections using a marker and write the initials of everyone in the group, date, and hour.
  4. Open the petri dish and streak the agar with 4 different objects. Streak the agar by rubbing the object over it without digging into the agar. Some suggestions: raw hamburger or chicken, clean lunch room fork, ear wax, hair, eyelash, pen, pencil, or anything else found in the classroom or on the students. Maintain an additional petri dish with agar as a "blank". Do not open this dish and incubate with the groups dishes.
  5. Close the petri dish and carefully write on the bottom of the petri dish what you put in each quadrant.
  6. To incubate the petri dishes overnight (if possible incubate over the week-end) put some moistened paper towels on the bottom of the tupperware container (rectangular container works best). Place the petri dishes upside down and cover. Leave at room temperature for 2-3 days. Record room temperature.
  7. After incubation look at the bacteria under the microscope (at least 40 x) for individual bacterium and determine what types of bacteria are present. Classify bacteria by shape of each individual bacterium or by gram staining.
    1. Make a bacterial smear. Place 1- 2 colonies of bacteria on a microscope slide. Spread the colonies over the slide. Allow the smear to dry at room temperature. Pass the slide over a flame several times.
    2. Cover the bacterial smear with crystal violet. Let stand for 20 seconds.
    3. Briefly wash off the stain for 2 seconds under running tap water. Drain off excess water.
    4. Cover the smear with Gram's iodine solution and let stand for 1 minute.
      Pour off the Gram's iodine and flood the smear with 95% ethyl alcohol for 10 - 20 seconds. Continue to decolorize with the alcohol until the solvent flows colorless.
    5. Wash the smear with running tap water for 2 seconds.
    6. Cover the smear with safranin and let stand 20 seconds.
    7. Wash the smear gently for 2 seconds, blot with paper towel, and let dry at room temperature.
    8. Examine the slide first under the low and high powered objectives, then you may switch to an oil immersion lens.
  8. CAUTION: Be careful of bright yellow, whitish, or pinkish red bacterial colonies. These could be pathogens!

T-Streak Test

Objectives

  1. Separate bacterial colonies and to determine the purity of pond water.
  2. Identify what types of microorganisms and bacteria can be found in pond water.

Materials (per group)

1 petri dish prepared with agar
1 paper clip or loop
  rubbing alcohol
  lighter or bunsen burner
  permanent marking pen
  pond /creek water

 

Procedure

  1. With the marking pen draw a "T" on the bottom of the petri dish, making 3 distinct areas ( Refer to Figure 1; Appendix A ). Also write your initials, date, and hour on the bottom of the petri dish. Do not open the petri dish!
  2. Bend the paper clip to form a loop.
  3. Dip the paper clip in rubbing alcohol. Use the lighter to burn off all the excess alcohol, sterilizing the loop.
  4. Dip the loop in the pond water making sure that there is a drop of water in the loop.
  5. Streak the agar with the loop. First streak on the agar with the loop over area 1.
  6. Dip the loop back into the rubbing alcohol and burn the excess alcohol off with the lighter again. CAUTIONS: Do not dip back into the pond water. Do not dig into the agar.
  7. Streak back over area 1, then into area 2 to separate the bacterial colonies.
  8. Dip the loop a third time into the rubbing alcohol and burn off the excess alcohol.
  9. Streak back over area 2 and then into area 3.
  10. Dip the loop in rubbing alcohol one last time and burn off the excess alcohol to sterilize the loop. This prevents the spread of bacteria in the classroom.
  11. Put the cover back on the petri dish and incubate overnight in the tupperware container with your diversity test plate. Remember to turn your petri dish upside-down.

At that the same time you are incubating the petri dishes from the Diversity Test and the T Streak Test, set up three good demonstrations.

Ph Test

Objective

  1. Demonstrate the effect of pH on growth of bacteria.

Materials

6 test tubes
  yogurt
  vinegar
  water
  ammonia
  litmus paper or pH meter
3 petri dishes prepared with agar
  loop or paper clip
  marking pen
  rubbing alcohol
  lighter

 

Procedure

  1. Set up the three test tubes as follows: Fill each test tube half way with yogurt. Add the specified liquid until the desired pH is reached ( Refer to step #3 ).
    1. Yogurt and vinegar
    2. Yogurt and water
    3. Yogurt and ammonia
  2. Let the test tubes sit for 1/2 - 1 hour.
  3. Recheck the pH of each test tube with litmus paper or a pH meter for the following:
    1. pH = 1.2
    2. pH = 6.7
    3. pH = 13
  4. Take 10 mL. of sample from test tubes #1, #2, #3 and pour into clean test tubes.
  5. Continue using the T Streak test to place the sample on the agar. Streak one plate for each sample. Write the pH on the bottom of the petri dishes.
  6. Incubate with the other petri dishes.
  7. Observe all three petri dishes the same time the other petri dishes are finished incubating.

 

Temperature Test

Objective

  1. Demonstrate how temperature affects bacterial growth.

Materials

3 petri dishes prepared with agar
  pond water
  loop
  rubbing alcohol
  lighter
  marking pen

 

Procedure

  1. Take 3 petri dishes and streak the agar with pond water as described in the T Streak Test procedures.
  2. Incubate one in the refrigerator. Be sure to mark bottom of the petri dish with the temperature. Try to keep the petri dish below 5 C.
  3. Incubate the second petri dish in the tupperware container or at room temperature. Record the temperature on the bottom of the petri dish.
  4. Incubate the third petri dish on a heater. Record the temperature on the bottom of the petri dish. Try to heat the petri dish above 30 C.
  5. Set up the demonstration and observe all three petri dishes on the same day when the experiment is complete.

 

Mouthwash Effectiveness Demonstration

Objective

  1. Demonstrate how effective different mouthwashes are in discouraging bacterial growth.

Materials

1 petri dish
4 different brands of mouthwash
1 single hole punch
  paper
  marking pen

 

Procedure

  1. Punch out holes from a piece of paper with a single hole punch.
  2. Soak the holes in the different mouthwashes. Use at least four different mouthwashes for a variety.
  3. Place the soaked holes on top of the agar.
  4. Label the bottom of the petri dish with the date and the brand name of the mouthwash.
  5. Incubate with the other petri dishes.
  6. Observe the petri dish on the same day when the rest of the experiments are finished incubating.

Evaluation

Lab Work: Lab forms have been included in appendices B and C.

Test: A test and answer key have been included in appendices D and E.

Evaluation: A student evaluation of the engineering and science lesson has been included in appendix F.


Alternative Environmental Engineering Laboratory

Removal of Sediment from Water

Objectives

  1. Agglomerate clay and silt and make them settle by chemical addition.
  2. Observe how much chemical you must add when processing water for drinking.

Background

This laboratory simulates the process of treating a surface water supply and converting it into drinking water. The steps involved in processing water for drinking are as follows: ( Refer to Figure 2; Appendix A )

Step 1: Water is removed from a lake or reservoir and sent to the processing plant. This water contains algae and sediments that need to be removed. To remove these materials, the water is sent to a flocculation tank.

Step 2: Part of the flocculation tank contains0a rapid mix tank. In the rapid mix tank, chemicals are added that allow particles to agglomerate. The flocculation tank is used to allow the particles to gently contact and grow in size.

Step 3: The water is then sent to a sedimentation tank. Here the particles formed in the flocculation tank settle to the bottom and cleaner water is taken off the top of the sedimentation tank.

Step 4: The water is now divided into two parts. The top of the tank has clean water and the bottom of the tank has sludge. The clean water goes to a sand filter. The filter removes any smaller particles that were not removed in the sedimentation tank.

Step 5: HOCl (bleach or chlorine) is added to the clean water to kill any disease causing pathogens. Chlorine is usually bought in a gaseous form so that the water plant can make their own HOCl solution. It is cheaper! The clean and disinfected water is finally distributed to towns and cities.

Materials

6 1000 mL. beakers
1 pH meter or litmus paper
1 magnetic stirrer or stirring rods
  water
  clay
  Alum solution ( 20 g. / 500 mL. tap water )
  clay solution ( 1 g. / 1000 mL. tap water )

 

Procedure

  1. In each beaker dissolve 1 g. of clay in 1000 mL. of tap water. This simulates a muddy surface water in need of treatment. Mix the clay and water together rapidly until the clay is dissolved and the water appears dirty. This dirty water should be mixed before class. Have the students begin with the addition of the chemical.
  2. Add Alum to each of the beakers with 1 - 2 minutes of rapid mixing in the following manner:

    Beaker 1: 0 mL.
    Beaker 2: 2 mL.
    Beaker 3: 4 mL.
    Beaker 4: 6 mL.
    Beaker 5: 8 mL.
    Beaker 6: 10 mL.

  3. After adding Alum to the muddy water mixture test the pH. Adjust the pH in a range of 7.0 - 8.0. If the pH is low add 2 N NaOH and if the pH is high add 2 N sulfuric acid until it reaches the desired pH. CAUTION: Be careful when handling NaOH and sulfuric acid. Gloves and safety glasses should be worn at all times.
  4. Gently mix the Alum and the water together with a stirrer for 20 more minutes (preferably at 20 rpms). This simulates the slow mix in the flocculation tank.
  5. After the mixing is complete allow the beakers to stand for 20 minutes. You should be able to observe floc particles settling to the bottom. This simulates the settling in the sedimentation tank.
  6. After the sedimentation period is complete, compare the six beakers to see which water is the cleanest.

Extensions

Determining Water Clarity by Absorption

Measure clarified water absorption with a spectrophotometer. Plot absorption versus Alum dose. Find the optimum low dose of Alum from the graph.

Calculating the Cost of Purification

Figure out the cost of purifying 1 mL. of water and 1,000,000 mL. of water. Compare your mathematical calculations. If possible graph the cost of purification versus the amount of water.


Appendix A

Diagram of Petri Dish and Wire Loop.

Figure 1: Diagram of "T" on the bottom of a petri dish and wire loop made from a paper clip.

Schematic of a Water Processing Plant.

Figure 2: Schematic diagram of water processing plant.


Appendix B

Name_____________________________________________ Date__________ Hour__________

Diversity Test

Purpose:

(Write a complete sentence about the purpose of this laboratory investigation).

__________________________________________________________________________________

__________________________________________________________________________________

 

Materials:

1 petri dish prepared with agar
4 different objects
1 marking pen
1 microscope, blank microscope slide, and cover slip
1 lighter

 

Procedure:

After your teacher has reviewed the lab procedures and safety precautions, the designated person should collect all the necessary materials for their entire group.

  1. Label the bottom of the petri dish with the initials of all the members of the group, date, and hour. Divide the petri dish into 4 sections too!
  2. Each person in the group should pick a section and streak the agar with any object in the classroom. Label the bottom of the dish with the name of the object you used.
  3. Cover the petri dish and bring it to your teacher for incubation.

Data:

Look at the petri dish under the microscope and sketch your observations. Be sure to include the name of the object, colors, and shape.

Prepare a smear plate and observe the bacteria under the microscope at 40 x. Record all observations including shapes and drawings of each shape


Appendix C

Name_____________________________________________ Date__________ Hour__________

"T" Streak Test

Purpose:

(Write a complete sentence about the purpose of this laboratory investigation).

__________________________________________________________________________________

__________________________________________________________________________________

 

Materials:

1 petri dish prepared with agar
1 paper clip
1 lighter
1 marking pen
1 microscope, blank microscope slide, cover slip
  rubbing alcohol
  pond water

Procedure:

After your teacher has reviewed the laboratory procedures and safety precautions, the designated person should collect all the necessary materials for your entire group.

  1. Keeping the petri dish covered write the groups initials, date, and hour on the bottom of the petri dish. Also divide the bottom of the petri dish into 3 sections by marking a "T".
  2. Streak your petri dish in the "T" formation with your paper clip loop and 1 drop of water.
  3. Dip the loop in the pond water only once and sterilize it after each section is streaked.
  4. Cover the petri dish and bring it to your teacher for incubation.
  5. Prepare a wet mount of the pond water for observations under the microscope.

Data:

Look at the petri dish under the microscope and sketch your observations. Be sure to include shapes and colors.

Prepare a smear plate and identify and record the types of bacteria present. Draw pictures of the bacteria.

From your wet mount identify the microorganisms present and draw a picture of them:

Name:

 

 

Picture:

 

 


Appendix D

Name_____________________________________________ Date__________ Hour__________

Bacteria Test

  1. Name the four different shapes of bacteria and draw a picture of each shape.

    1.

    2.

    3.

    4.

  2. Describe some different places where bacteria might be found.

  3. How can you isolate a single bacterium from a bacterial colony?

  4. What effect does temperature have on bacterial growth?

  5. What effect does pH have on bacterial growth?

  6. Why is it important to wash your hands after you finish observing bacteria?

  7. Are all bacteria harmful? Explain!


Appendix E

Bacteria Test ( key)

  1. Name Shape
    1. rod 1. rod
    2. cocci 2. round
    3. coccobacillary 3. oval
    4. spirillum 4. cork screw
  2. Bacteria are found everywhere. For example: clean silverware, door knobs, fingers, pens, pencils, hair, eyelashes, or food.

  3. Pathogens may be identified by their color. Stay away form bright yellow, opaque amoeba shaped bacteria and pinkish red bacteria.

  4. Bacteria grow best at room temperature. Most bacteria can not survive at either extreme cold or hot.

  5. Bacteria grow best in a neutral environment. If it is too acidic or too basic it will kill the bacteria.

  6. Washing hands prevents the spread of bacteria.

  7. No. Some bacteria are helpful like those found in the stomach that aid digestion.


Appendix F

Student Evaluation
Isolation, Observation, and Identification of Bacteria

Answer the following questions in complete sentences. Remember that theses questions are about the lesson, not the teacher. Please be honest. There are no incorrect answers.

  1. What did you learn about bacteria that you did not know about before?


  2. What did you learn about engineering that you did not know before?


  3. Did you prefer to learn science by listening to the teacher or by doing science experiments?


  4. Which teaching method helped you learn the most?


  5. How would you improve this lesson?


  6. Did the lesson make you more interested in science and engineering?