Microbial Discovery Activity

Is It Clean or Just Unseen? Dirty

Water and the Naked Eye

Authors
Kylie Weigel
Niagara University
Niagara University, NY
kweigel@mail.niagara.edu

Mark Gallo, Ph.D.

Associate Professor of Biology
Niagara University, NY
mgallo@niagara.edu

Intended Audience
K-4
5-8
9-12 X

Activity Characteristics
Classroom Setting x
Requires Special Equipment x
Uses Hands-on Manipulatives x
Requires Mathematical Skills x
Can Be Performed Individually x
Requires Group Work x
Requires More Than One Class Period (45 min) x
Appropriate For Students With Special Needs

American Society for Microbiology

Education Department
1752 N Street, NW
Washington, DC 20036
education@asmusa.org
Introduction
Abstract
In this experiment, serial dilution of a contaminated water source is examined for turbidity and
bacterial cell count. Because chemical and microbial contaminations are not always visible to the
naked eye, the microbial presence will be brought to the attention of the learner by use of viable
plate counts.

Core Themes Addressed

General Microscopy Concepts
Microbial Cell Biology x
Microbial Genetics
Microorganisms and Humans x
Microorganisms and the Environment x
Microbial Evolution and Diversity x
Other -Common properties of life; Cellular components

Keywords
Contamination, Serial Dilution, Turbidity, Microorganism Bacterial, Cell Count

Learning Objectives
At the completion of this activity the learner will be able to successfully perform serial dilutions as
well as be able to calculate the corresponding concentration of the dilutions. The student will also
have knowledge of how to conduct a bacterial cell count and thus bacterial concentrations.

National Science Education Standards Addressed

Standard 1: Unifying Concepts and Processes – students gather evidence to explain the relationship
between turbidity and cell number.

Standard 2: Science as Inquiry – the learner investigates cell counts in their particular water
sample.

Standard 4: Life Science – students follow populations and ecosystems.

Standard 6: Science and Technology - This experiment measures the number of bacteria in a water
sample as well the change in bacterial concentration throughout the series of dilutions.

Standard 7: Science in Personal and Social Perspective – students consider the safety of drinking
water.

ASM Microbial Discovery Activity

Is It Clean or Just Unseen? Dirty Water and the Naked Eye
Page 2
 Teacher Handout
Is It Clean or Just Unseen? Dirty Water and
the Naked Eye

Student Prior Knowledge

Students should be equipped with the skill set and knowledge of using transfer pipettes and
compound light microscope (if you choose to use one). They should also be able to use mathematical
concepts to prepare dilutions.

Teacher Background Information

Many disciplines and applications of science use serial dilutions in experiments. The purpose of this
experiment is to introduce serial dilutions as a laboratory tool and explain their use in practical
applications of science. Because many experiments require different concentrations of solutions the
use of serial dilutions are used frequently to save time. Mastering this tool will allow students to be
able to determine the number of microorganisms present in a sample. Determining the number of
microorganisms is a fundamental concern in public health issues, specifically in determining the
quality of water in our water systems. E. coli is a common microbe found in water and microbes that
are resistant to chlorine such as Cryptosporidium parvum and Giardia lamblia are becoming
increasingly common. This activity has been used in the middle school classroom with great success.
The only issue is reinforcing the point that the plates must not be opened after growth. We strongly
recommend using plates that are dry (no condensation on the lid). If there is liquid on the lid you
can quickly remove it, tap a corner on the bench, and flick the water droplets into a sink. After the
students spread the liquid on the plates, be certain to invert the plates.

Class Time
This experiment will take place over two sessions. The plates could be incubated at 37C over night
in between class sessions, however lower temperatures will require more time between sessions to
allow for bacterial growth. The first class period will consist of preparing the dilutions of the dye and
of the soil and the spreading of the bacterial plates. The second class will focus on determining the
bacterial count of the dilutions and of the original sample, and it will end by discussing the results of
the experiment.

Teacher Preparation Time

A blank (of just water) should be prepared ahead of time as well as a demonstration tube with a very
dilute (invisible) concentration of bacteria for students to analyze prior to starting the activity (~15
minutes). The paperclips need to be sterilized by baking at 350C for 10 minutes or by soaking in
70% ethanol and allowed to dry. The Petri dishes may be prepared well in advance and refrigerated
before use. After being spread with bacteria they should be incubated over night at 37C or longer if
at room temperature. The soil can either be collected ahead of time or students can collect soil from
outside in sandwich bags before starting the activity.

ASM Microbial Discovery Activity

Is It Clean or Just Unseen? Dirty Water and the Naked Eye
Page 3
Materials and Equipment
 Dye (a couple drops per person)
 Fresh Soil (1 sandwich bag per person)
 10 ml tubes (6 per person)
 1 ml pipettes (1 per person)
 Petri dishes with nutrient agar (1 per person)
 Sterilized Paperclips (1 per person)

*This experiment can be done in either small groups of 3 or 4 students per group or individually by
each student. The amounts of materials needed should be adjusted accordingly. If using plastic
pipettes, 2 (1 ml) pipettes per student (one for the dye and one for the soil experiment) should be
sufficient. If using micropipettes then only 2 tips per person should be necessary. It is strongly
recommended that a micropipette should be used to transfer the 0.1 ml of soil solution to the agar
medium and therefore an additional pipette tip would be needed per student. The dye utilized can be
anything available including food coloring or most laboratories stains. It would also be possible to
have students grow their samples on different media for example, minimal medium plus glucose,
nutrient agar + ampicillin, Actinomycete isolation agar, etc, if desired. It is advisable to not use
blood agar plates to avoid the isolation of pathogens.

Methods
Part A: (only performed by teacher as a demonstration)
1. Have 6 tubes of water (1 containing 10 ml and 5 containing 9 ml). Add two or three drops of dye
to the first tube containing 10 ml.
2. Take 1 ml out of the first tube and add it to another tube containing 9 ml of water.
3. Repeat this step 4 times or until no visible difference can be seen between the last prepared tube
and the blank, which contains no dye.
4. Ask the students if they think the water still contains any dye and is safe to drink. Would their
answer change if the dye were a chemical or bacterial agent?

ASM Microbial Discovery Activity

Is It Clean or Just Unseen? Dirty Water and the Naked Eye
Page 4
Part B:

1. Produce a series of six glass test tubes with caps (1 containing 10 ml and 5 containing 9 ml) of
sterile distilled water.
2. Allow the students to collect some soil into a small plastic bag, being careful not to contaminate
the soil with other objects that might contain microbes.
3. Scoop up some of the soil with the cap of the tube. Mix it thoroughly into the first tube by
inverting it and let it sit for a few minutes.
4. Take 1 ml from the first tube and transfer it to the second tube, which contains 9 ml of water.
Then mix the second tube and remove 1 ml of the liquid and place into the third tube. Continue
doing this until all the tubes have been used.
5. Place 0.1 ml of solution onto the individually labeled plates using a micropipette and individual
tips. Spread the liquid evenly across the plate using the paperclip spreaders. Allow the liquid to
be absorbed by the media before inverting the plates. This prevents liquid from condensing on
the lids.
6. Incubate overnight. If colonies are very small allow one more day of growth.
7. In the next session, have students compare and contrast their plates and calculate the number of
bacteria in the original sample (by counting the number of colonies grown on their plate and
performing the appropriate math to estimate the original count). A countable number on a plate
is between 1 and 300. Optional: Select certain groups or individuals to make wet mounts of their
bacteria and observe under the microscope.

Delivery
To start the lab, have students analyze the blank and the demonstration diluted bacterial tube to
decide which tube they would drink. After they have handed in their decisions, reveal what each
tube contained. Then allow them to perform the activity.

Technology
The only technology required for this experiment is a compound light microscope, however it is
optional.

Microorganisms Used
The microorganisms used in this experiment are any that can be found in the soil sample for
example, Bacillus spp.

Safety Issues
When working with microorganisms, a concern about safety always exists. Although nearly all soil
microbes are harmless, universal precautions should still be used when working with the cultures.
Take care in handling the organisms and have students collect soil from a safe area instead of places
containing potential pathogens such as manure piles. Because some students may be allergic to
molds, it is important not the open plates that appear to contain molds.

Suggestions for Assessment

If students have fully grasped the concepts introduced in this experiment the students should be able
to perform serial dilutions on other samples using 1/2, 1/50, or other dilution factors. Students
should also be able to effectively determine the concentration of bacteria in the original sample. For

ASM Microbial Discovery Activity

Is It Clean or Just Unseen? Dirty Water and the Naked Eye
Page 5
further assessment, have students perform 1/2, 1/4, 1/8, 1/16, and 1/32 dilutions on the same soil
sample to determine the bacterial concentration in the original sample and then compare these
results with their earlier results from this experiment. These calculations should reinforce the
concept that different dilution increments do not change the concentration of bacteria in the original
sample.

Grading for this experiment should be placed largely on student’s participation. If students are
writing a report, a rubric is attached in the supplement section. Minimal points should be deducted
for mathematical errors while larger deductions should be made for conceptual errors. Answers to
follow-up questions are open-ended and are subject to interpretation; however common answers can
be found below.

1. How would the environment from which the soil sample is taken affect the sample?
The environment from which the sample is taken would affect the types of bacteria found, their
pathogenicity or toxicity, and the number of bacteria found due to the presence of different
foodstuff, oxygen availability, pH and other environmental considerations.

2. Would you expect to find the same kinds of bacteria in samples taken from different areas?
You would not expect to find the same kinds of bacteria in samples of soil taken from different
areas, especially if the areas they are found in have different environmental conditions. The kinds
of bacteria isolated might be similar if the samples were taken from similar habitats but you
would not expect to find all the exact same types of bacteria.

3. What factors would affect the type of bacteria found in the sample?
The kinds of bacteria found in the soil sample are directly related to how close the soil is to sewage
treatment or chemical plants, moisture, available nutrients, types of plants, how heavily trafficked
the area is by animals and humans, temperature, pH, and the types of carbon sources found in the
soil.

4. Why should you not collect soil samples from areas that contain landfills or sewage treatment?
You should not collect soil samples from hazardous areas because it could contain human
pathogens and or toxins.

5. What factors could influence bacterial count in a sample?

Competition between bacteria and limiting factors such as available nutrients could influence
bacterial count.

6. What effect does dilution of a sample have on bacterial count?

The dilution of a sample will decrease the number of bacteria present per ml of sample.

7. Does diluting a sample change the concentration of bacteria in the original sample? Explain.
Dilution of a sample does not change the overall number of bacteria that was present in the
original sample; the same number of bacteria is present just in a larger volume of solution. For
this reason it is possible to calculate the number of bacteria present in the original sample from
streaking only a small amount of one of the dilutions.

ASM Microbial Discovery Activity

Is It Clean or Just Unseen? Dirty Water and the Naked Eye
Page 6
8. If you want to perform a 1/10 dilution on your stock sample to a volume totaling 50L, what
volume of water and what volume of your stock sample would you add to a new tube?

Total volume = 50L

Volume of stock= 5L
Volume of Water= 45L

9. Why is it inaccurate to perform a count on a Petri dish with many hundreds or thousands of
colonies?
Because you could not be sure if the colony was due to a single cell growing up or several landing
at the same location, you would underestimate the true number. It is also true that on a crowded
plate it would be difficult to count all colonies. Slow-growing cells would also be missed and
negative effects of cells growing too close together may inhibit the growth of some cells, also
leading to a lower than actual count.

Supplementary Information
With students’ newfound knowledge gained from this experiment it would now be
possible to have students perform dilutions on concentrations of nutritional requirements,
compounds, etc. and examine its effect on growth and metabolic processes. It is also
possible to have students perform dilutions of samples of unknown protein concentration
to determine the concentration from prepared standards.

ASM Microbial Discovery Activity

Is It Clean or Just Unseen? Dirty Water and the Naked Eye
Page 7
 Student Handout
Is It Clean or Just Unseen? Dirty Water and the
Naked Eye

Introduction
Often it is not always easy to see what is clean and what is dirty. In this experiment, you will be
diluting water contaminated with soil and dye to levels “visible” and ”invisible” to the naked eye. You
will then examine the contents of the “invisible” solution for microbial growth by culturing the
solution to see if bacteria will grow. If you experience a bacterial growth on your plate, you may
observe the bacterial presence using a microscope.

Student Background Knowledge

To perform this experiment, you should have a working knowledge of bacterial cell counts and the
concept of solution concentrations and dilutions. This knowledge is essential to understanding the
mathematical relationship between a diluted sample compared to one that is not. Also, you should be
able to properly use transfer pipettes.

Vocabulary
Serial Dilution- process of reducing the concentration of a solution by the addition of a solvent in a
stepwise fashion with the dilution factor being held constant
Dilution Factor- ratio of final volume divided by initial volume
Diluent- the solution with which the sample is being diluted
Aliquot- a measured volume of the original sample
Turbidity- the cloudiness or opaqueness of a liquid
Microorganism- an organism (usually singled celled) that is of microscopic size, too small to be seen
with the naked eye
Contamination- the presence of contaminants (organisms, particles) that make a substance impure
Bacterial Cell Count- number of microorganisms present in a population or sample

Material Checklist*
One 1 or 2 ml pipette
Six - 10 ml tubes
Soil Sample in a sandwich bag
One Petri dish with nutrient agar
One sterilized paper clip

*First determine if done individually or in a group; different media may be used

Procedure for Participants

1. Obtain six glass test tubes with caps (1 containing 10ml and 5 containing 9ml) of sterile distilled
water.

ASM Microbial Discovery Activity

Is It Clean or Just Unseen? Dirty Water and the Naked Eye
Page 8
2. Gather soil into a small plastic bag, be careful not to contaminate it with objects that could
contain other microbes.
3. Scoop up some of the soil with the cap of the tube. Mix it thoroughly into the first tube by
inverting it and let it sit for a few minutes.
4. Take 1 ml from the first tube and transfer it to the second tube, which contains 9ml water.
Continue doing this until all the tubes have been used.
5. Place 0.1 ml of solution onto the individually labeled plates using a micropipette and individual
tips. Spread the liquid evenly across the plate using the paperclip spreaders. Allow the liquid to
be absorbed by the media before inverting the plates. This prevents liquid from condensing on
the lids.
6. Incubate overnight. If very small colonies appear, allow one more day for growth.
7. Observe the next class. Calculate the number of bacteria in your sample by counting the number
of colonies on your plate. A countable number is between 1 and 300. Your teacher may ask you to
perform a wet mount of your bacteria.

1ml 1ml 1ml 1ml 1ml

Stock 9mlH20 9mlH20 9mlH20 9mlH20 9mlH20

Full 1/10 1/100 1/1000 1/10,000 1/100,000

Strength

Safety Considerations
 Assume every microbe is dangerous
 Do not collect soil from a hazardous area
 Do not open Petri dishes if they appear to contain mold

ASM Microbial Discovery Activity

Is It Clean or Just Unseen? Dirty Water and the Naked Eye
Page 9
 Is It Clean or Just Unseen? Dirty Water and the
Naked Eye
Name:___________________________________ Date:__________________________

Results________________________________________________________________

Dilution Recipe (ml % Fraction Scientific Decimal

of Previous Concentration of Notation Notation
Sln + ml of Original
H2O)

1 1 ml stock

Compare and contrast your findings with others in the class. Is the growth the same
on all media?

Number Number of Number Number Number Number Number

of Bacteria of of of of of
Colonies in Bacteria Bacteria Bacteria Bacteria Bacteria
on (1/100,000) in in in in (1/10) in
media Dilution (1/10,000) (1/1,000) (1/100) Dilution Original
Dilution Dilution Dilution Sample

Would you consider the smallest dilution of dye safe to consume? Why or why not?

ASM Microbial Discovery Activity

Is It Clean or Just Unseen? Dirty Water and the Naked Eye
Page 10
Follow-up Questions

1. How would the environment from which the soil sample is taken affect the
sample?
2. Would you expect to find the same kinds of bacteria in samples taken from
different areas?
3. What factors would affect the type of bacteria found in the sample?
4. Why should you not collect soil samples from areas that contain landfills or
sewage treatment?
5. What factors could influence bacterial count in a sample?
6. What effect does dilution of a sample have on bacterial count?
7. Does diluting a sample change the concentration of bacteria in the original
sample? Explain.
8. If you want to perform a 1/10 dilution on your stock sample to a volume totaling
50L, what volume of water and what volume of your stock sample would you
add to a new tube?
9. Why is it inaccurate to perform a count on a Petri dish with many hundreds or
thousands of colonies?

ASM Microbial Discovery Activity

Is It Clean or Just Unseen? Dirty Water and the Naked Eye
Page 11
 Lab Report: Is It Clean or Just Unseen? Dirty Water and the Naked Eye
Teacher Name: _________________________________________
Student Name: ________________________________________ Date _____________________
CATEGORY 4 3
Participation Used time well in lab and focused
attention on the experiment.
Procedures Procedures are listed in clear
steps. Each step is numbered and
is a complete sentence.
Calculations All calculations are shown and
the results are correct and
labeled appropriately.
Analysis The relationship between the
variables is discussed and
trends/patterns logically
analyzed. Predictions are made
about what might happen if part
of the lab were changed or how
the experimental design could be
changed.
Scientific Concepts Report illustrates an accurate
and thorough understanding of
scientific concepts underlying the
lab.
ASM Microbial Discovery Activity
Is It Clean or Just Unseen? Dirty Water and the Naked Eye
Page 12
2 1
Used time pretty well. Did the lab but did not Participation was minimal OR
Stayed focused on the appear very interested. student was hostile about
experiment most of the Focus was lost on participating.
time. several occasions.
Procedures are listed Procedures are listed Procedures do not accurately
in a logical order, but but are not in a logical list the steps of the experiment.
steps are not numbered order or are difficult to
and/or are not in follow.
complete sentences.
Some calculations are Some calculations are No calculations are shown OR
shown and the results shown and the results results are inaccurate or
are correct and labeled labeled appropriately. mislabeled.
appropriately.
The relationship The relationship The relationship between the
between the variables between the variables variables is not discussed.
is discussed and is discussed but no
trends/patterns patterns, trends or
logically analyzed. predictions are made
based on the data.
Report illustrates an
accurate Report illustrates a Report illustrates inaccurate
understanding of most limited understanding understanding of scientific
scientific concepts of scientific concepts concepts underlying the lab.
underlying the lab. underlying the lab.