Materials Included In Kit

Additional Materials Required

Prelab Preparation

Making and pouring the nutrient agar plates should be done at least one day before the lab. Add only enough nutrient agar to cover the bottom of each plate, about 20 mL.

Preparation of nutrient agar: Mix the 10 g of nutrient agar with 400 mL of water in an Erlenmeyer flask. Place a foam plug in the mouth of the Erlenmeyer flask to prevent evaporation of the water. Bring the solution to a boil, stirring occasionally to completely dissolve the agar. Sterilize the nutrient agar using an autoclave or pressure cooker. Sterilize at 121 °C at 15 psi for 15 minutes.

To prevent contamination before or after pouring the plates, follow these steps:

• Disinfect the work area using a spray bottle containing a 10% bleach solution or liquid Lysol^®.
• Open the sealed sleeve of plastic Petri dishes just before pouring the nutrient agar. (Keep this sleeve for storing the plates after they have been poured and cooled.)
• Without removing lids, put the dishes in a single line on the outer edge of the work area.
• Using insulated gloves, pour the nutrient agar into each Petri dish by picking the lid straight up, keeping the lid between you and the liquid agar. Hold your breath while pouring each dish to help prevent airborne contamination. Replace the lid and move to the next dish in the line. Avoid large, obvious movements to prevent creating air drafts.
• Allow the nutrient agar to cool without disturbing the dishes in any way. (Depending on room temperature, this could take up to 30 minutes.) When the dishes are completely cooled, tape them shut with two pieces of clear tape. Turn the dishes upside down and put them back in the plastic sleeve. Tape the sleeve shut and put the entire sleeve in a refrigerator or other cool location. Do not store in freezer!
• Prior to handing out the dishes to students, check for contamination on the nutrient agar surface. If present, tape the dish(es) shut and dispose of according to the instructions.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures governing the disposal of Biological Waste, Type I, and review all federal, state and local regulations that may apply, before proceeding. Obtain a container large enough to hold all of the contaminated plates as well as those used by students. Ensure that all plates are taped shut and then use the container to collect the plates. Carefully pour a 10% bleach solution into the container until all the plates are covered and let them soak overnight. Carefully drain off the used bleach into a laboratory sink with the water running. Using gloves, place all the plates into a plastic garbage bag. Tie off the bag and then place this bag inside a second bag. Label the bag and dispose of in the dumpster yourself or warn the custodian to avoid tearing the bag while transferring it to the dumpster.

Lab Hints

• Enough materials are provided in this kit for 30 students working in pairs or for 15 groups of students. The Prelab Questions and procedures for Day 1 can reasonably be completed in one 50-minute class period.
• Tyndallization can be used if no autoclave is available. Boil the nutrient agar for 20 minutes in the covered Erlenmeyer flask. Cool the agar and incubate at 37 °C for a day. Repeat the boiling, cooling, incubation procedure for three consecutive days followed by boiling again. The three incubations force many of the heat-resistant spores to grow due to heat shock. These newly growing bacteria and fungi are killed in the next boiling step.
• Discussion of student answers to the Prelab Questions and Day 2 procedures may be combined. Since analysis of all student plates should take place at the same time, do not give dishes to students until Day 2.
• The drawing and identification of colonies listed for Day 3, and answering the Post-Lab Questions, may require an extra day to complete. A minimum of one week may need to be set aside to complete the entire lab and discuss the results.

Teacher Tips

• Copy and laminate Identifying Descriptors page for future use.

• If available, compound microscopes set at the lowest power or dissecting scopes will help students identify a number of distinguishing microbial colony characteristics, such as texture and edge morphology, that may not be clearly visible with the naked eye.
• As extra credit, students could choose an especially interesting colony and do research to identify the microbe (by scientific name) that made the colony.

Answers to Prelab Questions

1. Write the pronunciation guide and definition of the italicized word in the subtitle—ubiquity.

Pronunciation: yōō bǐk'wǐ tē; Definition: Being or seeming to be present everywhere.

2. As a lab group, brainstorm a list of sites or surfaces at home that may contain microorganisms and write a minimum of 10 possible locations:

Student answers will vary.

3. From your list in Question 2, each person in the group selects a surface or site at home from which they will obtain a microbe sample and write the name of the location on the line below. Individuals in the same group should not select the same site. Caution: Do not choose a site that is commonly associated with human waste (i.e., inside toilets).

Selected microbial site/surface: Student answers will vary.

Answers to Questions

1. Was the source of the microbes found on each plate identical? If not, how did the locations differ?

All reasonable answers should be accepted.

2. Were there any similarities in colonies or in the descriptions of the colonies? Explain your answer using the information written on the page with your drawings.

All reasonable answers should be accepted.

3. Were there any differences in colonies or in the descriptions of the colonies? Explain your answer using the information written on the page with your drawings.

All reasonable answers should be accepted.

4. Were there more similarities or differences between the two dishes? Based on your comparison, do some microbes seem to prefer one habitat (surface) over another? Explain your answer.

All reasonable answers should be accepted.

Before answering Questions 5 and 6, observe the plates of all other class members and write down the sources of the microorganisms growing on their plates.

5. Which site or surface was the most surprising in terms of the number of microbial colonies that grew from that source? Why was this site or surface so surprising?

All reasonable answers should be accepted.

6. Briefly explain how this lab has helped you understand the ubiquity and diversity of microorganisms?

Microbes grew on most plates, regardless of their source, and few colonies were identical, supporting the idea that a diverse collection of microorganisms exists everywhere.

Teacher Handouts

10664_Teacher1.pdf

References

User Friendly Microdiversity. http://biology.beloit.edu/HHMI/99/projects.html (accessed April, 2005).

Norrell, S. A. and Messley, K. E. Microbiology Laboratory Manual: Principles and Applications. Upper Saddle River, NJ, Prentice Hall, 1997.

Introduction

Do you know the meaning of the italicized word in the subtitle? Due to the fact that they are more visible, the wide variety of macroorganisms on the Earth are generally more familiar to most people. However, because of microorganisms, at the completion of this activity you will realize that even when you think you are alone, you are never truly alone!

Concepts

• Microbes

• Biological diversity

Background

The term microorganism refers to any organism that cannot be clearly seen without the aid of a microscope. Even though they function much the same way as larger organisms, these microorganisms may exist as free-living, single cells or as part of a colony of single cells. Due to their size, colonies of microbes are much more visible than single cells. Nevertheless, unlike most macroorganisms, every single cell within the colony has the capacity to become independent and free-living, if necessary. Typical examples of microorganisms include bacteria, protists, fungi and some types of algae.

To grow and thrive, most microorganisms need the same basic living conditions that humans need, such as moisture/water, a place to live, food, air and reasonable temperatures. These needs are usually met using two of microbiology’s most familiar items. One is the round Petri dish, invented by J. R. Petri in 1877. A food supply is placed inside the dish. Nutrients, including vitamins and minerals, are combined in a variety of concentrations with agar, an extract taken from marine red algae. Agar has a gelatin-like consistency but remains solid at room temperature. After the agar is melted, poured into the Petri dishes, and allowed to cool, the microbes are “plated” (added) onto its surface. The dishes are then placed in a secure, preferably warm, environment to grow for 24 to 48 hours.

Experiment Overview

The focus of this activity will be to grow (culture) microorganisms that are collected from a variety of “habitats” outside of class, including the shower area in your own home. Since one never knows whether disease-causing microbes will be found on surfaces in any given area, it is critical for the health and safety of everyone that the Petri dishes are not opened after inoculation (adding the microorganisms).

Materials

Prelab Questions

1. Look in a dictionary and write the pronunciation guide and definition for the italicized word in the subtitle—ubiquity.
2. As a lab group, brainstorm a list of sites or surfaces at home that may contain microorganisms. Write a minimum of 10 possible locations:
3. From your list in Question 2, each person in the group select a surface or site from which they will obtain a microbial sample and write the name of the location on the line below. Individuals in the same group should not select the same site. Caution: Do not choose a site that is commonly associated with human waste (i.e., inside toilets) due to the greater likelihood of harmful microbes being present.

Selected Microbial Site/Surface:

Safety Precautions

Any of the microbes growing in the Petri dishes may be pathogenic (disease-causing). Therefore, once the dishes have been sealed, they should not be opened again. Wash hands thoroughly with soap and water before leaving the lab. When adding microorganisms to the agar, wearing latex gloves is recommended. Follow all other laboratory safety rules, such as wearing safety goggles.

Procedure

Day 1

A sterile swab applicator will be used to collect microorganisms from the site/surface you have chosen. To ensure the best results and prevent too much contamination, please read the steps below and then follow them.

1. Use a marker to label a zipper-lock bag with your name and class period.
2. With a partner, obtain one package of sterile swabs from your instructor, and carefully open the end that says, “PEEL.”
3. As your partner opens your zipper-lock bag, remove one swab from the package by slowing pulling on the “stick,” immediately put it inside the bag, and seal it up.
4. Repeat step 3 using your partner’s zipper-lock bag.
5. If available, obtain a pair of latex gloves from your instructor, wrap them inside a paper towel and take them home.
6. Before collecting any sample, put on the gloves and carefully remove the swab from the bag without touching the swab end. Note: If the sample collecting site is or contains a liquid or solid suspended in a liquid, after dipping the swab, press it against the inside of the container to force out excess liquid, return it to the bag and seal. If the sample is to be taken from a dry surface, dip the swab in a slow stream of tap water to moisten, and then rub the surface with the tip without rolling it. If nothing is visible on the swab, make a colored mark on the wooden shaft of the applicator on the same side used to collect the sample. Do not touch the tip! This will ensure that the correct side of the tip is used when applying the sample to the agar on Day 2.
7. Return the swab to the bag and seal.

Day 2

1. Obtain a Petri dish of sterile nutrient agar media from the instructor.
2. If working in a group of three, use a permanent marker to divide the bottom of the Petri dish (also called a plate) into thirds (see Figure 1). Each person in the group should then select one section and mark it with their initials on the edge, not across the middle. (If working with only one other person, draw one line across the bottom of the plate to make two sections.)

3. Wash hands with soap and water or put on gloves, then carefully remove the cotton swab from the bag without touching the end(s) used to collect the sample.
4. Open the lid of the Petri dish at a 45° degree angle with one hand while keeping the lid between you and the agar’s surface (see Figure 2).

5. With the other hand, gently swab a zigzag pattern on the surface of “your” section of the agar. Remember: Do not roll the swab or break the surface of the agar! Put the used swab back in the bag, seal it, and discard the bag in the trash. Do not lay it down on the counter or desktop.
6. After each group member has swabbed his or her section of the plate, use transparent tape to secure the lid in three places around the plate. This prevents the lid from being easily opened yet still allows air inside.
7. Put the plates in the designated area for 24 hours. Note: Store the plates upside down to prevent water droplets, which will condense on the lid, from dripping onto the growing cultures. Note: If no growth is visible on the plate after 24 hours, it may be necessary to let the plate incubate at room temperature for one or two more days.

Day 3

1. Without opening the lid of the Petri dish, turn the plate so the lid is on top and examine the surface of the agar. (Examining the agar from the side may be necessary if condensation on the lid blocks the surface.)
2. Use a dissecting microscope, if available, to help identify details not visible to the naked eye. Different microorganisms produce different kinds of colonies. Observe the size, shape, color, texture and any other distinguishing features of each colony in your section of the plate.
3. Obtain a sheet of unlined paper and place the Petri dish on the paper. Use a pencil to trace around the dish to make a circle.
4. Use a marker to divide the lid over your section of the plate into small quarters. This will help keep track of each colony’s location as they are drawn on the paper. (A pink or green highlighting marker may be useful for this so that the agar is visible through the lines. See Figure 3.)

5. Within the circle on the paper and to the best of your ability, draw and color the colonies the way they appear on the agar’s surface.
6. With the aid of the Identifying Descriptors page, write at least three descriptors, from the five categories on the page, to describe each of the colonies that were drawn.
7. On the back of the page used to make the drawings, record the information listed below for your plate or section of plate.

1. The source of the microbial population(s).
2. The total number of different colony types present on the plate.
3. The number of similar colonies present on the plate.

8. Consult your instructor for the appropriate disposal procedures of used Petri plates.

Student Worksheet PDF

10664_Student1.pdf