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BIOL1230 MICROBIOLOGY

MICROBIOLOGY
LABORATORY
MANUAL
Updated for fall 2010

Written and edited by Microbiology faculty and staff


Student Manual

Northeast State Technical Community College 2005

Updated May 2010

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MANUAL CONTENTS
INTRODUCTION TO MICROBIOLOGY LABORATORY

Welcome to the laboratory component of Microbiology! These laboratory sections have been designed to
enhance and bring to life the materials that will be covered in lecture by allowing direct observation,
experimentation, and application of techniques commonly used when studying the various microorganisms.
The student should expect this course to be challenging, informative, and hopefully enjoyable. The latter of
the expectations is achieved through preparation (rread each exercise prior to your scheduled lab) and active
participation in the laboratory exercises.
For the sake of time (and to retain sanity) it is imperative that you prepare before class. Read the
introduction to the scheduled exercise, and familiarize yourself with the steps in each activity process
(materials and methods section). It is not expected that you understand everything, just be familiar with the
activities.
You will be assigned a modified lab write-up as homework for each lab (the format is included in the
appendix). The introductory paragraph should include a purpose statement as well as an introduction to the
material in the background information. As you will soon realize, experimentation using microbes often
requires incubation time for growth in order to make a proper determination from your results. For most
exercises results will not be available until the following scheduled class. At that time a record should be
made of your results in the given area. Your results sections along with a conclusion paragraph will be
assigned as a post-lab.
Homework Policy
Homework Grade Sheet
Microscope Policy

EXERCISES
Exercise 1 Introduction: Microscopy and Cell types
Exercise 2 Basic Procedure in the Microbiology Lab
Exercise 3 Aseptic Technique and Media Preparation
Exercise 4 Morphological examination: Differential Staining Techniques Introduction to
Biochemical/Metabolic Differentiation
Exercise 5 Culturing: Media Selection (Defined, Complex, Selective, & Differential) and
Inoculation techniques
Exercise 6 Media Selection and Metabolic Characterization Continued
Laboratory Practicum I - Basic Laboratory Techniques
Laboratory Practicum II - Identification of an Unknown Bacterium
Exercise 7 Quantification of Microorganisms Bacteria
Exercise 8 Control of Microorganisms Testing and Evaluation Techniques
Exercise 9 Transformation of E. coli
Exercise 10 Parasitology

APPENDIX
Possible Organisms Chart (for Identification of Unknown)
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Homework Policy
Although class work is conducted cooperatively with your lab partners, the design of the
homework is for students to work independently. The purpose of the homework is to get
individual students to actively participate in the material covered with each exercise and to
allow the instructors to evaluate the level of understanding achieved by each student. For
this reason, it is imperative that students complete their homework on their own.
Students who are having difficulty with the homework assignments should discuss these
problems with their lab instructor.
Plagiarism is a serious concern for all laboratory homework. When writing the introduction
section it is important that you write this section in your own words rather than taking
phrases, sentences, or paragraphs directly from the lab manual. The best way to avoid
plagiarism is to read over the introduction and your notes prior to writing your introduction.
Make sure you have a complete understanding of the lab. Then close your lab manual and
notebooks and write your conclusion based on your own understanding. This will ensure
that you are using your own words. Then go back and double check to make sure you
have the information correct. Please see the plagiarism policy in the lab outline for
information on the consequences of plagiarizing.
Your instructor may require you to submit your lab write-ups electronically. Be sure and
save your file in the following format:
a. lastnameE#post.file extension
Homework Grade Sheet
Assignment

Lab Write-ups

Your Points
Exercise 1
Exercise 2
Exercise 3
Exercise 4
Exercise 5
Exercise 6
Exercise 7
Exercise 8
Exercise 9
Exercise 10

Points Possible

100*

Assessment I: Basic Microbiology Lab procedures

30

Assessment II: Identification of Unknown

30

Assessment III: Covers E7-E10

40

Total Points

200

*Post-labs will be graded the following way:


Submitting results pages on time...............................................................................2 points
Correct use of grammar, sentence structure, past tense, write-up format and correct use of
scientific nomenclature......2 points
Well-written introductory paragraph.2 points
Correct interpretation of results (drawing conclusions).4 points
Each Write-up is worth...10 points
Please see individual instructors policies for submission of late work, and how to handle
absences.
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Microbiology Microscope Policy


Use ONLY your assigned microscope. Your
assigned # is:
Check the microscope for problems such as:
o Slides left behind
o Broken glass on the stage
o Oil residue left on the ocular lens, objective lens or stage areas
o Other obvious problems such as the microscope has been
dropped or damaged

Report any problems detected to your


instructor
Before returning microscope make sure
you havent left any problems (see above)
Each instructor will devise and implement protocol for those
students who leave problem microscopes for subsequent
classes. Disciplinary actions include subtracting points from
homework, loosing replacement points, etc.

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EXERCISE 1
INTRODUCTION: MICROSCOPY AND CELL TYPES
Introduction
Microbiology is the study of very small organisms, microorganisms, which can only be viewed
with the aid of a microscope. There are several groups of organisms that fit into this category
including bacteria, cyanobacteria, fungi, and protists. Within this group there are several species
interesting to humans because of their ability to cause disease or their use in the food industry.
Many of these microorganisms are unicellular although some are multicellular. These organisms
are extremely diverse in cell type, size, color, and reproductive strategy.
When working with microorganisms one easy way to classify them is by their cell type. All cells
(including plant and animal cells) can be categorized as either prokaryotic or eukaryotic. The
primary difference between these two cell types is the presence of a membrane-bound nucleus.
All eukaryotic cells have a membrane-bound nucleus that houses the genetic material (DNA) in
addition to other membrane-bound organelles such as mitochondria and chloroplasts.
Prokaryotic cells lack a membrane-bound nucleus, their genetic material is located in a particular
region of the cell called the nucleoid. In addition to this difference, prokaryotic cells are much
smaller than eukaryotic cells. Examples of prokaryotic cells include bacteria and cyanobacteria
(photosynthetic prokaryotes). Eukaryotic microorganisms include fungi, protozoa, and algae.
In this lab, we will be getting some firsthand experience with the microorganisms described
above. Since the eukaryotic cells are larger than prokaryotic cells, this will be the best place to
start. Once you have a feel for these larger cells you are then ready to begin investigating the
smaller prokaryotic cells.

Fungi include unicellular and multicellular eukaryotic organisms. One thing all fungi
share is that they are non-motile heterotrophs that absorb dissolved organic material
through their cell walls and all but the yeasts metabolize aerobically. We will only be
observing yeast in lab. Yeasts are round unicellular microbes that are widely distributed.

Protists are for the most part unicellular, eukaryotic cells consisting of several groups.
The two groups of protists under investigation here are algae and protozoa. The
primary difference between these two groups is that algae are photosynthetic while
protozoa are described as animal-like because they are heterotrophs (consume other
protists, bacteria and detritus). There are several protozoa that cause disease including
Plasmodium vivax (malaria) and Giardia lamblia (gastroenteritis).

Prokaryotes will be the primary focus of the semester. In todays lab we will observe
some of the diversity of this group by looking at both bacterial cells and cyanobacteria.
Remember that these cells are much smaller than eukaryotic cells, and lack membranebound nuclei.

In order to investigate microorganisms we need to become intimate with our primary tool--the
microscope. This invaluable tool allows the viewing of objects/structures that otherwise would go
unnoticed by the unaided human eye.
The type of microscope shown below is called the light microscope. Light is conducted through
curved lenses in such a way that an object may be viewed larger than its actual size. You might
want to label the basic structures and take notes as your instructor goes over the microscopes
structure and function.

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The light microscopes used in this lab are binocular and


have ocular lenses with a magnification of 10X. In
addition to this magnification there are also four different
objective lenses to choose from 4X, 10X, 40X, and
100X. Magnification of the object being viewed is the
product of the ocular objective multiplied by the lens
objective currently in use. For instance when viewing
an object on the 4X objective lens, the object is
magnified a total of 40X.
Even the highest quality light microscope is limited in its
magnification abilities. The highest objective for our
microscopes is 100X, which has a magnification of
1000X. With this magnification even the slightest
distortion of light would greatly reduce the quality of the
image. In order to conduct the light properly at this
objective, it is necessary to place a drop of oil on top of
the slide and immerse the lens. The oil immersion lens
(100X) is specially sealed and is the only lens that
should be placed in oil. W e will learn more about the
oil immersion lens in E2.
For more information on microscopy visit the URL below.
http://biology.clc.uc.edu/fankhauser/Labs/Microscope/Microscope_Features&Care.htm
The importance of proper handling and use of the microscope is vital. You will find this to be
especially true as beginning microscopists. It is critical that you clean the microscopes before
and after use. Please take notes while your instructor goes over this information with you.
Materials Needed:
Blank Slides/Cover slips
Iodine
10% bleach soln.
Cultures:
Yeast
Yogurt
Gleocapsa

Oscillatoria
Anabaena
Volvox
Spirogyra
Amoeba
Paramecium

Activities
In order to observe the differences between these cell types and become familiar with the
microscope you will be preparing several slides primarily using the wet mount technique as
described below. In some cases prepared slides may be provided.

Remember to CLEAN your microscope before starting to view your slides. Use a new
KimWipe to gently wipe the ocular lenses and then wipe the 10x, 40x, and 100x objective lenses.
If there is any excess oil on the microscope be sure and remove that. If you have trouble
removing oil, use the microscope cleaner provided.
For each activity below, use the wet mount method to make your slides. Add the drop of iodine as
described for those cultures with an (*).
1. Add a drop of live culture
2. Add a cover slip and then add a drop of iodine* beside the coverslip.
3. Observe under the microscope up to 40x and sketch your results in table provided
4. Place slide in 10% bleach solution provided
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Activity I: Fungus
Yeast Observation*

(http://bugs.bio.usyd.edu.au/learning/resources/CAL/Microconcepts/images/Topics/Diversity/buddingYeastCells.jpg )

Activity II: Protists


A) Protozoa
Amoeba *

Paramecium *

http://www.hinsdale86.org/staff/kg
abric/DIMACS/amoeba.jpg

B) Green Algae
Volvox

http://www.lima.ohiostate.edu/biology/images/volvo
x.jpg

Activity III: Prokaryotes


A) Cyanobacteria
Gleocapsa

http://www.glerl.noaa.gov/seagrant/GLWL/Al
gae/Cyanophyta/Images/Gloeocapsa2.jpg

http://www.educa.madrid.org/web/i
es.alonsoquijano.alcala/carpetas/q
uienes/departamentos/ccnn/web_
1_ciclo_ESO/1eso/images/tema10/paramecium2.jpg

Spirogyra

https://www.msu.edu/course/bot/423/Spirogyr
a.jpg

Anabaena

Oscillatoria

http://www.bom.hik.se/n
esch/kac/anabaena.jpg

http://silicasecchidisk.conncoll.edu/Pics/Other%20
Algae/Blue_Green%20jpegs/Oscillatoria4.jpg

B) Bacteria
Lactobacillus (yogurt) *
No photo available
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Remember to CLEAN your microscope before putting it in the cabinet.


E1 RESULTS:

assigned microscope #:

Eukaryotic Cells
Type

Sketch on (40x)

Fungi: Yeast

Protist: Amoeba
Protist:
Paramecium
Protist: Volvox

Protist: Spirogyra

Prokaryotic Cells
Type

Sketch (40x)

Cyanobacteria: Gleocapsa
Cyanobacteria: Oscillatoria
Cyanobacteria: Anabaena
Bacteria: Lactobacillus (yogurt)
E1 Write-up
Introduction paragraph: include a description of the following items from the background
information:
Prokaryotic vs. eukaryotic cells
Introduction of types of cells viewed
Submit results from lab manual
Conclusion:
Draw conclusions about the importance of getting familiar with proper microscope
technique (for instance proper handling, focusing, and cleaning of the microscope).
Include a comparison of the prokaryotic and eukaryotic cells observed in this class.
This write-up must be typed and be in your own words.

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EXERCISE 2
BASIC PROCEDURE IN THE MICROBIOLOGY LABORATORY
Introduction
Microbes can serve as either our greatest allies or our worst enemies depending on their type
and location. This is why studying these organisms is so vital. Methods for studying microbes are
as diverse as the groups themselves.
Due to their size and ubiquity, microbes can be a challenge in the laboratory. This unit will focus
on the general techniques that are needed in order to achieve good experimental outcomes while
protecting the health and safety of everyone. We will be focusing on two important tools in the
microbiologists tool boxworking with bacteria cultures and microscopy.
The first part of the laboratory will introduce you to the materials and methods we routinely use
when working with bacterial cultures. The most important technique to learn today is aseptic
technique which will be explored in greater detail in E3. In addition to learning this basic
technique we will also focus on a key idea, that our world is full of bacteria present virtually
everywhere. Due to the ubiquity of microorganisms we will have to be extremely careful about
contamination of our lab materials and the cultures we are working with. You will see that it takes
very little exposure to introduce an unwanted organism to your materials.
The second part of the laboratory will continue our microscopic investigation into the microbial
world by introducing you to the variety of morphologies (shapes) and cellular arrangements that
are present among bacterial cells as well as comparing cell size. In addition, we will also focus
on using the oil immersion lens which is a critical skill in the microbiology lab.
Cellular morphology and arrangement refers to the cell shape and the association shared
between cells (if any). Although bacterial cellular morphology can be very diverse, there are
three basic shapes of interest in our lab. They are coccus (pl. cocci), rod (sometimes called
bacillus), and spiral. Based on how cocci cells divide they can designated as diplococci (pairs),
streptococci (chains), tetrads (groups of 4), or staphylococci (clusters). Rod cellular
arrangements can be described as singles, diplobaccilli (pairs), or streptobacilli (chains).
When comparing bacterial cell size, we will be using the metric system. The metric system is the
standard system of measurement used in the sciences, including microbiology. The system
makes measurement and unit conversions much simpler because all units and conversions are
based upon the number 10.
Three of the most commonly measured properties are length, mass, and volume. The standard
metric units for these variables are meter, gram, and liter. Prefixes are placed in front of each
units name to designate smaller and larger units. Lets use length as an example to see how
metric units work. One meter is equivalent to 39.37 inches, so it is roughly 1 yard in length. Now
imagine dividing the meter into 10 equal-sized pieces. Each piece is 1 decimeter (dm) in length
th
(1/10 of a meter). Imagine dividing the meter into 100 equal-sized pieces. Each piece is 1
th
centimeter (cm) in length (1/100 of a meter). Finally, divide the meter into 1000 pieces. Each
th
piece is 1 millimeter (mm) in length (1/1000 of a meter).

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Note the following:


1 m = 10 dm
1 m = 100 cm

1 m = 1000 mm
1m = 1,000,000 m

Most microorganisms are smaller than 1 mm, so we need to intr


introduce
oduce an additional unit called
the micrometer (m).. Imagine dividing 1 mm into 1000 equal
equal-sized
sized pieces. Each of these
extremely small pieces is 1 micrometer in length. A ttypical bacteria cell is about 0.2-2.0
0.2
m in
diameter and 2-8 m in length.
Morphology/Arrangement

Species

Rods Singles

Escherichia coli

streptobacilli

Bacillus subtilis

Tetrads

Micrococcus
luteus

staphylococci

Staphylococcus
aureus

Photo

Photographs copied from URL:

http://faculty.mc3.edu/jearl/ML/mL-5http://faculty.mc3.edu/jearl/
2.htm

Materials per pair:

3 TSA plates
2 sterile swabs

sterile water
prepared bacterial slides

Activity I: Ubiquity of Microorganisms


The specific media type used in this lab is Trypticase Soy Agar (TSA) which is a complex media
providing a wide range of nutrients supporting a diversity of microorganisms.

Ubiquity of Microorganisms: Swab Samples


37oC

25oC

1. Obtain
btain 3 TSA plates
plates, flip the plates over, and label the bottom
Definition of Fomite:
with your group initials, lab day/time.
an inanimate object
2. Label one plate air. Remove the lid and leave it exposed to
that can transfer
the air until the end of the lab period.
disease i.e. door
3. Add the following information on the remaining 2 plates:
knob
a. Divide each plate into quadrants

b. Label one plate 37 C and the other 25 C.


c. Number each se
section 1-4, and make a key below of the four samples you
would like to swab. You must use the same four sample areas for both plates
and these should include one fomite sample, two body samples,
sample and one
surface sample.
Source 1: dirty finger
Source 3:
Source 2: clean finger
Source 4:
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4. In order to inoculate sources 1 and 2 start with dirty hands. Moisten your index finger
with water then press your dirty index finger into quadrant 1 of both plates.
5. Now wash your hands following these directions from the Center for Disease Control:
a.
b.
c.
d.
e.

Wet your hands with warm running water and apply soap.
Rub hands together to make lather and scrub all surfaces.
Continue rubbing hands for 20 seconds. (Imagine singing "Happy Birthday" twice)
Rinse hands well under running water
Dry your hands using a paper towel and use your paper towel to turn off the faucet.

6. Moisten your index finger again and press the clean index finger into quadrant 2 of
both plates.
7. For sources 3 and 4: obtain two sterile swabs and one micro vial of water from the
cart.
8. For source 3, moisten one swab and streak the area of interest thoroughly. Proceed
to streak each of your TSA plates in the designated quadrant for that sample. Be
sure to remember aseptic technique! (repeat for source 4)
9. Place the 37 C plate upside down (bottoms up) inside the incubator. Place the 25
C plate upside down on top of the incubator. Since microbes are so small, it is
necessary to allow time (24-48 hours) for them to multiply into populations so large
we can see their colonies unaided. The plate incubated at room temperature will
need a longer incubation (5-7 days).
We will be discussing throughout the semester basic requirements microbes have in order to live
and replicate. These requirements include nutrition and environmental conditions such as
temperature, pH, moisture, and oxygen. Microbes vary in their nutritional needs and their levels of
tolerance to environmental conditions. Since both plates were streaked with the same samples
and supplied the same nutritional media, it is interesting to expose each plate to a different
o
o
variable in this case temperature (either 25 C or 37 C). Both plates will be incubated in aerobic
atmospheric conditions.

Activity II: Cellular Morphology and Arrangement (prepared slides)


The purpose of this second activity is 2-fold:
1) introduction to cellular morphology and arrangement
2) proper use of the oil immersion (100x) objective lens.
1. Obtain 3 prepared slides representing different morphologies and arrangements.
2. Begin focusing each slide on the 4x and then move to the 10x and 40x objective
lenses. Once you have your image focused clearly on 40x move the turret in
between the 40x and 100x objectives.
3. Without moving the stage or changing focus, drop a small, single drop of
immersion oil onto the slide.
4. Move the oil immersion (100x) objective lens directly into the spot of oil.
5. View slideyou might have to do some minimal focusing with the fine
adjustment knob to clear up your image.
6. Sketch results in table provided.
7. Clean oil off 100x object lens before moving to the next slide.

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AIR PLATEyou should have left this plate uncovered on your lab benchothe entire
lab. At the end of the period be sure to close your dish and invert and incubate at 37 C.

Clean-up and Disposal follow instructors directions.


Once all items have been put in their assigned places wipe your work area with ethyl
alcohol and wash your hands well.

E2 Results:
ACTIVITY I RESULTS: Obtain your
two sample plates you swabbed last
lab session and look for growth. Use
the colony description figure taken
from Science Buddies website
( http://www.sciencebuddies.org/me
ntoring/project_ideas/MicroBio_img_
003.gif) to assist you in describing
bacterial growth for each quadrant.
The results tables provided below
are to be used to briefly describe
what you observe. List each sample
area in the space provided. The
lower portion is for growth
description. Include color, colony
sizes, amount of growth, and colony
description for each listed sample
area.

Colony Description by Source for 37C Plate


Source 1: dirty index finger

Amount

Description

Source 2: clean index


finger
Amount

Description

Source 3:

Amount

Description

Source 4:

Amount

Description

Colony Description by Source for 25C Plate


Source 1: dirty index finger

Amount

Description

Source 2: clean index


finger
Amount

Description

Northeast State Technical Community College 2005

Source 3:

Amount

Description

Source 4:

Amount

Updated May 2010

Description

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AIR Plate Results: Did you have growth?


ACTIVITY II RESULTS: For each slide sketch enough cells to demonstrate the morphology and
arrangement of the bacteria viewed using the oil immersion (100x) objective lens.
assigned microscope #:
Slide 1

Slide 2

Slide 3

SKETCH
Describe
morphology and
arrangement

E2 Write-up
Introduction paragraph: include a description of the following items from the background
information:
Ubiquity of microorganisms and aseptic technique
Discussion of culturing conditions provided including temperatures, media used, and
atmospheric requirements
Description of different morphologies and arrangements
Submit results from lab manual
Conclusion:
Use your data to support use of aseptic technique
Use your data to address the use of optimal temperature during incubation
Discuss your observations of cellular morphology and arrangement.
This write-up must be typed and be in your own words.

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EXERCISE 3
ASEPTIC TECHNIQUE AND MEDIA PREPARATION
Introduction
Asepsis means without contamination. The ability to carry out procedures without the
introduction of unwanted organisms, or contamination, is paramount to obtaining correct
results/identification. In addition, since some of these microbes are potential pathogens
(organisms that cause disease), contamination could expose you and any other person you may
come into contact with the possibility of infection. That is why vigilance in proper technique is
necessary to reduce risk.
Aseptic technique can be further divided into four categories including work area preparation,
media preparation/handling, culture transfer, and clean-up and disposal.
Work Area Preparation
Since microbes are everywhere, even on us, it is necessary to begin to minimize the potential for
contamination before we begin to work with the microbes. Prior to beginning each laboratory
session:
Remove all you personal items away from the workbench except for your lab
notebook and writing instrument.
Wipe down your workbench with ethyl alcohol (ETOH) and paper towels.
Wash your hands using CDC method as described in E2.
Begin gathering materials as instructed and outlined for that particular laboratory.
Media Preparation/Handling
When culturing bacteria you must provide them with all of the conditions they need for growth
including nutrients, temperature, atmospheric requirements and more. Media is the nutrient
mixture that is used to grow and keep microbes. Media is usually in broth or solid forms and will
vary in content based on the goal of its use. These nutrient mixtures are normally in powdered
form to increase their shelf life. When needed, the nutrient mix is added in a specific amount to
water, boiled, transferred to containers, and autoclaved to sterilize. As long as the media remains
unopened in the original container, it should remain a sterile environment. If the media is
transferred from the original container, care should be taken to avoid contamination. In the case
of the plastic Petri plates we will be using extensively in this lab section, media must be
transferred. Plastic used for these plates are unable to withstand the temperature of the autoclave
for sterilization so they are shipped and remain in sterile packaging until their time of use. The
agar will be poured hot and quickly into the Petri dish to further avoid contamination.
Culture Transfer
All of microbiology work involves transferring cultures to different growth media or onto slides. It
is critical when transferring bacteria that aseptic technique is maintained. A large portion of
todays lab procedure will emphasize how to move bacteria to different media types while
preventing contamination.
Clean-up and Disposal After EACH Lab (last step of aseptic technique)
Unless otherwise instructed items used in the lab session should be treated in the following
manner:
Glassware - will be autoclaved and cleaned for re-use.
Test tubes/flasks place in the designated test tube rack on the lab cart after the
labeling has been removed. If an adhesive label is present, simply peel it off and
place in the autoclave bag. If a marker has been used, wipe off with acetone prior to
placement in the rack.
Slides If the slide has been heat fixed, wash with sponge and dish soap and place
in designated container.

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Disposable Items items intended for one time use and items that cannot maintain their form
and function after being autoclaved should be placed in the autoclave bag. These items will be
sterilized prior to their disposal to avoid contamination during and after waste collection. These
items include but are not limited to Petri plates and any paper or plastic items that have come into
contact with microbes.
Goggles clean with diluted dish soap, dry and then put away.
Once all items have been put in their assigned places wipe your work area with ETOH and
wash your hands.
Activities
Materials:
4 test tubes of TSB
1 TSA plate
plates from E2
stirrer/hotplate
magnetic stirrer

hot pads
flasks with filtered water
powdered media
thermometers

Aseptic Transfers
A.) Sterile Broth to Sterile Broth:
1. Obtain two sterile Trypticase Soy Broth (TSB) test tubes.
Label one tube A and one tube B and label both with your group initials and lab day/time.
2. Flame loop until the loop and some of the wire is red hot then let the loop cool.
3. Open test tube A using the pinky finger techniquehold the lid of the test tube with your
pinky finger of your dominant hand, and use your other hand to twist the test tube away
from the lid.
4. Flame the lip of the test tube
5. Get loopful of broth; make sure that only the sterilized portion of the loop makes contact
with the broth.
6. Flame and close test tube.
7. Open test tube labeled B using the same technique as above.
8. Flame the lip of the test tube.
9. Inoculate test tube B with the loopful of sterile broth from tube A. Be sure that only the
sterilized portion of the loop makes contact with the broth.
10. Flame and close test tube B.
11. Flame loop red hot.
12. Place both test tubes in a rack in the incubator.

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B.) Sterile Broth to Sterile Plate:


1. Obtain one sterile TSB test tube and a sterile Trypticase Soy Agar plate (TSA). Label both
with your group initials and lab day/time. Label the test tube C and the plate D. Be sure to
put your labels on the bottom of the Petri dish.
2. Flame loop until the loop and some of the wire is red hot then let the loop cool.
3. Open the test tube using the pinky finger technique (see above)
4. Flame the lip of the test tube
5. Get loopful of broth; make sure that only the sterilized portion of the loop makes
contact with the broth.
6. Flame and close test tube.
7. Open the Petri dish just enough to get the loop in.
8. Very gently move the loop across the Petri dish. Be careful not to dig into or make any
breaks in the agar bottom.
9. Close the Petri dish.
10. Flame loop red hot.
11. Invert the Petri dish and add it to your class stack in the incubator. Put the test tube in the
incubator rack too.

C.) Colony to Broth Transfer


1. Obtain one sterile TSB test tube and a plate with growth from E2. Label with your group
initials, lab day/time and E on the TSB tube.
2. Flame loop until the loop and some of the wire is red hot. Allow loop to cool.
3. Open the Petri dish just enough to get the loop in and very gently use your loop to pick up an
isolated colony of bacteria. Be sure not to disturb the agar or any neighboring
colonies.
4. Close the Petri dish.
5. Open the test tube using the pinky finger technique (see above).
6. Flame the lip of the test tube
7. Inoculate the broth; make sure that only the sterilized portion of the loop makes contact with
the broth.
8. Flame and close test tube.
9. Flame loop red hot.
10. Put the test tube in the incubator rack.

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Media Preparation
Each group will be asked to prepare media. The type of media and the specific instructions will be
assigned by the instructor. It will take just a few minutes to get the media measured and added to
the flask. It will take about 20-30 minutes for the media to reach boiling before it is ready to pour
into test tubes. Instructors will need to make sure that each class tubes are autoclaved as soon
as possible and then stored in the refrigerator as room allows. The basic procedure to be used
by all classes is as follows:
1. Measure out the proper amount of media powder for L of media using the electronic
balance.
2. Fill up your flask with the proper volume of filtered water (1/2 L) and place on the
stirrer/hotplate. Drop in a magnetic stirrer and turn the stirrer on 6-8.
3. Slowly pour in the media powder into the water.
4. Turn the hotplate on high. Agar can superheat so it is important to keep an eye on your
boiling agar. Also agar will not dissolve in water that is hot, so DONT heat the water until
after adding the powder.
o

5. Once the agar or broth has reached boiling point (use thermometers to register 100 C). You
will notice that the liquid is clear rather than cloudy indicating that the media has dissolved.
Also there is often a frothy head that forms on the top of the boiling media (when TSA and
TSB start to look like beerthey are ready).
6. Follow instructors directions on how to pour the media into the test tubes given. Loosely cap
the test tubes and put autoclave indicator tape on the rack. Be sure to provide a label on the
rack with the type of media, date made, and group initials. Your instructor will help you get
your rack autoclaved with the rest of the class tubes.
7. Test tubes need to be autoclaved as soon as possible after being poured. Once sterilized
test tubes can be stored in the refrigerator.

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E3 Results
For each item record whether or not you had growth. If you have growth in TSB then the test
tube will be cloudy. You might need to vortex your TSB to see growth as the bacteria can settle.
Procedure

Materials

Growth? Yes or No

Tube A
Sterile TSB

Sterile TSB
Tube B
Tube C

Sterile TSB

Sterile TSA
TSA plate D

Isolated colony

sterile TSB

Tube E

E3 Write-up
Introduction paragraph: include a description of the following items from the background
information:
Aseptic technique define, state purpose, give some examples of
Submit results from lab manual
Conclusion:
Did your results turn out as expected? Explain.
Describe some procedural errors that could negatively affect your results.
This write-up must be typed and be in your own words.
.

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EXERCISE 4
DIFFERENTIAL STAINING TECHNIQUES GRAM STAIN AND SPECIAL
STRUCTURE STAINS
Introduction
As we have seen in the previous exercises, light microscopy and the use of a stain are valuable
tools for viewing bacteria. This allows us to see the morphology of a microbe of interest. A
differential stain technique allows additional discrimination and helps to narrow down the list of
possibilities with regard to its identity. A differential stain would be one that allows determination
of differences between species having similar morphologies based on their ability to take the stain.
In multiple stain procedures, the initial stain is retained by cells that are termed positive while
the negative cells loose the initial stain. This necessitates the addition of a secondary or
counter stain, to make the negative cells visible under the microscope. Differential stains show a
difference in chemical composition, metabolism, and/or the presence of a special structure.
The first step in making a slide is to smear the bacteria onto the slide. Preparation procedures for
the smear will vary according to cell concentration in the culture. Generally a broth culture will be
less concentrated than surface cultures and can be smeared directly onto the surface of a clean
slide and allowed to air dry. Surface cultures (ones growing on agar) need to be diluted in order to
discriminate single cell shapes once stained. A loop of water is added to the slide and the loop of
sample is mixed in and distributed on the surface of the slide and allowed to dry.
The second step in making most differential stains is to heat fix the slide. A heat fixed slide is
one where the organisms are placed on the slide and the slide is allowed to dry. After the slide is
free of visible moisture, it is passed through an open flame 2-3 times before the staining begins. It
is imperative that the slide is completely dry before the heat-fix step; otherwise the bacterial
proteins can be denatured during the heat-fix step and cause a distortion in the cell morphology.
The purpose of the heat-fix process is to affix them to the surface of the slide so that they are not
washed off during the staining process. When done correctly, heat-fixing will also kill the
organisms.
The most well-known and used differential stain is the Gram stain. This stain process
determines differences in peptidoglycan (a chemical found only in bacteria) on the outside of the
cell wall. Bacteria can be divided into one of two categories based on their gram reaction, either
gram positive or gram negative. Gram positive cells have a thicker peptidoglycan cell wall than
gram-negative cells, and retain the initial stain, crystal violet (purple), in the gram stain procedure.
Gram negative cells are decolorized during the gram stain procedure which requires the
application of a counter stain, Safranin (pink), so that these cells can be easily viewed.
The acid-fast stain is used in a limited number of cases to stain organisms that have an addition
of mycolic acid in their cell walls which prevents them from Gram staining. The Mycobacterium
species require this type of stain process in order to view them. Due to the nature of the waxy
mycolic acid, these cells must first be steamed in order for the stain to penetrate the cell wall. In
this procedure acid-fast cells retain the initial stain, carbolfuchsin (reddish-purple), while negative
cells are stained by the secondary stain, Methylene blue (blue).
Some species have cellular structures that are uncommon and can be used in identification.
Endospores are examples of specialized cellular structures produced by some Bacillus and
Clostridium species in response to stressful environmental conditions. Endospores are dormant
cells able to survive hazardous conditions that later germinate to form vegetative (metabolically
active) cells when conditions improve. The endospore stain, employs a multiple stain
procedure that stains both endospores and vegetative cells. The endospore stain involves steam
similar to the acid-fast stain. Without steam the tough spore coat prevents the endospore from
taking up stain. During this procedure, endospores keep the initial stain, Malachite green (green),
while the vegetative cells retain the secondary stain, Safranin (pink).
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Other important structures that can be detected with stains are flagella. These structures are
responsible for the motility of an organism. While the presence of flagella alone can be important
in characterizing bacteria, the ability to describe the arrangement of the flagella can be even more
useful. For direct observation of flagella a tricky staining procedure is used. Motility media is an
alternative method to staining that allows for indirect observation of flagella. Due to the dilute
nature of this media microbes are able to travel in the media if they are mobile. This takes time
and incubation will be necessary. In addition the media has a chemical added, TCC, which
microbes take into their cells. When metabolized, the TCC changes from being colorless to a
pink color allowing easier viewing of the placement of the growth.
There are plenty of resources on the internet that can help you visualize these processes a little
better. Check out the following links:
Gram Stain: http://faculty.mc3.edu/jearl/ML/mL-5.htm
Acid-fast Stain: http://faculty.mc3.edu/jearl/ML/mL-6.htm
Endospore stain: http://www.slic2.wsu.edu:82/hurlbert/micro101/pages/101lab6.htmL
Activity
Materials:
Per Class:
Blank slides
Hotplates/beakers

Per team:
M. smegmatis (pathogenic)
B. subtilis
E. coli

K. pneumonia
S. epidermidis
P. aeruginosa
3 motility media test tubes

Students will have 2 lab periods to complete E4.

Each group should start by inoculating the 3 motility tubes


1.

Obtain 3 test tubes of motility media and label 1-3, group initials, and class period.
1.
2.
3.

Escherichia coli
Pseudomonas aeruginosa
Klebsiella pneumoniae

2. Using aseptic technique, stick the sterile inoculation needle into the broth culture (be
sure to flame the entire length of the needle).
3. Insert the needle straight down into the motility media and pull the needle straight
back out.
4. Fire the tube and quickly replace the lid. Fire the needle
5. Place in the test tube rack in the incubator for 48 hours.

Each student should obtain a total of 8 slides and label slides a-h (see
list of bacteria below). Make all 8 slides using the heat-fix procedure. After
heat-fixing all 8 slides begin staining slides starting with the 4 gram stain
slides.

Slide label
a and f
b
c and h
d and g
e
i

Organism
Staphylococcus epidermidis
Pseudomonas aeruginosa
Escherichia coli
Bacillus subtilis
Mycobacterium smegmatis (PATHOGENIC)
MIXED slide of S. epidermidis and E. coli

Northeast State Technical Community College 2005

Stain Technique
Gram stain and acid-fast stain
Gram stain
Gram stain and endospore stain
Gram stain and endospore stain
Acid-fast stain
Gram stain

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BIOL1230 MICROBIOLOGY

For All slidesmake smear using heat-fix procedure:


From Solid Sample:
1.
Clean and label bottom of slide
2.
Add loopful of water
3.
Flame loop red hot
4.
Aseptically obtain bacteria (isolated
colony if possible)
5.
Smear (spread-out) bacteria on
slide
6.
Flame loop red hot

7.

AIR DRY COMPLETELY

8.
9.

Heat-fix (pass slide through flame)


Proceed to directions below for the
specific differential stain.

From Liquid Sample:


1. Clean and label bottom of slide
2. Flame loop red hot
3. Vortex Sample
4. Flame test tube opening
5. Aseptically obtain bacteria
6. Flame test tube opening
7. Smear (spread-out) bacteria on
slide
8. Flame loop red hot

9. AIR DRY COMPLETELY


10. Heat-fix (pass slide through flame)
11. Proceed to directions below for the
specific differential stain.

Under oil immersion, observe your slides--remember you are looking for differences in
morphology, size and arrangement in addition to whether the organism is positive or negative for
the differential stain. Then sketch your observation in the results section. (Dont forget to clean
your microscope BEFORE & AFTER use.)
Procedure I

Gram Stain Procedure


Organisms:
a. Staphylococcus epidermidis
b. Pseudomonas aeruginosa
c. Escherichia coli
d. Bacillus subtilis
i. MIXED slide of S. epidermidis and E. coli
1.

Prepare slides for Gram stain as instructed above.

2.

Attach a clothespin to the end of the slide and hold it over the sink.

3.

Cover the slide with Crystal Violet stain and allow it to react for 1 minute

4.

Rinse the slide with a small amount of running water and remove the excess water by gently
shaking the slide.

5.

Cover the slide with Grams Iodine--allow it to react for 1 minute.

6.

Rinse the slide with water as in Step 4.

7.

Hold the slide at an angle and apply 1 drop of decolorizer

8.

Immediately rinse the slide thoroughly and remove any excess water as in Step 4.

9.

Cover the slide with Safranin for 1 minute.

10. Rinse the slide thoroughly.


11. Blot the slide dry by placing it in between the sheets of the bibulous paper and lightly pat

with your hand.

Apply immersion oil when ready to view & record in Results section
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Procedure II

Acid-fast Stain Procedure


Organisms:
e. Mycobacterium smegmatis (pathogenic)
f. Staphylococcus epidermidis

1. Prepare slides as instructed above.


2. Cover the slides with a piece of paper towel the same size
as the smear.
3. Place clothespins at both ends of the slide to form a rack
and place it on top of the steaming water beaker.
Steam

4. Flood the slide with Carbolfuchsin stain.


5. Gently steam the slide for 10 minutes, reapplying the stain
as needed to prevent the slide from drying out.
6. Remove the paper towel carefully with forceps and place in trash.
7. Rinse the slide with a small amount of running water until the excess stain is removed.
8. Hold the slide at an angle and apply acid alcohol by making drops at the highest part of the slide
(near the clothespin handle) and allow it to drip down the slide. Do this for 25-30 seconds.
9. Rinse the slide.
10. Apply several drops of Methylene Blue stain and leave for 45 seconds.
11. Rinse the slide thoroughly and blot dry.

Apply immersion oil when ready to view & record in Results section

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Procedure III

Endospore Stain Procedure


Organisms:
g. Bacillus subtilis
h. Eshericia coli

Vegetative (A); Endospores (B)

1. Prepare slides as instructed above


above.
2. Apply clothespins to each end of the slide and place
over a steaming beaker of water (like acid-fast).
3. Apply a piece of paper towel cut to the size of the
slide on the surface of the slide.

http://www.sp.uconn.edu/~terry/229sp03
/lectures/structure.htmL
mL

4. Flood the paper towel with Malachite Green stain.


5. Allow the slide to steam for 5 minutes and reapply the stain as needed to
prevent the paper towel from drying out.
6. Remove the paper towel gently using forceps and remove one clothespin.
7. Rinse the slide with a small amount of running water.
8. Hold the slide over the sink and apply Safranin and allow it to react for 1 minute.

9. Rinse and blot dry.


Apply immersion oil when ready to view & record in Results section

Motile

N
Non-motile

Motile

http://a-s.clayton.edu/furlong/BIOL2250LAB/Reviews/mediastudy.htm
s.clayton.edu/furlong/BIOL2250LAB/Reviews/mediastudy.htm

E4 Write-up
Introduction paragraph: include a description of the following items from the background
information:
Gram stain, endospore stain, acid
acid-fast, and motility media
Submit results from lab manual
Conclusion:
Use your results to describe the cellular characteristics of each organism tested
This write-up
up must be typed and be in your own words.
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E4 Results
Procedure I-Gram Stain
Slide Label
Organism

assigned microscope #:
Sketch & Indicate Color

a.

Staphylococcus epidermidis

b.

Pseudomonas aeruginosa

c.

Escherichia coli

d.

Bacillus subtilis

i.

MIXED S. epidermidis and E. coli

Procedure II-Acid-fast
Slide Label
Organism

assigned microscope #:
Sketch

e.

Mycobacterium smegmatis
(pathogenic) (pathogenic)

f.

Staphylococcus epidermidis

Procedure III-Endospore Stain


Slide Label
Organism

g.

Bacillus subtilis

h.

Escherichia coli

assigned microscope #:
Sketch & Color

Procedure IV-Motility Results: Draw the regions where growth was observed.
Organism

1.) Escherichia coli

2.) Pseudomonas
aeruginosa

3.) Klebsiella pneumoniae

Sketch

See previous page for E4 write-up assignment.

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EXERCISE 5
CULTURING: MEDIA SELECTION AND INOCULATION
TECHNIQUES
Introduction
In the previous four exercises we became familiar with visual techniques used in microbiology.
You learned to describe various colony characteristics that, although not often, may aid in the
determination of a particular microbe. We also examined various types of bacteria under the
microscope to record cell morphology and any biochemical/structural differences that could be
obtained through the use of staining procedures. Although helpful, determination of cell type and
structure is limited when it comes to correctly identifying a microorganism. There are many
species that, for example, are Gram negative rods with motility. When studying a bacterium or
when trying to diagnose a disease for proper treatment, the exact species (or even strain of that
species) must first be determined.
For further characterization we look to various metabolic differences that are specific to that type
of bacteria. All living organisms must acquire energy from their environment. There are
differences in energy sources and metabolic pathways between groups of bacteria. There are
also ranges of environmental conditions that are particular to groups of bacteria (pH, temperature,
atmospheric requirements, etc.) where they best function (refer to E2 for temperature differences).
Knowing and manipulating these conditions provide excellent tools for correctly identifying
bacteria. The next two exercises will focus on various procedures that have been developed to
manipulate differences in energy needs and environmental condition ranges.
Exercise 5 will be focusing on the use of media. For solid media, agar base (solidifying agent) is
added to a broth containing nutrients that provide energy for microbes so that they may
metabolize and replicate. The ingredients that are present in the broth will determine what groups
of microbes are able to metabolize and grow. There are some metabolic differences among
similar microbes that can be used for identification (see differential media below). Metabolic
differences are often observed using colorimetric tests which typically incorporate either a pH
indicator that will change the color of the media if there is a change in pH or a chemical that
attaches to the metabolite of interest and concentrates in the microbes changing the normal
colony color.
In general there are three different groups of media type used in this lab:
Complex media is comprised of partially digested chemical compounds from organic
substances such as yeast, meats, dairy products, tissues, or vegetable materials. The
amount of each cannot be known due to differences between the organic compounds
and the amount of digestion that has occurred. This knowledge is not necessary when
culturing most types of microbes. This media type is useful when trying to grow out
bacteria, or when culturing a mixed diverse sample (see E2). An example of a complex
media is Trypticase Soy Agar (TSA).

Selective media is used to select, or isolate, specific groups of bacteria. This is usually
based on a known environmental condition range they can tolerate that most groups
cannot.

Differential provides chemical compounds that bacteria metabolize differently. This


difference is observed by colony or media color change once the microbe has been
introduced to the media and allowed to grow and metabolize. Often this type of media is
used to differentiate between similar groups of organisms with the same morphology and
biochemical resemblances.

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There are several medias that are both selective and differential these include Eosin
Methylene Blue and Mannitol Salt Agar.
Eosin Methylene Blue (EMB) is used to differentiate between gram-negative,
enteric, rods because this media primarily supports (or selects for) the growth of
these organisms. Growth on EMB can be used to differentiate organisms based
on their ability to ferment lactose. During the fermentation process, acid is
produced as a waste product. When grown on EMB this acid production results
in a colony color change ranging from pink to purple. In the case of E. coli and
Klebsiella pneumoniae, colonies can appear dark purple with a metallic green
sheen. One exception to EMB selectively growing gram-negative rods is the
gram-positive cocci Enterococcus faecalis. E. faecalis is able to grow on this
media because it is an enteric bacterium.
Mannitol Salt Agar (MSA) contains salt that most organisms cannot tolerate due to their
osmolarity ranges. Microbes that normally exist in an
environment with this condition, such as microbes of the skin, are
able to grow. For those halotolerant organisms, MSA is also
used to differentiate species based on their ability to ferment
mannitol. Organisms that can ferment mannitol produce an acid
S. aureus
S. epidermidis
by-product causing the pH indicator in the media to turn from pink
to yellow.
In addition to using color changes to differentiate bacteria,
location of growth in a special media can be useful as we have already seen with motility media
(E4). Media can be used to determine whether a microbe is an obligate
aerobe (A), obligate anaerobe (B), or a facultative anaerobe (C) species
by observing location of growth in special media (see figure). An oxygen
A
B
gradient is formed in the oxygen requirement media once the test tube is
boiled. An aerobic environment remains in the top 1-2 cm of the test tube,
while anaerobic conditions are present below this point.

When working with media it is important that you place the


Determination of Oxygen
bacteria on the media in an informative way. When trying
Requirements
to simply grow bacteria on a media the best method to
use is a streak-to-grow technique. With this method
T-Streak
you are simply trying to get the bacteria on the media and you can either use a
back-and-forth motion on the plate with your loop or one distinct streak. If you want
to obtain isolated colonies for use in working with a pure culture, than a T-streak
method would be more appropriate. The idea is to dilute or disperse the cells so
when incubated, they will form isolated colonies that are separated to such a degree
the colonies do not touch. This will allow viewing of the individual colonies for any
distinguishing culture characteristics as well as for creating and working with a pure culture.

Activities
Materials
Per team:
4 TSA plates
2 EMB plate
2 MSA plates
4 Oxygen Requirement tubes

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Per table:
Test tube rack containing Escherichia coli, Staphylococcus epidermidis, Staphylococcus aureus,
Pseudomonas aeruginosa, Enterobacter aerogenes, Enterococcus faecalis, Klebsiella
pneumoniae, Clostridium perfringens, and Micrococcus luteus

Activity I: Inoculation and Isolation of Colonies

Isolation of a Colony: T-Streak Technique


1.

Obtain a TSA plate and label on the back your groups initials, YOUR NAME, class
time, 1-4, and divide your plate by drawing a T (see below).
1. Staphylococcus epidermidis
2. Pseudomonas aeruginosa
3. Escherichia coli
4. Enterobacter aerogenes
Sterilize your transfer loop and allow it to cool.
Aseptically obtain a loop of broth from one of the cultures (see box below).
Streak the top section with closely spaced streaks that DO NOT overlap
FLAME loop
Drag bacteria from top section into right hand section, streak as in step 4
FLAME loop
Drag bacteria from right section to left section, streak as in step 4
FLAME loop
o
Invert and Incubate at 37 C

2.
3.
4.
5.
6.
7.
8.
9.
10.

Flame loop

Flame loop

1
2

Activity II: Media Selection (MSA & EMB)


1. For each type of media plate (2 MSA & 2 EMB) label with your initials, class day/time,
and label one set of MSA and EMB plates as a-d (see figure) and the second set of
plates e-h.
2. For each plate aseptically streak to grow with the appropriate culture. Remember to
always practice good aseptic technique, especially between the transfers of the different
species.
a. Staphylococcus epidermidis
A B
b. Staphylococcus aureus
c. Micrococcus luteus
C D
d. Enterococcus faecalis
e.
f.
g.
h.

Pseudomonas aeruginosa
Escherichia coli
Enterobacter aerogenes.
Klebsiella pneumoniae

3. Invert and incubate all 4 plates 37 C.


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Activity III using media to determine O2 requirements:


1. Obtain 4 Oxygen Requirement test tubes. Label with initials, day/time, and 5-8.
2. Allow the oxygen requirement tube to cool to body temperature (it will feel comfortably
warm to the touch). DO NOT allow your media to solidify before inoculation. Be sure to
sterilize the full length of the loop and wire to prevent cross contamination of the
tubes.
3. Inoculate the test tube with the appropriate microbe. To do this, put the loop with
inoculate straight down to the bottom of the tube and pull out. Repeat for all 4 species.
1. Klebsiella pneumoniae
2. Pseudomonas aeruginosa
3. Escherichia coli
4. Clostridium perfringens

E5 Results:
Activity I: Inoculation and Isolation of Colonies
Obtain your best T-Streak plate and observe the growth. Sketch your
results in the diagram below, and then count the total number of isolated
colonies you observe.
Total Isolated colony count: _____

Dont forget you will be using these T-streaks for your E6 procedure.
Activity II: Media Selection (TSA, MSA, & EMB)
For each microbe - record the presence of growth on each media. Also record the appearance of
colony and/or media. The highlighted organisms are gram () rods, and the remaining are gram
(+) cocci.
Label
a.
b.
c.
d.
e.
f.
g.
h.

Organism

Growth
Yes or No?

MSA
Media
Appearance

Notes

Growth
Yes or No?

EMB
Colony
Appearance

Notes

Staphylococcus
epidermidis
Staphylococcus
aureus
Micrococcus luteus
Enterococcus
faecalis
Pseudomonas
aeruginosa
Escherichia coli
Enterobacter
aerogenes
Klebsiella
pneumoniae

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BIOL1230 MICROBIOLOGY

Activity III using media to determine O2 requirements:


For each tube, observe the areas of growth and sketch your results in the tubes provided below.
Organism

1. Klebsiella
pneumoniae

2. Pseudomonas
aeruginosa

3.Escherichia coli

4.Clostridium
perfringens

Sketch

E5 Write-up
Introduction paragraph: include a description of the following items from the background
information:
EMB, MSA, and oxygen requirements
Obligate aerobes, obligate anaerobes, and facultative anaerobes
Submit results from lab manual
Conclusion:
Use your results to describe the cellular characteristics of each organism tested
This write-up must be typed and be in your own words.

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BIOL1230 MICROBIOLOGY

EXERCISE 6
MEDIA SELECTION & METABOLIC CHARACTERIZATION
CONTINUED
Introduction
As described in the previous exercise, identification of bacteria is limited to visual observation.
Knowledge of individual energy requirements and specific environmental condition ranges were
introduced in Exercise 5 with the various media types. Within these there are a variety of ways by
which groups of microbes metabolize. These differences will be the focus of this exercise.
There are numerous procedures presently practiced, however for the purposes of time, we will be
conducting only a couple of tests that focus on the ability to use a certain carbon source, the
waste products produced, and the enzymes present.

1. Carbohydrate fermentation is a test often used to help with the identification of


enteric bacteria. Fermentation is a common practice of enteric organisms (as well as
many others) allowing them the ability to still metabolize even in the absence of oxygen.
For this test, tubes contain a sugar source, a pH indicator, and an inverted Durham tube
in the solution. Once the tube has been inoculated it will be incubated at body
temperature and observed for any color change and/or gas production. A phenol red (pH
indicator) change from red to yellow indicates an acid has been produced as an end
product of fermenting the sugar source. For a color change to be visible with phenol red
pH indicator, the pH must be below 4.3. The inverted Durham tube allows us to observe
whether a gas has been formed during catalysis of the sugar. Gas will be trapped in the
tube and appear as a bubble. As pressure of gas increases, this tube will travel upward.
Any Carbohydrate can be used to analyze metabolic activity of a microbe. We will be
testing the disaccharide lactose. Not all organisms can metabolize this sugar. Only
organisms with a special enzyme called -galactosidase are able to break the glucosegalactose bond.

2. Citrate Utilization Test will indicate whether or not the microbe in question is
able to use citrate as its carbon source. Simmons Citrate agar is used for this test. This
differential agar contains a pH indicator, bromothymol blue, which will turn from green to
dark blue if there is a rise in pH. The premise behind this test is that citrate is the only
carbon source in the media and only microbes that have the enzyme citrate permease
will be able to transport citrate inside the cell for catalysis. The end product of citrate
breakdown is carbon dioxide gas. This gas will be released and combine with salts and
water in the media to form sodium carbonate, a base (Stukus,1997).
Enzymatic indicators are chemicals that allow determination of how a microbe of interest respires.
The following two tests we will be observing today are mainly for bacteria that can respire
aerobically.

3. Oxidase Test- Aerobic organisms must have an enzyme that is able to carry
electrons to oxygen in order to produce a usable energy source through aerobic
respiration. One of the four different enzymes that have this ability is cytochrome c. The
Oxidase test uses a reagent, Tetramethyl-p-phenylenediamine dihydrochloride, to
determine the presence of this enzyme. A positive test result would be a dark color
change in the area of the colony where the test was conducted.

4. Catalase Test- Most cells that respire aerobically have an enzyme to combat
hydrogen peroxide, a by-product of aerobic respiration. A few but important anaerobes
also produce catalase. The catalase test may be used for differentiating among aerobes
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(e.g., Staphylococcus from Streptococcus and Enterococcus species) and among


anaerobes (e.g., Propionbacterium acnes from Actinomyces meyeri). Catalase breaks
down hydrogen peroxide into water and oxygen gas. To test for the presence of catalase
hydrogen peroxide is dropped on a colony. Bubbling indicates presence of catalase.
Materials
Per Team:
4 Durham tubes Phenol Red lactose broth (black lids)
4 Simmons Citrate agar slants
T-streak plates made in E5 (Pseudomonas aeruginosa, Enterobacter aerogenes, Escherichia coli
& S. epidermidis)
Test tube rack containing: Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter
aerogenes, Escherichia coli)
Per class:
TSA plates of (Klebsiella pneumoniae, B. subtilis, & E. faecalis)
Oxidase reagent, filter paper, wooden sticks
Hydrogen peroxide (H2O2)

Activities
I. Carbohydrate Fermentation Test (Phenol Red & Durham Tubes)
1. Label 4 Phenol Red and Lactose tubes (black lids) a-d, groups initials, and class
day/time.
2. Aseptically inoculate each of the tubes with a loop of designated bacteria.
a. Pseudomonas aeruginosa
b. Enterobacter aerogenes
c. Klebsiella pneumoniae
d. Escherichia coli
3. Place in the test tube rack in the incubator.

II. Citrate Utilization Test (Simmons Citrate agar)


1. Label 4 Simmons citrate slants a-d, groups initials, and class day/time.
2. Aseptically streak-to-grow with the microbe that corresponds with the label and
incubate.
a. Pseudomonas aeruginosa
b. Enterobacter aerogenes
c. Klebsiella pneumoniae
d. Escherichia coli

III. Oxidase Test:


TSA streak plates will be located up front with pure cultures of several known organisms.
1. Place a drop of oxidase reagent on an piece of filter paper located in a disposable
Petri dish.
2. Obtain a colony with a wooden applicator and rub onto filter paper.
3. Record any purple color change within the first 10-15 seconds. After 60 seconds
color changes are NOT considered positive. The color change might be faint so look
carefully.

IV. Catalase Test:


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Hydrogen peroxide and droppers will be available for the catalase determination.
1. Place a drop of hydrogen peroxide on an area of growth on the TSA streak plates
provided.
2. Record whether or not bubbles are present for each.

E6 Results
Organism

Results by organism
Lactose Fermentation

Simmons Citrate

a. P. aeruginosa
b. E. aerogenes
c. K. pneumoniae
d. E. coli

Catalase and Oxidase Determination


Source of sample

Organism

E5 T-streak #3

E. coli

E5 T-streak #4

E. aerogenes

E5 T-streak #2

P. aeruginosa

Demo plate

K. pneumoniae

Demo plate

B. subtilis

E5 T-streak #1

S. epidermidis

Demo plate

E. faecalis

Catalase
Reaction

Oxidase
Determination

E6 Write-up
Introduction paragraph: include a description of the following items from the background
information:
Carbohydrate fermentation test, Simmons citrate test, oxidase test, catalase test
Submit results from lab manual
Conclusion:
Use your results to describe the cellular characteristics of each organism tested
This write-up must be typed and be in your own words.

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Microbiology Lab Practical Assessment I: Basic Microbiology Lab


Procedures (30 points)
STUDENT STUDY SHEET:
Your instructor will ask you to perform a series of tasks and will verify the proficiency with which
you conduct each task. Tasks will be assessed by a combination of watching students perform the
task, and/or looking at the students results from each task. You should be prepared to do the
following procedures:

1. Aseptic Techniqueaseptically transfer sterile broth


2. Gram Stainsuccessfully stain a known organism and interpret result (you will NOT
need to memorize the gram stain procedure, but you will need to know how to make a
smear from memory)
3. Microscopysuccessfully focus and maintain a microscope
4. T-streaksuccessfully isolate a colony

Each item of the test will be worth 7.5 points for a total of 30 points.

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PRACTICAL ASSESSMENT II:


IDENTIFICATION OF AN UNKNOWN ORGANISM (30 points)
Each student will select an unknown culture and use the lab procedures and data collected from
exercises 1-6 to correctly identify the culture. Students will be able to use their lab manual, lab
notes, textbook, and Internet research materials to help with this project. Students will need to
review the Possible Organisms Chart (see Appendix ) in order to help with the identification of the
unknown cultures. Students will be given time in 3 class periods to complete this project.
Students must attend and be prepared to use the time provided in class.
**If possible bring a digital camera (or use your cell phone) to take photographs of your results as
you get them. Include these images in your write-up.**
Class 1 (1 hour 50 minutes):
1. Obtain numbered culture:
2. Describe the color of the growth on TSA
3. Complete Gram stain and record results below
assigned microscope #:
Pour Plate Directions:
4. Use chart in Appendix to help you narrow down your possible organisms.
1. Boil to melt media
5. Based on gram reaction and research inoculate the minimum number of tests to
2. Once melted
be able to confidently identify your organism:
aseptically pour
Motility Media (E4)
into sterile plate
EMB slant or pour plate (E5)
3. Allow media to
MSA pour plate (E5)
cool and solidify
Oxygen Requirement (E5)
4. Inoculate plate
Simmons Citrate (E6)
Phenol Red and Lactose (E6)
Oxidase (E6)
Catalase (E6)
Class 2 (30 minutes):
1. Obtain results from Class 1, record in a results table and draw conclusions
2. Inoculate additional tests as needed.
Class 3 (10-15 minutes):
1. Get results from tests inoculated in class 2.
2. Write-up due following class periodsee instructor directions.
RESULTS TABLE:
Date Test Completed

Test

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Date Result
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EXERCISE 7
QUANTIFICATION OF MICROORGANISMS
Introduction
Determination of a microbial population is an important aspect in microbiology. It is used in
industrial applications, municipal and federal standards for our water and food, and the health and
safety fields. Prior to this exercise, the majority of microbes we have been working with in the
laboratory have employed pure cultures purchased by suppliers and reanimated when needed.
In the real world, microbes are not only abundant, they also cohabitate and function in mixed
populations. The dynamics for many of these groups are not well understood. Studies that have
been conducted on some of these populations have indicated that some species may be
dependent on others for metabolic activities, growth factors, etc{For example, the US Army
Corps of Engineers is currently researching biofilm dynamics and their potential applications.} In
order to study these environmental microbes it is necessary to take a sample of the
environmental media. Samples can be obtained from virtually anywhere. Environmental samples
may include those from the soil, air, and water. They may also be obtained from surfaces,
inanimate objects, food, drink, and any other products. It is also important to understand that not
all microbes can live in the same places. Their presence in a certain system is dictated by their
metabolic requirements and environmental ranges.
Sampling is necessary for a variety of reasons:
a. To identify an infectious agent
b. To monitor the health status of an ecosystem
c. To monitor the status of bioremediation activities
d. For Quality Assurance/Quality Control (QA/QC) activities in industry and
food/beverage production processes
e. To find new microbes and/or their metabolic products (novel antibiotics, for
example).
A sample is only as good as the techniques employed. It is vital that some sort of aseptic
technique is utilized throughout the entire process. This includes but is not limited to the use of
sterile containers, proper sampling strategies, and handling.
Prior to this lab, you were asked to bring in a water sample. You will need to collect 1/2
Liter of untreated, recreational water from a source of your choice. Be sure to make sure
the container you collect the water in is clean.
Quantification methods for can be direct or indirect. Direct methods count cells. Microscopic
counts and electronic counters such as the flow cytometer count every cell. Other direct methods
are concerned only with viable cells cells capable of multiplying. Methods used for this include
the viable plate count method (serial dilution), most probable number method (MPN) and filtration.
Some samples may have so many viable organisms that it would be impossible to determine
individual colony forming units (CFUs). If this is suspected to be the case, serial dilutions are
necessary to obtain a countable plate. A serial dilution is a series of 1:10 (or 1:100 if highly
concentrated) dilutions in hopes of obtaining a countable number of CFUs. Since these counts
will be derived from a known amount of the original sample it will be easy to establish the number
of organisms in the original sample. Usually the last three to four tubes will be plated and
incubated to increase the odds of obtaining a plate where it is possible to count CFUs in an
amount that is considered statistically significant (25-250 colonies).
The most probable number (MPN) method can be used in water testing and uses a series of
phenol red and lactose tubes with a certain amount of sample in each series. Evidence of lactose

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fermentation, as indicated by color change and gas production, in each series of tubes is
observed and recorded. The results are then compared to a chart to determine the MPN.
Some samples, like air or well water, may have such a dilute amount that the CFU results would
be indeterminate. To remedy this, a known amount of sample is taken and with the aid of vacuum,
pulled through a membrane filter, this method is referred to as membrane filtration. The pores
are small enough to trap cells while the water passes through. Once complete, the sample
microbial population, if present will be concentrated onto a membrane that can be removed and
placed on growth media and incubated. Colonies may then be counted to establish the number of
microbes present in the original sample amount.
In this exercise, both MPN and Membrane filtration methods are testing for indicator organisms
designated coliforms or total coliforms that may indicate fecal contamination. Water
contaminated with fecal matter may contain pathogens, including bacteria, viruses, and parasites.
Total coliforms or other indicators are easier to detect than pathogens and therefore are used to
screen for possible contamination. Coliforms are lactose fermenting gram negative rods of
environmental or fecal origin, including among others: Enterobacter aerogenes, E. coli, and
Klebsiella pneumoniae. E. coli, and Klebsiella pneumoniae are also designated as fecal
coliforms based on their ability to grow at 44.5 C and are more specific for fecal contamination
than total coliforms. If drinking water is found to contain total coliforms it should be tested for fecal
coliforms and/or E. coli, which is the most specific indicator of fecal contamination since it is found
in the intestine of most humans and many warm-blooded animals. Other indicator organisms
which will not be tested in this lab include fecal streptococci, enterococci, and/or bacteriophage.
Lactose broth with added phenol red indicator is used in the MPN test and EMB in the membrane
filtration test to detect total coliforms in our lab. Lactose broth is an alternative to the standard
media (lauryl tryptose broth, which is more selective) in the first (presumptive) step of a three step
procedure for detecting total coliforms. Tubes demonstrating gas are normally subjected to
confirmed and completed steps to rule out false positives caused by other lactose fermenting
bacteria, but will not be performed in our lab due to time and material restraints. Total coliforms
(lactose fermenters) on EMB are pink to purple with or without a green metallic sheen. The
standard media for testing by membrane filtration for total coliforms is mEndo media at 35 C
where they detected as red colonies with a green metallic sheen.
References
http://www.epa.gov/volunteer/stream/vms511.html
http://www.norweco.com/html/lab/WhatTests.htm
http://www.epa.gov/safewater/disinfection/tcr/index.html
http://www.water-research.net/coliform.htm
http://filebox.vt.edu/users/chagedor/biol_4684/water.html
http://www.bd.com/ds/technicalCenter/inserts/m_Endo_Broth_MF.pdf
http://www.bd.com/ds/technicalCenter/inserts/Eosin_Methylene_Blue_Agar.pdf

Activity I Serial Dilution


E. coli culture
4 9.9 mL tubes
8 pipettes

1 pipetter
4 sterile Petri plates
4 TSA deeps
-2

-4

-6

-8

1. Label your 4 sterile water blanks as 10 ,10 ,10 ,and ,10


2. Change pipettes with each transfer:
a. Vortex E. coli culture

-2

b. Transfer 0.1 mL of E. coli culture into the 9.9 mL test tube labeled 10 and vortex.

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c.

-2

Transfer 0.1 mL from 10

-4

d. Transfer 0.1 mL from 10

test tube into the 9.9 mL tube labeled 10

-4

and vortex

-6

test tube into the 9.9 mL tube labeled 10 and vortex

-6

e. Transfer 0.1 mL from 10 test tube into the 9.9 mL tube labeled 10
-6

-7

-8

-8

-9

3. Label the 4 sterile Petri dishes as 10 , 10 , 10 and 10 , group initials, and class
day/time. Obtain the 4 TSA deeps and allow them to cool to body temperature. While the
TSA is cooling move on to step 4.
-8

-9

4. Use the same pipet to add 0.1 mL from 10 test tube into the 10 Petri plate and then
-8
add 1.0 mL from the 10-8 test tube into the 10 Petri plate. Add 1 test tube of cooled
TSA to each.
-6

-7

5. Use a new pipet to add 0.1 mL from 10 test tube into the 10 Petri plate and then add
-6
-6
1.0 mL from the 10 test tube into the 10 Petri plate. Add 1 test tube of cooled TSA to
each.
6. Incubate the 4 Petri dishes and autoclave the water blanks.

0.1 mL

0.1 mL

10-2

E.
coli

0.1 mL

10-4

10-6

1.0 mL

Serial
Dilution

10-6

0.1 mL

10-8

0.1 mL

10-7

1.0 mL

10-8

0.1 mL

10-9

Activity II. Membrane Filtration You will use your water sample for membrane filtration. Your
instructor will provide a visual for the use of the filtration apparatus. Be sure to keep the grid-side
up when collecting bacteria on the filter and when plating on the EMB plate.
1. Obtain 3 EMB plates label them with your group initials, day/time and 100 mL, 10 mL and
1 mL.
2. Filter 100 mL of sample onto a sterile membrane (grid up).
3. Use sterile tweezers to transfer the membrane onto an EMB plate (labeled 100 mL)be
sure and tap the filter down (grid up) so that it sticks to the media.
4. Repeat for using 10 mL and 1 mL of the same sample. With the 1 mL sample, you might
need to follow with 10 mL of sterile water.
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5. Invert and incubate 37 C all three plates

Activity III. Most Probable Number (MPN) You will need to use the same water sample you
used for the membrane filtration procedure for the MPN.
Materials per group:
water sample
6 tubes of single strength lactose phenol red fermentation tubes with black lids
o Label 3 as 0.1 mL
o Label 3 as 1.0 mL
3 tubes 2X strength lactose phenol red fermentation tubes with white lids
o Label all 3 as 10 mL
**You can use the same pipet for your 0.1 and 1.0 mL transfers as long as you start with the 0.1
transfer first.**
1. Add 0.1 mL of your water sample to the three tubes labeled 0.1 mL.

2. Add 1.0 mL of your water sample to the three tubes labeled 1.0 mL.
3. Add 10 mL of your water sample to the three tubes labeled 10 mL.
4. Incubate all the inoculated tubes 24 48 hrs.

EXERCISE 7 RESULTS
Serial Dilution Results
Use a marker and a tally to help you count the number of colonies on each plate. Be sure and
count all growth (large and small colonies) equally. For those plates with <25 colonies designate
as Too Few to Count (TFTC) and for those plates >250 colonies designate as Too Numerous to
Count (TNTC).
Dilution Number of colonies counted per plate
-6
10
-7
10
-8
10
-9
10
Now calculate the number of viable cells/mL in your original sample (X):
# of colonies x (1/sample volume added to plate) x (1/dilution) = # organisms/1mL of sample
-6

-6

Example 1) 275 colonies on 10 plate =275 x (1/1.0) x (1/10 ) = 275 x 10 or 275,000,000 organisms/mL.
-1

-6

Example 2) 135 colonies on 10-7 plate =135 x (1/10 ) x (1/10 ) = 135 x 10 or 1,350,000,000 organisms/mL
-1

Note: 0.1 = 10 in scientific notation


Note: the plate labels indicate final dilution

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Most Probable
Number
Record the number of
positive tubes (acid and gas)
in the table provided below,
then use the chart provided
to determine the MPN for
your sample type.

2X Lactose
Broth/10mL

Lactose
Broth/1mL

Lactose
Broth/0.1mL

MPN

Water Sample:

Filtration Test
Sample
100 mL

# Coliforms/plate

# coliforms/100 mL

10 mL
1 mL
E7 Write-up
Introduction paragraph: include a description of the following items from the background
information:
Serial dilution, coliform bacteria, membrane filtration and MPN
Submit results from lab manual
Conclusion:
Did you have a countable sample for serial dilution? Which plate? What can you
conclude about the sample based on these data?
Discuss how you could change the serial dilution procedure in the event that you didnt
get a countable plate.
What can you concluded about your water sample based on your MPN and membrane
filtration results?
This write-up must be typed and be in your own words.

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EXERCISE 8
CONTROL OF MICROORGANISMS
INTRODUCTION
Microorganisms are important for so many functions on earth, yet they can be a detriment to
human health and our economy when they are in the wrong place at the wrong time. From very
early on humans have learned various mechanisms of control of unwanted microbes. Presently
there are two major types of control physical and chemical. Physical control measures include
the use of temperature that is outside the range of the organisms we want to control, radiation,
and filtration. Chemical control would include the use of agents on areas in the environment to
kill/reduce the number of microbes in hopes of preventing contamination that should ultimately
reduce the risk of infection. Contamination is the presence of unwanted agents on a surface or
within a substance. Infection is the entrance of a microorganism into the host. Chemical agents
may also be used to treat a disease within the host. When a chemical is used in this manner it is
referred to as chemotherapeutic agent. A chemotherapeutic agent used to fight an infection is
an anti-microbial agent. An antibiotic is an anti microbial agent that has been naturally
produced by a living organism. Often the two are used interchangeably.
Like the old adage An ounce of prevention is worth a pound of cure, the best selection of a
control design is one that is successful in controlling microbes in that particular environment.
Infection cannot occur unless microbes are present. Although simple sounding, it is quite the
challenge. Choices must be made based on many different variables. These variables include but
are not limited to the following:
Mechanism of action
Cost
Surface to be cleaned
Toxicity or toxic residue
Location and activity
Depending on these variables one should decide what level of control is appropriate, in other
words, how clean is clean? Levels range from bacteriostatic to sterile.
Chemical effectiveness will be measured by a diffusion susceptibility test known as the KirbyBauer Test. This test uses a streak plate of a known microbe with various disks each containing a
chemical of interest. The plate is incubated and observed for growth. Growth should appear
everywhere on the agar unless there is something that is preventing it. If a microbe is susceptible
to a particular chemical, there will be a zone of inhibition (no growth) around the disc. A microbe
is considered resistant if there is no observed impediment to growth around that chemical. We
can also use this test to compare effectiveness of chemicals by measuring the zone of inhibition.
The diameter of the zone of inhibition is measured and recorded in mm. The larger the diameter,
the more effective the chemical agent is for that particular microbe. Zones of inhibition for
chemotherapeutic agents are measured and recorded. The results are compared to an
interpretation table that has been developed by the National Committee for Clinical Laboratory
Standards to establish whether the microbe of interest is susceptible, intermediate, or resistance
to a particular antimicrobial agent.
To gain additional information this test uses multiple plates each with a different organism. The
test usually includes representatives from the four major types of infections Upper respiratory
tract (URI), Uro-genital tract (UTI & STD), Infections of the skin and wounds, and gastroenterics.
The results of this aids in the determination of an appropriate choice of an anti-microbial that
would be the most effective with that particular type of microbe.
The last activity allows focus on a particular area and not a known organism. Microbes are
present in mixed populations. In some situations it is more important to focus on whether or not

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any microbes are present after a control procedure has been implemented. An In-Use Test tests
the efficacy of a cleaning agent by swabbing an area of interest prior and post cleaning.
ACTIVITY
Procedure I: Kirby-Bauer Disk Diffusion Test - Antibiotic
Per team:
4 Mueller Hinton Agar (MHA) Plates
1 beaker containing 6 different antibiotics with dispensers
4 sterile swabs
Procedure:
1. Using a permanent marker on the bottom of the plate, divide each plate into
six equal sections as shown.
2. Label each plate with a different representative organism that will be
streaked onto the plate.
a) Escherichia coli
d) Mycobacterium smegmatis
b) Pseudomonas aeruginosa
(pathogenic)
c) Staphylococcus epidermidis
3. Each plate should be streaked with the microorganism that is labeled on the back. Use a
nd
sterile swab and streak to grow swabbing in one direction and then in a 2 direction.
4. Place a different antibiotic disc onto each section of a plate. Once a disk has been
dispensed onto the surface, it must be lightly tapped with tweezers so it will stick during
incubation. Always remember to sterilize you tweezers between touching disks to
prevent contamination! Repeat this process for each plate.
5. Insert the Nystatin disk in the center of each plate.
6. Invert and incubate.
Procedure II: Kirby-Bauer Disk Diffusion Test Disinfectants/Antiseptics
Per Team:

2 MHA plates
2 Sterile swabs

Procedure:
1. Divide each of the two plates into quadrants using a permanent marker as shown below
and number 1-4.
1 2
Disinfectants/Antiseptics used
3

2. Label one plate E. coli and streak to grow using


a sterile swab. Label the other plate S.
epidermidis and streak to grow.
3. Obtain a blank disc, dip the disc into the test
chemical, shake off the excess liquid, and then
place disc in the labeled area for both plates.

1.
2.
3.
4.

4. Invert plate and incubate.


Procedure III: In-Use Test
Per team:

2 MHA plates
2 sterile swabs

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Procedure:
1. Swab an area of interest and streak to grow. Label this plate before.
2. Clean the area thoroughly and allow drying.
3. Swab the area again and streak. Label this plate after.
4. These plates will also be incubated inverted in the incubator.

EXERCISE 8 RESULTS
Procedures I and II Disk-Diffusion Tests:
Observe each plate for the presence of growth and record each result by organism in the
appropriate table. Measure each zone of inhibition and record in millimeters. For the antibiotic
plates, use the chart provided to determine the effectiveness and indicate whether the organism
is Sensitive (S), Intermediate (I), or Resistant (R) to the antibiotic.
Antibiotic Susceptibility Test Results
Escherichia
coli (a)

Antimicrobial Agent
Penicillin

Disk
Code
P

Erythromycin

Streptomycin

Chloramphenicol

Tetracycline
Trimethoprim /
Sulfamethoxazole
Nystatin

Te

Zone
Size
(mm)

S, I,
or R

Pseudomonas
aeruginosa (b)
Zone
Size
(mm)

S, I, or
R

Mycobacterium
smegmatis (d)

Staphylococcus
epidermidis (c)
Zone
Size
(mm)

S, I, or R

Zone Size
(mm)

Sxt

Did you have any plates that had colonies growing within a clear zone?
Explain this growth.
For the Disinfectant/antiseptic plates, record your disk choices in each column and provide zone
measurements (mm) for each per microbe. In this analysis there is not a standard zone size to
use for comparison, however, you can draw conclusions by comparing the data collected.
Disinfectant/Antiseptic Susceptibility Tests Results
Disinfectant/Antiseptic Used
Organism
1)
2)
3)
E. coli

4)

S. epidermidis

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S, I,
or R

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In-Use Test
Plate
Source

Cleaner Used

Growth
Amount

Appearance

Before
After

E8 Write-up
Introduction paragraph: include a description of the following items from the background
information:
Methods of control, Kirby-Bauer Disk diffusion test, in-use test
Submit results from lab manual
Conclusion:
Refer to your results, which antibiotic, disinfectant, cleaner, etc. were most effective at
inhibiting the growth for each organism tested?
This write-up must be typed and be in your own words.

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EXERCISE 9
Transformation Handout
Print from Link: http://northeaststate.edu/~pmglass/exercise9.pdf
E9 Results

Plates

GROWTH? TRANSFORMATION? # OF
TRANSFORMANTS

LB LB/AMPLB+
LB/AMP+

Did you see any satellite colonies?

What plate?

E9 Write-up
Introduction paragraph: include a description of the following items from the background
information:
Transformation, competency, steps required to achieve competency, discussion of the
genes added
Submit results from lab manual
Conclusion:
What were you able to conclude about E. coli from the growth or absence of growth on
each plate?

What evidence do you have that competency was achieved?


This write-up must be typed and be in your own words.

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EXERCISE 10
Parasitology (Protozoa and Helminths)
INTRODUCTION
Human parasitology is the science that studies parasites that infect humans. A parasite is an
organism that obtains nutrients through its close contact with another living organism, its host.
Parasites are unicellular or multicellular eukaryotic organisms. Examples of these organisms that
transmit diseases include fungi, algae, protozoa, parasitic helminthes, and arthropods. In this
exercise we are going to concentrate on protozoa and helminths.
Protozoa of Clinical Importance
Protozoa are eukaryotic unicellular heterotrophic organisms. The protozoa constitute a numerous
group of organisms in which relatively few species cause human diseases. These organisms
have a variety of shapes. All members, except for one group (the Apicomplexa), move by using
different types of structures including flagella, pseudopods, and cilia. Protozoa exhibit two
stages in their life cycle: a feeding and growing stage called a trophozoite and a resistant stage
called a cyst. The trophozoites feed on bacteria and small nutrients. Under certain conditions,
some species of protozoa produce a protective form called a cyst. A cyst enables the parasitic
protozoa to survive outside the host--once it has been excreted from an infected or carrier-- and
to infect a new host. Infection occurs through fecal-oral route, i.e. the ingestion of cystcontaminated food or water. The cysts resist the acid of the stomach. Once cysts reach the
intestines, they undergo cellular division and turn into trophozoites. These trophozoites parasitize
the intestinal mucosa causing it damage. Trophozoites are rarely seen in the direct stool smears.
Protozoa are classified as:
1. Phylum Amoebozoa include protistst that move via pseudopods
Entamoeba histolytica: The invasive E. histolytica causes amoebic dysentery.
Cyst: The cyst measures 12-15 m. It has 4 nuclei. Refer to figure 1.
Trophozoite: The trophozoite measure 15-20 m. It has a single nucleus. It
moves by means of pseudopods. Trophozoites do not have a definite shape.
Refer to figure 2.
2. Phylum Archaezoa include protists that lack mitochondria

Giardia lambia causes giardiasis. Cyst: On average a cyst measures 10-14


m. Cysts are ovoid with 2 or 4 nuclei visible depending on the degree of
maturation. Refer to figure 3. Trophozoite: It measures 10-20 m. G. lambia
has a heart shaped trophozoite with four pairs of flagella. Refer to figure 4.

3. Phylum Apicomplexa. Members of this group do not move.

Plasmodium spp. causes malaria. The worldwide prevalence of malaria is 100


million cases, with 1 million deaths per year. Plasmodium spp. are non-motile
parasites with a sexual reproductive stage that takes place in the intestinal tract
of the Anopheles mosquito; and an asexual phase that occurs in the human host.
Refer to figure 5.
Toxoplasma gondii causes toxoplasmosis. It has a particular propensity to infect
the CNS in humans. According to the CDC Division of Parasitic Diseases, the
only known definitive hosts for T. gondii are domestic cats and their relatives. It
has both sexual and asexual stages. The sexual stage occurs in the intestines of
cats. Small oocysts are excreted in the cat feces. These oocysts may
contaminate food or water. The sexual stage commonly occurs in animals both
herbivorous and carnivorous and also in humans. In the human host, the
parasites form tissue cysts, in the brain, skeletal and cardiac muscles. Refer to
figure 6.

Helminths of Clinical importance

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BIOL1230 MICROBIOLOGY

All helminths are multicellular eukaryotic organisms. These organisms have some organs and
organ systems including the reproductive, digestive, excretory, nervous, and muscular systems.
The reproductive system is the most developed organ in parasitic helminths. The helminths
include the roundworms, tapeworms, and flukes. Adult worms can be seen with the naked eye.
They are included among microorganisms because part of their life cycle includes microscopic
eggs and larvae.
1. Nematodes (roundworms). The species of intestinal nematodes that most commonly
infect humans include: Ascaris lumbricoides, Enterobius vermicularis (pinworm),
Hookworms (Necator americanus and Ancylostoma duodenale). The life cycles of these
nematodes vary in complexity and mode of infection. The life cycle includes an egg form,
which is the resting stage and serves as the diagnostic or infective form. The adult
worms male and female reside in the intestines or contiguous duct systems.
Ascaris lumbricoides cause ascariasis which is the most common nematode
infection worldwide. Laboratory diagnosis is made by observing the adult
worms as they protrude from body orifices, in-situ, or detecting eggs in the
stool sample. Refer to figures 7 and 8.
Enterubius vermicularis. Humans are considered the only host of E.
vermicularis. The adult pinworms male and female live in the human large
intestine. The female pinworm migrates to the anus to deposit her eggs.
These eggs can be ingested by the host -self infection or person-to-person
through contaminated clothing or bedding. Refer to figures 9 and 10.
Ancylostoma duodenale (Hookworm) live in the human small intestine and
the eggs are excreted in the feces. The larvae hatch in the soil. The
infective larvae can penetrate the skin of humans and they travel through
blood vessels until they arrive at the heart and lungs. In the lungs, the
larvae penetrate the pulmonary alveoli, ascend the bronchial tree to the
pharynx where they are swallowed. The larvae reach the small intestine and
mature into adults. Adult worms attach to the intestinal wall. Refer to figure
11.

2. Class Trematoda (flukes). A fluke has a flat, leaf shaped body. Flukes attach to the host
using ventral and oral suckers.
Fasciola hepatica (the sheep liver fluke). Contaminated humans or
sheep excrete eggs in the feces. The eggs release larvae and after several
stages in the life cycle may contaminate freshwater plants, which are
consumed by humans. The larva migrate to the biliary ducts where they
mature. The adult fluke measure up to 30 mm by 13 mm. Refer to figure 12.

3. Class Cestoda (Tapeworms). Tapeworms lack a digestive system. They attach to the
human intestinal mucosa using suckers and hooks on the scolex (head). Eggs are found
in the stool samples.
Taenia saginata (the beef tapeworm). Laboratory diagnosis is made by finding
eggs or proglottides passed in feces. Adult worms may measure 5 m. The eggs
measure 30 um. Refer to figure 13.
Materials Needed:
Blank slides/cover slips
Prepared slides: Entamoeba histolytica, Giardia lambia, Toxoplasma gondii, Plasmodium spp.,
Ascaris lumbricoides, Ancylostoma duodenalis, Enterubious vermicularis, Fasciola hepatic,
Taenia saginata, and others.
Activities
Prepared slides will be provided to observe the different unicellular and multicellular parasitic
organisms. Remember to clean your microscope before and after viewing slides.

Northeast State Technical Community College 2005

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BIOL1230 MICROBIOLOGY

Activity 1: Protozoa
Microscopic observation of Entamoeba histolytica, Giardia lambia, Toxoplasma gondii,
Plasmodium spp. using prepared slides.
1. Observe under the microscope up to 40X and sketch your results in the table
provided.
2. Place slide in the appropriate tray.

Activity 1: Helminths
Microscopic observation of Ascaris lumbricoides, Ancylostoma duodenalis, Enterubious
vermicularis, Fasciola hepatica and Taenia saginata.
1. Observe under the microscope up to 40X and sketch your results in the table
provided.
2. Place slide in the appropriate tray.
--------------------------------------------------------------------------------------------------------------Entamoeba histolytica: all pictures taken from: http://www.dpd.cdc.gov/DPDx/html/Amebiasis.htm

Figure 2 E. histolytica trophozoite in a direct wet mount stained with iodine.


Figure 1 E. histolytica cyst.

Giardia lambia: all pictures taken from: http://www.dpd.cdc.gov/DPDx/html/Giardiasis.htm

Figure 3 G. lambia cyst


Figure 4 G. lambia trophozoite in a wet mount stained with iodine.

Plasmodium falciparum: all pictures taken from: http://www.dpd.cdc.gov/dpdx/HTML/Malaria.htm

Figure 5 P. falciparum Ring-form trophozoites in a blood smear

Toxoplasma gondii: all pictures taken from: http://www.dpd.cdc.gov/dpdx/HTML/Toxoplasmosis.htm

Figure 6 T. gondii organisms stained with Giemsa, from a smear of peritoneal fluid.

Ascaris lumbricoides: all pictures taken from:

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http://www.dpd.cdc.gov/dpdx/HTML/Ascariasis.htm

Updated August 2010

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BIOL1230 MICROBIOLOGY

Figure 7 Fertile eggs range from 45 to 75 m in length.

Figure 8 Adult A. lumbricoides

Enterubious vermicularis: All pictures taken from: http://www.dpd.cdc.gov/dpdx/HTML/Enterobiasis.htm

Figure 10 Adult E. vermicularis (1.4 mm)


Figure 9 Eggs of in a wet mount (50-60 m by 20-30 m)

Ancylostoma duodenale: All pictures taken from: http://www.dpd.cdc.gov/dpdx/HTML/Hookworm.htm

Figure 11 The eggs are thin-shelled, colorless and measure 60-75 m by 35-40 m.

Fasciola hepatica: All pictures taken from:

http://www.dpd.cdc.gov/dpdx/HTML/Fascioliasis.htm

Figure 12 Eggs are operculated and measure 130-150 m long by 60-90 m wide.

Taenia saginata All pictures taken from: http://www.dpd.cdc.gov/dpdx/HTML/Taeniasis.htm

Figure 13 Eggs measure 30-35 micrometers in diameter and are radially-striated.

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E10 RESULTS:

BIOL1230 MICROBIOLOGY

assigned microscope #:

PROTOZOAN ORGANISMS

Name

Sketch on 40X

Entamoeba
histolytica
Giardia lambia

Plasmodium spp

Toxoplasma
gondii

HELMINTHIC ORGANISMS

Name

Sketch on 40X

Ascaris lumbricoides
Ancylostoma
duodenalis
Enterubious
vermicularis
Fasciola hepatica

Taenia saginata

E10 Write-up
Introduction paragraph: include a description of the following items from the background
information:
Parasite, host, trophozoite, cyst
Submit results from lab manual
Conclusion:
Research protozoan and helminth life cycles and discuss from a public health perspective
how you would prevent human contamination.
This write-up must be typed and be in your own words.

Northeast State Technical Community College 2005

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BIOL1230 MICROBIOLOGY

Possible Organisms Chart


Notes: (*)indicates slow growth/incubate 4-5 days, (**) requires anaerobic conditions during incubation, (***) tests that require fresh (24 hr old)

Catalase ***

Oxidase***

cocci/tetrads

OA

staphylococci

FA

Y (A)

staphylococci

F.A

Y (A)

streptococci

FA

Y (A)

streptobacilli

OA

streptobacilli

OAN

rod/singles

OA

rod/singles

FA

Y (AG)

Y*

Y*

rod/singles

FA

rod/singles

FA

+ (little to no color change)

Northeast State Technical Community College 2005

Y (AG), metallic
green
Y (A), metallic
green

Updated August 2010

Citrate

Lactose
Fermentation

Klebsiella pneumoniae

O2
Requirements

Bacillus subtilis
Clostridium
perfringens**
Pseudomonas
aeruginosa
Enterobacter
aerogenes
Escherichia coli

Flagella

Micrococcus luteus*
Staphylococcus
epidermidis
Staphylococcus
aureus
Enterococcus faecalis

X
Gram stain

Possible Organisms

X
Morphology
Arrangement

Write in the media or


medias required to test
each

Salt
Tolerance
Mannitol
Fermentation

culture, Obligate Aerobe (OA), Facultative Anaerobes (FA), Obligate Anaerobes (OAN), inapplicable test (X), acid production (A), gas production (G)

Page 51 of 51

Northeast State Technical Community College 2005

BIOL1230 MICROBIOLOGY

Updated August 2010