National Teachers Enhancement Network: Lesson Plan

Lesson Plan: Life in Streams and Ponds
Subject: Earth and Space / Biology / Microorganisms
Classtime:

*Note: Approximately 4 days lead time prior to Gram stain technique is necessary (for collection of microorganisms and subsequent incubation of plates require to grow cultures).

Total: 4 class periods (50 minutes each)

2 (design tables, observe, and code specimens)
2 (gram stain cultures)
2-3 days time for incubation of plates to grow larger colonies
One-half to one day in the field to collect samples from stream site(s)

Grade Level: 9-12

Materials/Technology:

Suggested per group-

science journal/learning log
pencil
wading boots
scraper
vials
microscope
depression slides
glass slides
sterile pipettes
glass spreader rod
inoculating loop
nutrient agar plates
Gram stain materials (iodine solution, crystal violet, alcohol, safranin)
Bunsen burner
incubation facilities
bleach

Safety, Handling, Disposal:

Safety near the water is essential. Review safety techniques prior to entering the field. It is suggested that a stout rope and/or personal flotation devices be readily at hand (particularly if the water source is near a larger river).

Work with partners-- keep track of each other.
Use extreme caution in rapidly moving water areas.
Leave as little impact on the area as possible (pack it in--pack it out).

Aseptic technique must be rigorously applied when working with microbes. In this particular experiment, after the culture has been examined, teachers should sterilize petri plates by immersing them in a bucket of 5 percent bleach for 20 minutes to kill hazardous bacteria.

Learner Outcomes:

Students should be able to:

Collect microorganisms.
Demonstrate correct laboratory/Gram stain procedure.
Design and construct tables to organize data.

Problem/Purpose:

Students will gather, culture, and Gram stain microorganisms from an aquatic environment, then create a data table detailing and organizing their information.

Background/Inquiry:

Visit a local stream or pond. The waterways are teeming with life not visible to the naked eye, called microorganisms (microbes, "bugs").

In order to study them effectively it is necessary to reveal them. Growing a culture of the microorganisms, then coloring the microbes, and finally using a microscope to observe the colonies makes it easier to sort and categorize them. Using a technique called "Gram stain" will facilitate effective study.

Vocabulary List: Here.

Hypothesis:

N/A

Procedure:

This is an inquiry lesson in which a specific technique is taught. It is through this technique that the students will be immersed in an effective inquiry process. As the National Science Content Standards suggest, "In grades 9-12, students should develop sophistication in their abilities and understanding of scientific inquiry. Students can understand that experiments are guided by concepts and are performed to test ideas some experiences begin with little meaning for students but develop meaning through active involvement, continued exposure, and growing skill and understanding."

Lecture introduction (or review) for students on Microbiology basics, including protozoa, plankton, bacteria, algae, etc.

*Note: The procedure for collecting microorganisms does not take very much time in the field. It is suggested that the collections be a part of some other activities planned for the same trip to the site, for instance, the collection of macroinvertebrates.

The students in the class will be divided into groups of 3-5 students. Each group will collect their own microorganism samples. Prior to the collection, the teacher will have discussed and demonstrated the appropriate approach to microorganism collection.

At a water site, carefully wade into the water and find a rock. Keeping the bottom side down, scrape the upper surface of the rock and place the material in a vial filled with stream water. Have groups of students stay at least 100 feet apart to avoid stirring up too much sediment that could contaminate other group collections.
When in the lab, place one drop of your material into the well of a depression slide. Place the slide under the microscope and sketch the images under low and high power.
Have students keep track of the various drawings of microorganisms found in their science journal/learning log. Be sure to record the date(s) specimens were gathered and note descriptions of the area gathered from, including pertinent names of the waterway and township.
Using a sterile pipette, transfer one drop of the stream water to a nutrient agar plate. Dip the glass spreader rod into alcohol and then flame the rod to sterilize it. After letting the rod cool, spread the drop of water over the plate (use a back and forth motion to spread the colonies out, then turn the plate 90 degrees and repeat the same motion).
Incubate the plate until large colonies develop (37 degrees Centigrade: 2-3 days). *Note: If an incubator is not available, leave the cultures out at room temperature. The cultures will take longer to grow but the results will be similar.
Design a table to describe the different colonies. Use color, texture, shape of edges, and odor to help categorize colonies observed.
Gram stain organisms from different colonies using the following protocol:

Place a drop of water on the microscope slide. Using the inoculating loop, pick up a colony and mix it into the water.
Holding the slide by the edges, quickly pass it through the flame of a Bunsen burner to evaporate the water and fix the microorganisms to the slide.
Cover the heat-fixed smear with crystal violet. After 60 seconds rinse with water.
Cover the smear with Iodine solution for 60 seconds and then rinse with water.
Decolorize with alcohol for about 10 seconds.
Counterstain the smear with safranin for 30 seconds. Follow with a water rinse.
Blot dry. BE CAREFUL to not wipe off the smear. Observe the microbes under oil immersion or high/dry (if the microscope doesn´t have an oil immersion objective).
Record student´s results in the table(s) created in step #6.

Results/Analysis:

Students will report and compare their results with the rest of the class. Hold a general discussion of the techniques used to create the data tables.

An open ended inquiry discussion should then ensue.