The Microbial World Around (and In!) Us
Grade level(s):Middle School (6-8), High School (9-12), Grade 8, Grade 9, Grade 10
Subjects(s):Biology/Life Science, Science Skills
Microorganisms are the dominant form of life on earth. Microbes have evolved very diverse lifestyles, much more diverse than those of macroorganisms. Microorganisms help sustain macroscopic life on earth, such as through nutrient cycling and direct symbioses. Only a minority of microbes causes disease. (This lesson will be followed by a lesson in antimicrobial resistance, see “Antibiotic Resistance” lesson.)
Before this lesson students should be familiar with the terms:
microorganism, petri dish, agar, media, sterile/aseptic technique
Vocabulary introduced during lesson includes:
prokaryote, eukaryote, phylogenetic tree, colony, transfer, streak technique, differential media, gram-negative, gram-positive, enteric
What you need:
- Notebooks to collect data and worksheets
- Petri dishes
- LB agar (nutrient agar) (can order from Carolina Biological Supply)
- MacConkey agar (can order from Carolina Biological Supply)
- Cotton swabs
- Disposable loops, or re-usable loops
- Bunsen burners, and flint lighters
- Access to a fridge (to store agar petri dishes before use)
- Access to a 30°C or 37°C incubator (for microbial growth)
- Access to projector
- Capabilities to show an online video
- 3% hydrogen peroxide, sharpies
- (optional) microscope slides
Groups of 3 students
Classroom or laboratory space.
After inoculating petri dishes, cultures need to incubate: overnight (12-24 hrs) at 37 ºC or (24 to 48 hrs) at 30 ºC. The lessons can be finished over a period of 2 days. If colonies are transferred and microbes are to be isolated, additional incubation times (12 to 48 hrs) may be necessary. This lesson is followed by a lesson in antimicrobial resistance, and not too much time should pass between the 2 lessons (collectively these activities can take a week's time).
In the first lesson in this two-part series, students are introduced to the concept of microbes by collecting samples to grow on agar Petri dishes. They will isolate colonies and perform two biochemical tests that microbiologists regularly use to identify bacteria.
Overall Lesson Plan Layout:
1. Students will complete the pre-lab worksheet to assess prior knowledge and to address misconceptions.
2. The attached presentation will be used to accompany the lesson (See attachment).
3. Microbial diversity is introduced by:
- Showing a video
- Doing an activity (as a class)
4. The students are divided into groups of three for the hands-on work. Within these groups students pass around the petri dish so that each student can analyze it closely, and then they alternate between transferring colonies onto fresh agar media. Those students who are not transferring colonies can either watch the student who is doing so or draw the streak technique in their notebooks as practice until their turn. (Details for this activity are described below)
- Students should be aware of general lab safety.
- Students should know what a living cell is and its components.
- Students will have a better awareness and appreciation of the microbial world.
- Students will better understand the importance of microorganisms for the health of the planet and humans.
- Students are able to perform basic laboratory techniques.
- Students make observations creatively, carry out experiments, and record and interpret data.
NOTE: This lesson is followed by a lesson in antimicrobial resistance in which students will use similar lab techniques.
Microbes refer to microorganisms (too small to see) that include members of archaea, fungi, bacteria and protozoa domains. They were among the first living forms on earth. They can be unicellular or multicellular, eukaryotes or prokaryotes. Their habitats expand the entire biosphere. Microbes are found deep in the earth’s crust, on the ocean floor, in hot springs (extremophiles), inside and on other living organisms, the atmosphere and everywhere in between. They function as recyclers and decomposers. Only a small fraction of known microbes are pathogens. Many have been used to the production of various foods and beverages (through fermentation), and in biotechnology (genetic engineering, waste treatment, production of chemicals…). The study of these microbes is called microbiology.
Bacteria are prokaryotes because they do not contain a membrane-bound nucleus, and lack most organelles. Their size range from 0.5 μm to 5 μm, and their morphology vary from spheres to oblongs, rods to spirals, and more. In a phylogenetic tree, species are arranged and classified on a branching diagram according to their genetic similarities and differences, rooted by nodes representing a common ancestor. Bacteria make up one of three branches, along with archaea and eukaryotes. Although they are very similar in appearance to archaea, the latter have genomes more similar to that of eukaryotes. Their position on the tree is therefore closer to the eukaryote branch.
In humans, bacteria have beneficial roles, such as aiding digestion (by breaking down food so it is easier to absorb) and working with the immune system to defend against pathogens. Bacteria that live in the gut are called enteric bacteria, and they make up the gut flora. Many of these symbionts are acquired within hours of birth and throughout life. Pathogenic bacteria can cause serious diseases (such as cholera, syphilis, anthrax, tuberculosis etc.) and are treated with antibiotics. Recently, a renewed appreciation of beneficial bacteria and concern over the emergence of “superbugs”, bacteria that are insusceptible to current antibiotics, have spurred debates over the proper use of antibiotics.
In the laboratory, only about half of known microorganisms can be grown in vitro. Most commonly, bacterial samples are plated (“inoculated”) on Petri dishes filled with agar mixed with LB medium (lysogeny broth, containing nutrients necessary for growth). Inoculation is done by streaking the toothpick/needle across the plate multiple times to spread the culture. The bacteria that thrive on the agar form round colonies that can be picked up with a toothpick or needle and inoculated onto another plate to isolate it (called a transfer). Sterile/aseptic techniques are very important to avoid cross-contamination. The Petri dishes are lidded and stored upside down. The working station must be cleaned thoroughly before and after the procedure. Ideally, the equipment and reagents are sterilized as well. Bunsen burners can be used to sterilize needles and other small metals. Use of personal protective equipment (PPE), such as lab coats, goggles, and gloves, is recommended. When finished, place plates in autoclavable bags and dispose as biohazard. Liquids can be disposed of in the sink with plenty of water. Please check with local laws and guidelines for more information on handling and disposal procedures.
The Gram stain is used to classify bacteria according to the properties of their cell wall. Gram positive bacteria have peptidoglycans (sugar and amino acid polymers) in their cell wall that stain purple with the crystal violet dye in the stain. Gram negative bacteria have much thinner walls and are stained (counterstained) pink with the safranin dye.
Differential media are used to visually distinguish different types of bacteria. For example, MacConkey agar can grow Gram negative bacteria on plates. It is used to classify bacteria on whether they are lactose fermenters or non-fermenters. The medium contains lactose (sugar commonly found in milk) as food. Lactose fermenters will digest the lactose and produce acid which will lower the pH of the medium. As a result, the colonies will be red/pink. Non-fermenters will use peptone (amino acids) as food instead and produce ammonia which will raise the pH. The colonies will appear white.
The catalase test identifies bacteria that can grow in the presence of oxygen (aerobic). The catalase enzyme can convert hydrogen peroxidase (which is toxic to cells) into water and oxygen. In the procedure, a bacterial colony is mixed with a drop of hydrogen peroxide on a slide. Bubbles of oxygen will appear if the bacteria have catalase.
Prepare agar petri dishes in advance. Be careful when handling molten agar and using a Bunsen burner.
Lesson Implementation / Outline
- Demonstrate streaking technique for inoculating bactria on agar petri dishes.
- After discussion, students identify sources and swab microbes onto LB agar petri dishes.
- Agar petri dishes are incubated overnight at 37 °C.
- Students are assigned pre-lab worksheet as homework (or done in class).
1) Circle where microbes can be found: everywhere on Earth
2) True or False: Most bacteria cause diseases. False
3) True or False: Antimicrobials and antibiotics are great ways to kill bacteria. Antimicrobials kill microorganisms such as fungi, archaea, bacteria and protists. Antibiotics kill or inhibit the growth of bacteria.
4) What do bacteria eat? Bacteria absorb nutrients from their environment. Different types of bacteria will feed on a wide variety of compounds from oil, sugar, sulfur, waste products… even electrons.
5) Guess how many bacteria can fit in a drop of water? Depends, over a million bacteria can be found in a drop of seawater.
In a pinch of soil? Over 50 billions microbes, or about a billion bacteria in 1 tablespoon of soil.
6) Write the name of one bacterium you know: Common bacteria are Escherichia coli, Lactobacillus acidophilus (in yogurt), Salmonella etc.
7) How is a bacterium similar to and different from a cell (ie. A human cell)?
8) List 5 similarities: Many bacteria are shaped round or oblong similar to most mammalian cells; a bacterium is a living cell; they both have a plasma membrane; they both contain DNA as genetic material, they both use ribosomes.
9) List 5 differences: A bacterium is much smaller (0.5 μm – 5 μm, compared to 1 to 100 μm for most cells); they lack organelles such as a mitochondria or a membrane-bound nucleus, they are prokaryotes while mammalian cells are eukaryotes, bacteria have a circular chromosome, often have a complex cell wall, no cytoskeleton, they can transfer DNA to each other directly.
Day 2 (Following day):
UNDERSTANDING YOUR AUDIENCE
1. How would you prove that the samples you collected are alive? What conditions for life have been demonstrated? (Living organisms are made of cells, they need nutrients to grow, propagate and eventually die)
2. (Show pictures of bacteria, protists, fungi, archaea.) How are they similar? How are they different? How could you identify the colony you picked?
(More biochemical tests, DNA isolation.)
INTRODUCTION TO MICROBIAL DIVERSITY (See attachment)
Explain the phylogenetic tree of microbes and bacteria.
Show video “The Invisible Universe That Lives On Us And In Us” (5:25)
- Go over the pre-lab worksheet
- Give overview of activity (picking colonies, catalase test)
- Show petri dishes from class via overhead projector
What do you expect to see on your plate? How will you know what microbes are present?
- Divide the class into groups of three. Pass a plate to each group to look at.
- Students draw what they see in the petri dishes and record their observations. Yeast, for example, has a very particular smell. Tests are needed to identify the microorganisms.
- Catalase test: have each group choose and pick different colonies. Place each in a drop of hydrogen peroxide solution on a slide. Record what happens.
- If bacteria will be isolated, have each group pick a colony to inoculate on a MacConkey plate for further characterization. Incubate overnight and store in fridge until ready to observe.
- What bacteria are present on a MacConkey plate? Gram-negative bacteria. Why do they have different colors? Pink/red for lacto fermenters, white for non-fermenters. They eat different foods in the media.
(NOTE: Follow this lesson plan up with the “Antibiotic Resistance” lesson)
The pre-lab worksheet is the first opportunity to assess students' knowledge and understanding. Working with students in small groups we can further evaluate their understanding. Post-lab discussions and assessment of lab notes will also be valuable. After the antimicrobial resistance lesson, a quiz is given to evaluate knowledge gained by both activities.
How many different colonies was the class able to isolate? Can any conclusions be drawn from the tests? What conditions could be changed to grow other types of microbes?
(Liquid media, different incubation conditions (temperature, time, pressure), different foods. If time is available, investigations can be performed to identify more microbes.)
Extensions and Reflections
A beautiful animated exploration of the human microbiome.
The students particularly enjoyed the hands-on component of this lesson. It may be a good ideal to give the microbial diversity background and show the video (or assign watching the video as homework) before the hands-on work. With additional time the class could explore liquid vs. agar cultures and incubation conditions. One key bit of information that was left out of this lesson is that the majority of microorganisms are unculturable and therefore what is seen on petri dishes represents a small fraction of the bacteria in the natural environment. It would also be great to add a microscope component to this lesson. By using a microscope, students can see both individual bacteria in addition to the colonies of bacteria. Additionally they could earn that similar looking colonies may not represent the same type of bacteria. Another idea that an instructor can explore using this lesson plan would be for students to monitor the growth of their bacteria by looking for changes in colonies on their petri dishes over several days.