LABORATORY 6

Exploring Bacteria
PART B
OVERVIEW
Last week, we inoculated agar plates to carry out tests to further characterize our two test species of
bacteria E. coli and B. subtilis. We also set up an experiment to compare two bacterial communities in
terms of the abundance and different species of bacteria. This week, we will analyze the results from
these tests and from our experiment. In addition, you will pick some interesting bacterial species from
your communities to further characterize using some of the tools that you have learned.

LEARNING OBJECTIVES
When you complete this lab, you will be expected to:
1. Discuss how and why bacteria are diverse in their metabolic capabilities.
2. Discuss where antibiotics come from and some mechanisms they employ.
3. Describe the difference between Gram-positive and Gram-negative bacteria.
4. Discuss why the identification of a Gram-positive or Gram-negative is important in medicine.
5. Explain the theory behind the starch agar and the disk diffusion tests.
6. Analyze data from both of these bacterial characterization tests.
7. Analyze data from an experiment performed to compare the number and types of bacteria in
two test areas.

ANALYZING RESULTS FOR LAB EXERCISE A

Characterization of Test Species

EXERCISE A1
Characterizing Metabolic Capabilities: The Starch Agar Test
As a group, bacteria are very diverse in their metabolic pathways. Several tests can be used to
characterize bacteria based on the molecules they can breakdown and the by-products that are
produced in its metabolism (e.g., acid or gas). The ability of a bacterium to breakdown a particular
molecule reflects the suite of enzymes it possesses to use resources and survive in its environment.

In this exercise, you will characterize the ability of E. coli and B. subtilis to break down starch into
glucose monomers. A positive result indicates that this bacterial species produces the enzyme
amylase. Iodine will form a dark complex when it interacts with starch. A clearing around the bacteria
indicates that the bacterial species is able to hydrolyze starch.

Procedure A1
Either the plate that you set-up last week or a demo plate will be flooded with iodine to observe.
1. Observe whether there is a clearing around the strip culture of the test species.
2. Complete Table A1. and answer the questions on this exercise in the Lab Report section.

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EXERCISE A2
Characterizing Sensitivity to Antibiotics: The Disk Diffusion Test
Many bacteria and fungi produce antibiotics, which are chemicals that either kill or prevent the division
of bacteria cells. Some examples are penicillin (produced by fungal genus Penicillium) and
streptomycin (produced by bacterial genus Streptomyces). Production of antibiotics in nature can be
considered as an evolutionary adaptation since chemoheterotrophic bacteria and fungi compete as
decomposers. Most antibiotics that we use in medicine, however, are semi-synthetic (modified from
molecules found in nature) or synthetic (not found in nature).
Antibiotics have specific targets in bacterial cells. These targets are, in many cases, enzymes that
help build certain molecules or carry out processes important for life in the bacteria cell. Many
antibiotics are classified as broad-spectrum because they can be effective against a wide range of
species. Other antibiotics are classified as narrow-spectrum because they target a process specific
to some bacterial species and are only effective against a subset of species. Narrow-range antibiotics
can be helpful in characterizing bacterial species. For instance, penicillin is quite effective against
Gram-positive bacteria but less effective against Gram-negative bacteria. Penicillin is effective
against Gram-positive cells because it inhibits the enzyme that crosslinks the peptidoglycan polymers.
This is very important in Gram-positive but less important in Gram-negative cells. Therefore, if a
bacterial species is sensitive to penicillin, it would indicate that it is Gram-positive. Sensitivity or
resistance of a bacterial species to antibiotics can tell us something about the bacterial species based
on the molecules present or structure of the cell.
Several mechanisms can lead to a bacterial cell’s resistance to an antibiotic. A cell may not possess
the target of the antibiotic or produce a modified version of the target that is not recognized by the
antibiotic. In addition, the antibiotic may be stopped from getting into the cell. If the antibiotic does
enter the cell, it may be inactivated by enzymes or be pumped out of the cell by membrane proteins. A
resistant strain of a normally susceptible bacterial species can also be produced by mutation and
natural selection.
In this exercise, you will measure the zone of inhibition created by the antibiotic discs in the E. coli
and B. subtilis lawns. The zone of inhibition is the diameter of the circular clearing in the lawn of
bacteria around the antibiotic disc. A large diameter indicates high susceptibility to the antibiotic, a
small diameter indicates a low susceptibility to the antibiotic, and no zone of inhibition (diameter is the
size of the disk) indicates that the bacterial species is not susceptible to that antibiotic (i.e. resistant).

Procedure A2

1. Take the nutrient agar plates from last week’s Exercise A2.
No zone of
2. Using the ruler provide, measure the zone of inhibition. Report
inhibition for each antibiotic tested in each as 6mm (diameter
experimental plate (E. coli and B. subtilis). When of the disk).
measuring this diameter, include the diameter of
the disk in your measurements (see Fig. 1). For
example, if your disk has a diameter of 6 mm and
the clear area has a width of 3 mm beyond the
disk, the diameter of the zone of inhibition that you
have measured is 12 mm (6 mm + 3 mm + 3 mm). Figure 1. Zone of inhibition.

3. Complete Table A2 and answer the questions on this exercise in the Lab Report section. We
will compile all of our class data and take the mean zone of inhibition for each bacterial
species for each antibiotic.

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EXERCISE A3
Characterizing Colony Morphology
A particular bacterial species will produce colonies on agar with particular characteristics (see Fig. 1 in
Lab 4 Part A).

In this exercise, you will describe the colonies formed by E. coli and B. subtilis from your culture
plates. If you were not successful in producing colonies, a demo plate is available for observation.

Procedure A3
1. Each team of 2 will take a stereoscopic microscope to their bench. Observe the characteristics
of the colonies formed by the test species (see characteristics compared in Table A3).
The stereoscopic microscope is a great tool to help differentiate colony morphologies.

2. Complete Table A3 in the Lab Report section.

ANALYZING RESULTS FOR LAB EXERCISE B

Comparison and Characterization of Bacterial Communities

EXERCISE B
Collection of Data
In this exercise, you will measure your dependent variables for the experiment you designed last week
and analyze the results.

Procedure B

1. Take the nutrient agar plates from last week’s Exercise B: Ecosystem Comparison

2. Estimate the number of colonies in each section of the plate. Record your data in Table B.
For a large number of colonies on a plate, count the colonies in a quarter or eight of the
plate and multiply by 4 or 8 for your estimation.

3. Using macroscopic observation and the aid of the stereoscopic microscope, estimate the
number of different colonies (different colony morphologies) in each section of the plate.
Complete Table B and answer the questions on this exercise.

LAB EXERCISE C
Cell-Level Characterization of Bacterial Test Species

EXERCISE C
Observation of Cell Characteristics
Two sets of demonstration microscopes have four (4) bacterial test species: B. subtills, E. coli, S.
aureus, S. volutans under high magnification (oil immersion mount). Observe the various species in
one of these sets and record your observations in Table C in the Lab Report section. The various
species can also be found on a poster of images on display over the microscopes.
N.B. Please DO NOT adjust any setting on these demo microscope when viewing except for the
interpupillary distance between oculars and the FINE focus.

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 Lab 6 - Bacteria (Part B)

LABORATORY 6 (Part B)
Exploring Bacteria
Name: ___________________________________________ Lab Section: _____

Lab Report

Introduction
1. List two reasons why biologists want to characterize and identify bacterial species.

Last week, you collected bacteria from various habitats on a swab and inoculated agar plates. This
week we will hopefully have several colonies on our experimental plate that represent the community
of bacteria living in these habitats.

2. You will want to determine the total number of bacterial cells that were living in your test areas.
What data from the plate will you collect to determine this?

3. You will want to determine the total number of different species that were living in your test areas.
What data from the plate will you collect to determine this?

4. How will you know that the bacteria cultured are from your experimental area and not due to
contamination from other sources? Briefly explain.

5. Describe ONE (1) way (be specific) that you will characterize an unknown bacterial species from a
community at the:

A. Colony Level:

B. Cell Level:

C. Molecular/Metabolic Level:

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6. The habitat of a particular bacterial species might give you insight into some of its
characteristics. What is the main habitat of E. coli? B. subtilis? Be specific!

E. coli:

B. subtilis:

7. Which important process in bacterial cells is inhibited by the following antibiotics? Match the
process inhibited to the antibiotic.

Processes: A. Cell wall synthesis, B. Protein synthesis, C. DNA replication.

Norfloxacin (NOR) interferes with: _______ Penicillin (P) interferes with: _______

Streptomycin (S) interferes with: _______

8. Streptomycin specifically interferes with the activity of the bacterial ribosome. It does not affect our
cells and does not affect archaeal organisms to a large extent. However, there is some effect on
the mitochondria of our cells when we take this antibiotic to treat a bacterial infection.
a) Why would mitochondria have ribosomes and require their activity?

b) Would you expect the structure and function of ribosomes in mitochondria to more closely
resemble the ribosomes of bacteria, archaea, or eukaryotes? Briefly explain.

9. Complete the following table to compare Gram-positive and Gram-negative bacteria.

Gram-Positive Gram-Negative
Number of cell membranes
[1 or 2]
Thickness of peptidoglycan layer
[thin or thick]
Color after Gram stain
[Purple or Red]

10. Why is knowing whether a bacterial species is Gram-positive or Gram-negative important when
treating a bacterial infection?

Exercise A1. Characterizing Metabolic Capabilities: The Starch Agar Test

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Table A1. Breakdown of starch by E. coli and B. subtilis.

Starch Agar Test Result
Bacterial Species
(Positive or Negative)
E. coli

B. subtilis

1. A positive test indicates that the bacterial species has a gene for and produces which enzyme?

2. Briefly describe what this enzyme does in the bacteria’s metabolism.

3. Consider the habitats where you find these two species of bacteria. Do the results of the starch
agar test make sense? Briefly explain.
Hint: would starch be present as a source of food for these species where they live?

Exercise A2. Characterizing Sensitivity to Antibiotics: The Disk Diffusion Test

Table A2. Sensitivity of E. coli and B. subtilis to various antibiotics.

E. coli B. subtilis
Mean Zone Sensitivity Mean Zone of Sensitivity
Antibiotic Target of Inhibition (- , +, ++, Inhibition (- , +, ++,
(mm) or +++)* (mm) or +++)*
Norfloxacin

Penicillin

Streptomycin
* “-” indicates not sensitive (diameter of disk); “+” indicates low sensitivity (> diameter of disk to ≤15 mm); “++”
indicates medium sensitivity (>15 to ≤25 mm); “+++” indicates high sensitivity (>25 mm).
1. Based on the results, which antibiotic would be BEST to use in order to differentiate between these
two bacterial species? Briefly explain your response.

2. Based on the results for penicillin, which bacterial species is Gram-positive? Gram-negative?
Briefly explain.

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3. How can you explain your results for norfloxacin? Hint: Think of the cell process that it interferes
with (see pre-lab exercise).

Exercise A3. Characterizing Colony Morphology

Table A3. Characteristics of unknown bacterial species.

Test Species B. subtilis E. coli

Colony Shape
[circular/non-circular]

Colony Color
[if pigmented: list color
if non-pigmented: white/tan]

Light Reflection of Colony

[matte/glossy]

Transparency of Colony
[translucent/opaque]

Exercise B. Comparing the bacterial community in two different ecosystems

Table B. Number and types of colonies in experiment plates

Readout 2:
Readout 1:
Plate # of Different
# of Colonies
Colonies*
Ecosystem 1:
_____________
Ecosystem 2:
_____________

Contamination Control
*based on colony morphology

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1. Which specific dependent variable can each readout be used to measure for your bacterial
community comparison?

Readout 1:

Readout 2:

2. a) Describe the results you obtained for your contamination control. b) What information does this
provide you with in this study?

3. Based on your results from this comparison study, what are your conclusions with respect to the
specific question that you asked (last period when you set up this study)? Explain.
Reminder: you are only interested in one of these readouts for the dependent variable you chose in
your question.

Exercise C: Characterizing Cell Characteristics in Bacterial Test Species

Record your data from your observations at stations 1 and 2 in the table below.

Table C. Cell characteristics of various bacterial species

E. coli B. subtilis S. aureus S. volutans

Cell Shape
[coccus/bacillus/spirillum]
Cell Arrangement
[single/diplo-/strepto-/staphylo-]
Gram Stain
[positive/negative]

Other Defining Cell Characteristic?

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