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MCB 1000L

Applied Microbiology
Laboratory Manual

By
Frances Duncan
With contributions from
Valerie Walker and Neil Clark
Fourth Edition 2005
Table of Contents

Topic Page Number

Safety 3

Aseptic Technique 8

Microscopy 11

Smears 17

Staining 20

Culturing and Isolation Techniques 25

Staphylococcus 33

Streptococcus 38

Throat Culture 45

Oxidase 48

Urea 50

Triple Sugar Iron Agar (TSI) 52

Motility 56

IMViC 58

Disc Diffusion Susceptibility Methods 61

Guidelines for Identification of Unknowns 67

References 70

2
Safety
Introduction

In the laboratory individuals are exposed to hazards not found in a regular


classroom. It is essential that students follow all rules established by the lab instructor,
lab manager, or lab assistant to ensure the safety of all individuals in the class. Failure
to follow established rules may result in dismissal of the individual from the class.

Laboratories have certain standard safety equipment. These typically include a


general-purpose fire extinguisher, eyewash, safety shower and cut off switches for
electrical and gas outlets. It is the responsibility of the student to locate and know how
to use the general safety equipment in the laboratory. Additionally, students should be
aware of exits from the room in case of emergency, the location of the nearest fire call
box, how to summon Campus Security, and how to obtain emergency medical
assistance.

The microbiology lab has some additional safety considerations. Since


individuals work with potentially pathogenic organisms care must be taken to prevent
possible infection or transmission of the organisms from the laboratory. Students must
wear protective clothing (lab coats) while working the laboratory. Lab coats may not be
worn outside the laboratory. Intact skin is an adequate barrier against microorganisms
so gloves are not necessary in lab. Gloves will be provided and students may wear
gloves when handling cultures if they so desire. Tabletops must be disinfected before
and after lab using the disinfectant provided. Instruction in aseptic technique will be
provided. Aseptic technique must be followed while working with microorganisms.

Handwashing is a simple and effective way to prevent the transmission of


disease. While antibacterial soap may provide some additional protection the major
effect of handwashing is the mechanical removal of microbes from the skin. Friction
when washing hands is important to mechanically remove organisms from the surface
of the skin. Using a paper towel to turn off the water prevents recontamination of the
hands with microorganisms. Hands must be washed whenever the student leaves the
lab.

Two copies of the Laboratory Safety Rules are included. One must be signed
and returned to the laboratory instructor at the end of class. The additional copy is for
your reference.

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Microbiology Laboratory Safety Rules
1. All materials and clothes other than those needed for the laboratory are to be kept away from the
work area.

2. A lab coat or other protective clothing must be worn during lab. The lab clothing is not to be worn
outside of the laboratory.

3. Clean the lab table before and after lab with the disinfectant solution provided

4. Wash hands before leaving lab.

5. Any item contaminated with bacteria or body fluids must be disposed of properly. Disposable
items are to be placed in the BIOHAZARD container. Reusable items are to be placed in the
designated area for autoclaving prior to cleaning. Sharps are to be disposed of in the appropriate
container

6. Reusable items should have all tape and marks removed by the student before being autoclaved.

7. Because organisms used in this class are potentially pathogenic, aseptic technique must be
observed at all times. NO eating, drinking, application of cosmetics or smoking is allowed. Mouth
pipetting is not allowed.

8. Cuts and scratches must be covered with Band-Aids. Disposable gloves will be provided on
request.

9. Long hair should be tied back while in lab.

10. All accidents, cuts, and any damaged glassware or equipment should be reported to the lab
instructor immediately.

11. Sterilization techniques will involve the use of Bacticinerators that are fire and burn hazards.
o o
Bacticinerators reach an internal temperature of 850 C or 1500 F. Keep all combustibles away
from the Bacticinerators. Do not leave inoculating loops or needles propped in the Bacticinerator.

12. Microscopes and other instruments are to be cared for as directed by the instructor.

13. It is the responsibility of the student to know the location and use of all safety equipment in the lab
(eyewash, fire extinguisher, etc.)

14. Cultures may not be removed from the lab. Visitors are not allowed in the lab.

15. Doors and windows are to be kept closed at all times.

16. For the best lab experience, read labs before coming to class. Make notes as necessary. Wait
for a laboratory introduction by the instructor before starting work.

I have read and understand the above rules and agree to follow them.

Signed_____________________________________________ Date_________________

Name (Please print)________________________________________________________

4
Microbiology Laboratory Safety Rules
1. All materials and clothes other than those needed for the laboratory are to be kept away from the
work area.

2. A lab coat or other protective clothing must be worn during lab. The lab clothing is not to be worn
outside of the laboratory.

3. Clean the lab table before and after lab with the disinfectant solution provided

4. Wash hands before leaving lab.

5. Any item contaminated with bacteria or body fluids must be disposed of properly. Disposable items
are to be placed in the BIOHAZARD container. Reusable items are to be placed in the designated
area for autoclaving prior to cleaning. Sharps are to be disposed of in the appropriate container

6. Reusable items should have all tape and marks removed by the student before being autoclaved.

7. Because organisms used in this class are potentially pathogenic, aseptic technique must be observed
at all times. NO eating, drinking, application of cosmetics or smoking is allowed. Mouth pipetting is
not allowed.

8. Cuts and scratches must be covered with Band-Aids. Disposable gloves will be provided on request.

9. Long hair should be tied back while in lab.

10. All accidents, cuts, and any damaged glassware or equipment should be reported to the lab instructor
immediately.

11. Sterilization techniques will involve the use of Bacticinerators that are fire and burn hazards.
o o
Bacticinerators reach an internal temperature of 850 C or 1500 F. Keep all combustibles away from
the Bacticinerators. Do not leave inoculating loops or needles propped in the Bacticinerator.

12. Microscopes and other instruments are to be cared for as directed by the instructor.

13. It is the responsibility of the student to know the location and use of all safety equipment in the lab
(eyewash, fire extinguisher, etc.)

14. Cultures may not be removed from the lab. Visitors are not allowed in the lab.

15. Doors and windows are to be kept closed at all times.

16. For the best lab experience, read labs before coming to class. Make notes as necessary. Wait for a
laboratory introduction by the instructor before starting work.

I have read and understand the above rules and agree to follow them.

Signed_____________________________________________ Date_________________

Name (Please print)________________________________________________________

5
Safety Review Questions

1. List all emergency exits from the laboratory.

2. Describe how you would reach Campus Security.

3. Describe how you would obtain emergency medical assistance.

4. What protective clothing must be worn during lab?

5. What is a simple and effective way to prevent disease transmission?

6. What general safety equipment is found in the laboratory?

7. Where is the nearest fire call box?

8. What do you need to bring to the lab table for lab?

9. How do you dispose of material that may be contaminated with bacteria?

10. How do you dispose of a broken slide?

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11. You have just disinfected your lab table. Where do you dispose of the paper
towels you used?

12. After washing your hands, where do you dispose of your paper towels?

13. When discarding reusable contaminated material where do you put it? What
must be done to it before it is discarded?

14. It is the end of lab. What must you do before you leave lab? List the tasks in
order of performance.

7
Aseptic Technique
Introduction

When working with microorganisms it is desirable to work with a pure culture. A


pure culture is composed of only one kind of microorganism. Occasionally a mixed
culture is used. In a mixed culture there are two or more organisms that have distinct
characteristics and can be separated easily. In either situation the organisms can be
identified. When unwanted organisms are introduced into the culture they are known as
contaminants.

Aseptic technique is a method that prevents the introduction of unwanted


organisms into an environment. When changing wound dressings aseptic technique is
used to prevent possible infection. When working with microbial cultures aseptic
technique is used to prevent introducing additional organisms into the culture.

Microorganisms are everywhere in the environment. When dealing with


microbial cultures it is necessary to handle them in such a way that environmental
organisms do not get introduced into the culture. Microorganisms may be found on
surfaces and floating in air currents. They may fall from objects suspended over a
culture or swim in fluids. Aseptic technique prevents environmental organisms from
entering a culture.

Doors and windows are kept closed in the laboratory to prevent air currents
which may cause microorganisms from surfaces to become airborne. Once these
microbes are airborne they are more likely to get into cultures. Transfer loops and
needles are sterilized before and after use in the Bacticinerator to prevent introduction
of unwanted organisms. Agar plates are held in a manner that minimizes the exposure
of the surface to the environment. When removing lids from tubes, lids are held in the
hand and not placed on the countertop during the transfer of materials from one tube to
another. These techniques are the basis of laboratory aseptic technique.

In this laboratory exercise the location of environmental organisms will be


explored and how microorganisms can be transmitted through contact with
contaminated surfaces.

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Laboratory Procedure
General Instructions
1. Students will work in groups.

Materials/Equipment
2 blood agar or nutrient agar plates per student plus one plate per group
Markers

Instructions
1. Label one plate “Open”. Write on the agar containing side of the plate, not on the
lid. Remove the lid from the plate and place in on the lab table, agar side up,
until the end of lab.

2. Obtain one agar plate per student and draw a line on the agar containing side of
the plate to divide the plate in half. Label one side “dirty” and one side “clean”.
Remove the lid and gently touch your fingertips to the agar on the “dirty” side.
Replace the lid. Wash your hands or clean your hands with hand sanitizer and
gently touch your fingertips to the agar on the “clean” side of the plate

3. Obtain one agar plate per student and using a marker divide the plate into
quadrants. Label the quadrants “1”, “2”, “3”, and “4”.

4. Put on gloves and try to touch as few surfaces as possible. The lab instructor will
swab the left gloved palm of each student.

5. Remove the lid from your agar plate and touch the first two fingers of your right
hand to the agar in quadrant 1.

6. Replace the lid on the agar plate and touch the first two fingers of your right hand
to the left palm of another student in your group.

7. Remove the lid from your agar plate and touch the first two fingers of your right
hand to the agar in quadrant 2.

8. Repeat steps 6 and 7 with two other students in your group and inoculate
quadrants 3 and 4.

9. Carefully remove your gloves and place them in the biohazard container. Wash
your hands.

10. Replace the lid on the “open” plate. Stack all plates agar side up and incubate
them until the next lab period.

11. During the next lab period examine plates for growth and record results on page
10. Discard all plates in the biohazard container.

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Conclusions

1. Describe the growth on the plate labeled “Open”.

2. Are organisms found in the air? What results support your conclusions?

3. Record your results from the first plate you inoculated with your hands in the
chart below.

Side of Plate Results


“dirty”
“clean”

4. What effect does hand washing have on microorganisms? Should you ever
touch a sterile surface?

5. Record the results from the second plate you inoculated with gloved hands in the
chart below.

Quadrant Results
1
2
3
4

6. One person in your group had microorganisms swabbed on their glove. The
others did not. From you results can you determine who had the “contaminated”
glove?

7. What conclusions can you draw from your data concerning where microbes are
found in the environment?

10
Microscopy
Introduction

Microorganisms are too small to be seen with the naked eye so a microscope
must be used to visualize these organisms. While a microscope is not difficult to use it
does require some practice to develop the skills necessary to use the microscope to its
maximum capabilities. Bacteria and other cellular microorganisms are measured in
micrometers (µm) or 1 x 10-6 meters. Viruses are even smaller and are measured in
nanometers (nm) or 1 x 10-9 m. When carrying a microscope always use both hands.
One should be on the arm of the microscope and one should be under the base of the
microscope.

Discussion

There are several types of microscopes but the only one used in this laboratory is
the compound light or bright-field microscope. Individual microscopes will vary
depending on the manufacturer but all microscopes have the same basic features.
Ocular

Nosepiece

Arm
Objectives

Stage Coarse Adjustment


Knob
Iris Diaphragm
Fine Adjustment
Stage Adjustment Knob Knob

Base Light Source

These microscopes are known as compound microscopes because there are two
magnifying lenses in the microscope. One magnifying lens is in the ocular and one is in
the objective. Each contributes to the magnification of the object on the stage. The
total magnification of any set of lenses is determined by multiplying the magnification of
the objective by the magnification of the ocular. The nosepiece rotates allowing the
objectives to change and thus change the magnification of the microscope.

The stage is where the slide is placed. The stage adjustment knobs allow the
slide to be moved easily. Light provides the illumination for the specimen. To control
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the amount of light reaching the eye the iris diaphragm may be opened or closed using
the lever just under the stage. On low magnifications less light is need than on higher
magnifications. Too much light on low magnification may mask the specimen,
particularly something as small as a bacterial cell.

The coarse and fine adjustment knobs are used to focus on the specimen. When
a slide is on the stage there is a space between the objective and the slide. This space
is known as the working distance. The coarse adjustment knob will cause the working
distance to visibly change while the fine adjustment knob is for final, fine focusing.

The ability to see things using a microscope is limited by the resolving power of
the microscope. The resolving power of a microscope is the distance two objects must
be apart and still be seen as separate and distinct. For the light microscope this is 0.2
µm. Objects closer together than 0.2 µm will not be distinctly seen. Increasing the
magnification will not make the objects more distinct, just bigger.

Each objective has the magnification of the objective written on the objective.
The magnification of the ocular is also inscribed on the ocular. Low magnifications are
used for quickly examining the slide to find an appropriate area to examine. Higher
magnifications allow the examination of a particular object on the slide. Examine your
microscope and complete the table below.

Objective Magnification of Magnification of Total


Objective Ocular Magnification
Scanning
Low Power
High Power
Oil Immersion

When you look through the ocular you will see a lighted circle. This is known as
the field of view or the field. While looking through the microscope move the iris
diaphragm lever and notice how the brightness of the light changes. As you move the
objectives to provide increased magnification you will look at a smaller section of the
slide. Be sure you move the object you want to view into the center of the field before
moving to the next objective.

These microscopes are parfocal. Once you have focused on an object using one
objective the object will be approximately in focus on the next objective. Use of the fine
focus knob will sharpen the focus.

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Procedure for Focusing

1. Obtain a slide.

2. Use the coarse adjustment knob to obtain maximum working distance.

3. Place the slide on the stage. The slide should fit into the slide holder but is not
placed under the slide holder. Use the stage adjustment knob to move the slide
over the hole in the stage.

4. Rotate the low power (10X) objective in place.

5. Use the coarse adjustment knob to obtain the minimum working distance.
Develop the habit of watching this process to be sure the objective does not
crash into the slide.

6. Look through the ocular. Adjust the light with the iris diaphragm lever if
necessary. Slowly turn the coarse adjustment knob until something comes into
focus. Use the fine adjustment knob to sharpen the focus.

7. Using the stage adjustment knob move the slide around until you find an area
you wish to examine more closely. Move the slide until the object you wish to
examine is in the center of the field.

8. Rotate the high power objective into place. Use the fine adjustment knob to
sharpen the focus. Do not use the coarse adjustment knob. Adjust the light
using the iris diaphragm lever if necessary.

9. Rotate the high power object halfway to the next position. Place a drop of
immersion oil on the slide, then rotate the oil immersion objective into place. The
objective should be immersed in the oil on the slide. Use the fine adjustment
knob to sharpen the focus. Adjust the light using the iris diaphragm lever if
necessary.

10. When finished viewing the slide use the coarse adjustment knob to maximize the
working distance and remove the slide from the stage. If you want to look at
another slide, begin the process over. If you are finished with the microscope
clean the microscope and return it to storage.

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Procedure for Cleaning a Microscope

1. Turn off the light and unplug the cord. Store the cord appropriately.

2. Using the coarse adjustment knob to obtain maximum working distance and
remove the slide from the stage.

3. Using lens paper clean all the lenses starting with the cleanest first—ocular, low
power, high power and oil immersion. Use lens cleaner if necessary.

4. Clean any oil off of the stage using Kimwipes or paper towels.

5. Rotate the scanning objective into place. Use the coarse adjustment knob to
obtain minimum working distance.

6. Return the microscope to the appropriate storage area.

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Review Questions

Label the drawing of the microscope.

Define:

Resolving power

Parfocal

Field

Working distance

Tell the function of each of the following.

Coarse adjustment knob

Fine adjustment knob

Iris diaphragm

Stage adjustment knob

What unit of measurement is used for measuring bacteria?

How do you determine the total magnification of a set of lenses?


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Describe the process for focusing on a slide.

Describe how to properly clean a microscope.

16
Smears
Introduction

The microscopic examination of microorganisms is a valuable identification


technique. In order to view microbes it is necessary to prepare slides of the organisms.
Microscopic preparations may be either wet mounts or smears. Wet mounts involve
placing cells in a drop of water, adding a coverslip and viewing the material under the
microscope. In microbiology most of the organisms viewed are bacteria which are small
and difficult to see without staining. Wet mounts are temporary preparations and the
ability to stain is limited. A smear is a thin preparation of cells allowed to dry on a slide.
This material is then fixed to the slide using heat or a chemical. A smear is a more
permanent preparation and may be stained using a variety of techniques.

Smears are made using plain tap water. While tap water is not sterile it has too
few organisms in it to interfere with a bacterial smear. At least 500,000 cells per
milliliter must be present in order to see one cell per oil immersion field. Bacteria are
mixed in water and allowed to dry on the slide to make a bacterial smear. This is then
fixed to the slide using heat. Heat fixing helps attach the cells to the slide so they are
not washed off during the staining process, kills the cells so the slide is not hazardous to
handle, and alters the cell wall for staining. The number of cells placed on the slide is
important for viewing the cells. Too few organisms and it is ha rd to find them on the
slide. Too many organisms and it is difficult to see individual cells to determine their
morphology or shape.

Laboratory Procedure

General Instructions

1. Students work individually.

2. To sterilize an inoculating loop or needle insert the loop or needle into the
Bacticinerator and observe it. It must glow red for three seconds to be sterilized.
Loops and needles should never be propped in the Bacticinerator. The handles
are aluminum and will melt. Also they conduct heat readily a nd can cause burns
if the handles heat up. A hot loop or needle must cool slightly before touching a
bacterial colony to prevent killing the cells.

3. To aseptically remove a lid from a bottle or tube, grasp the lid with the little finger
of the dominant ha nd. Twist the bottle or tube to loosen and remove the lid. Do
not put the lid on the table but keep it in your hand while removing material from
the bottle or tube. Return the lid to the bottle or tube by turning the bottle or tube
to tighten the lid.

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Materials/Equipment

Clean glass slides


Prepared cultures of Staphylococcus aureus and E. coli
Inoculating loop
Bacticinerator
Laboratory marker

Instructions

1. Glass slides should be relatively clean and grease free. Slides that do not
appear clean may be washed in soap and water and dried with a paper towel.
Label two slides across one end Staph. and two slides E. coli.

2. Work with one slide at a time. Sterilize an inoculating loop. Aseptically remove
the lid from the water bottle and remove a loopful of water from the bottle.
Return the lid to the bottle.

3. Tap the loopful of water onto the center of one of the labeled slides.

4. Sterilize the loop.

5. Obtain a slant culture of one of the organisms. Aseptically remove the lid. Insert
the sterile loop into the tube being careful not to touch the lip of the tube. Touch
the loop to the surface of the agar. DO NOT scrape or dig into the agar.
Remove the loop and return the lid to the tube.

6. Mix the material on the loop in the drop of water on the appropriately labeled
slide. Spread the drop over the surface of the slide making a uniform preparation
of bacteria and water. The thinner the smear the quicker it will dry.

7. Allow the smear to air dry.

8. Heat fix the slide by passing it 10 times over the top of the Bacticinerator.

9. The slide is ready for staining. It may be stored until needed.

10. Repeat Steps 2 -8 to make two smears of Staphylococcus aureus and two
smears of E. coli. Store the slides in slide boxes for use in future lab exercises.

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Smear Review Questions

1. Describe two preparations that may be used to observe microorganisms.

2. What is the purpose of heat fixing?

3. Outline the procedure for making a smear?

4. Why must slides used in smear preparation be grease-free?

5. Is it necessary to use sterile water when making a smear? Why or why not?

6. List two reasons for not propping inoculating loops and needles in the
Bacticinerator during sterilization.

7. How long does it take to sterilize an inoculating loop or needle?

8. When removing a lid from a lid or a bottle using aseptic technique, what do you
do with the lid?

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Staining
Introduction

Bacteria have almost the same refractive index as water. This means when you
try to view them using a microscope they appear as faint, gray shapes and are difficult
to see. Staining cells makes them easier to see.
Simple stains use only one dye that stains the cell wall of bacteria much like
dying eggs at Easter. Differential stains use two or more stains and categorize cells into
groups. Both staining techniques allow the detection of cell morphology, or shape, but
the differential stain provides additional information concerning the cell. The most
common differential stain used in microbiology is the Gram stain.
Bacteria have three basic shapes or morphological types. Round cells are
known as cocci, rod-shaped cells are bacilli, and spiral-shaped cells are spirilla.

Cocci Bacilli Spirilla

Principle

Simple Stain: The simple stain consists of one dye. The dye adheres to the cell
wall and colors the cell making it easier to see.

Gram Stain: The Gram stain is a differential stain. Four different reagents are
used and the results are based on differences in the cell wall of bacteria. Some
bacteria have relatively thick cell walls composed primarily of a carbohydrate known as
peptidoglycan. Other bacterial cells have thinner cell walls composed of peptidoglycan
and lipopolysaccharides. Peptidoglycan is not soluble in non polar or organic solvents
such as alcohol or acetone, but lipopolysaccharides are nonpolar and will dissolve in
nonpolar organic solvents.

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Crystal violet acts as the primary stain. This stain can also be used as a simple
stain because it colors the cell wall of any bacteria. Gram’s iodine acts as a mordant.
This reagent reacts with the crystal violet to make a large crystal that is not easily
washed out of the cell. At this point in the staining process all cells will be the same
color. The difference in the cell wall structure is displayed by the use of the decolorizer.
A solution of acetone and alcohol is used on the cells. The decolorizer does not affect
those cell walls composed primarily of peptidoglycan but those with the lipid component
will have large holes develop in the cell wall where the lipid is dissolved away by the
acetone and alcohol. These large holes will allow the crystal violet-iodine complex to be
washed out of the cell leaving the cell colorless. A counterstain, safranin, is applied to
the cells which will dye the colorless cells.
The cells that retain the primary stain will appear blue or purple and are known
as Gram positive. Cells that stain with the counterstain will appear pink or red and are
known as Gram negative. The lipopolysaccharide of the Gram negative cell not only
accounts for the staining reaction of the cell but also acts as an endotoxin. This
endotoxin is released when the cell dies and is responsible for the fever and general
feeling of malaise that accompanies a Gram negative infection.
When reporting a Gram stain you must indicate the stain used, the reaction, and
the morphology of the cell. Round, purple (blue) cells would be reported as Gram
positive cocci and rod-shaped, purple (blue) cells would be reported as Gram positive
bacilli. There are standard abbreviations that may be used for these reports.

GPC Gram positive cocci


GNC Gram negative cocci
GPB Gram positive bacilli
GNB Gram negative bacilli

The spiral-shaped bacteria of medical importance do not Gram stain well and are
usually demonstrated using a dark-field microscope. There are no standard
abbreviations for Gram stain reactions for the spirilla.

Procedure

Simple stain

Materials
Heat-fixed bacterial smears
Methylene blue, Crystal violet, or Safranin to act as simple stain
Bibulous paper or paper towels
Microscope

1. Cover the label on the slide with tape.

2. Place the slide on the staining rack and flood the slide with stain for 1 minute.

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3. Rinse the slide with tap water, tilting the slide slightly to rinse all the stain from
the slide. Tap the slide gently to remove excess water.

4. Place a piece of bibulous paper or paper towel on the lab table and put the slide
on it. Fold the paper over the slide and gently blot the slide to remove the water.

5. Examine the stained smear with the microscope and record your results in
the chart below.

Organism Results
Staphylococcus aureus
E. coli

Gram Stain

Materials
Heat-fixed bacterial smears
Gram stain reagents
Crystal violet
Gram’s iodine
Acetone-alcohol decolorizer
Safranin
Bibulous paper or paper towels
Microscope

1. Cover the label on the slide with tape.

2. Place the slide on the staining rack and flood with crystal violet for 1
minute.

3. Rinse the slide with tap water, tilting the slide slightly to rinse all the stain
from the slide.

4. With the slide slightly tilted, drop a few drops of Gram’s iodine on the slide
to rinse off the last of the rinse water. Place the slide flat and flood with Gram’s
iodine for 1 minute.

5. Rinse the slide with water as in step 3.

6. With the slide tilted slowly drop acetone-alcohol decolorizer on the slide.
Blue color will run from the smear. Continue to apply decolorizer drop-by-drop
until the blue stops running from the smear.

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7. Immediately rinse with water.

8. With the slide slightly tilted add safranin to the slide to replace the rinse
water then lay the slide flat and flood the slide with safranin for 30 seconds.

9. Rinse safranin from the slide with tap water. Gently tap the slide to
remove excess water.

10. Place a piece of bibulous paper or paper towel on the lab table and put
the slide on it. Fold the paper over the slide and gently blot the slide to remove
the water.

11. Examine the stained smear with the microscope and record your results in
the chart below.

Organism Results
Staphylococcus aureus
E. coli

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Review Questions

What is the purpose of staining bacteria?

List and describe the three basic bacterial shapes.

What are the differences between a simple stain and a differential stain?

What is the most common differential stain used in microbiology?

What is the basis for Gram stain results between different bacteria?

List the reagents used in the Gram stain and tell the function of each.

What would be the proper way to report each of the following if they had been Gram
stained?

Purple (blue), round cells

Pink (red), rod-shaped cell

Pink (red), round cells

Purple (blue), rod-shaped cells

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Culturing and Isolation Techniques
Introduction

Microorganisms must have a constant nutrient supply if they are to survive.


Free-living organisms acquire nutrients from the environment and parasitic organisms
acquire nutrients from their host. When trying to grow microbes in the lab adequate
nutrition must be provided using artificial media. Media may be liquid (broth) or solid
(agar). Any desired nutrients may be incorporated into the broth or agar to grow
bacteria.

Agar is the solidifying material used in solid media. It is an extract of seaweed


that melts at 100o C and solidifies at about 42o C. Most pathogenic bacteria prefer to
grow at 37o C so agar allows for a solid medium at incubator temperatures. Since agar
remains solid until reaching 100o C, thermophiles (heat-lovers) that prefer temperatures
above 50o C for growth can still be grown on solid media.

Organisms grown in broth cultures cause turbidity, or cloudiness, in the broth.


On agar, masses of cells, known as colonies, appear after a period of incubation.
Certain techniques will allow bacterial cells to be widely separated on agar so that as
the cell divides and produces a visible mass (colony), the colony will be isolated from
other colonies. Since the colony came from a single bacterial cell, all cells in the colony
should be the same species. Isolated colonies are assumed to be pure cultures.

Principle

A mixed culture contains two or more bacterial species that are known and can
be easily separated based on cultural or biochemical characteristics. Culturing
techniques provide a means for maintaining adequate nutrition for the organisms so
they can continue to survive. As organisms grow in a culture they consume the
available nutrients and periodically need to be transferred to fresh media to continue to
grow. Certain culturing techniques not only provide the organisms with a fresh supply of
nutrients but also allow for the separation of bacterial cells to obtain isolated colonies.
These culturing procedures are known as isolation techniques.

Streak plates allow for the growth of isolated colonies on the surface of the agar.
An isolated colony is a colony that is not touching any other colonies and is assumed to
be a pure culture. These colonies are easily accessible for performing staining and
identification procedures. They also show colonial morphology that may be useful in
identifying the organism. Part of the identification of any organism includes a
description of colonial morphology. Since organisms may grow differently on different
media, the type of media used must be included as a part of any colonial morphology.
Other elements of a colonial description include colony color, hemolysis (if grown on
blood agar), form, elevation and margin.

25
Form refers to the overall appearance of the colony. Elevation is the height the
colony achieves on the surface of the agar. The appearance of the edge of the colony
is referred to as the margin.

FORM

Circular > 1mm Irregular


Punctiform < 1 mm

ELEVATION

________________________________________________________
Flat Convex Umbonate

MARGIN

Entire Undulate Curled

The pour plate is used for counting organisms in a solution. A standard volume
of solution is mixed in the liquefied agar. Each organism in the solution is separated
from all others. When the agar solidifies the cells are trapped in the agar and develop
into colonies. Each colony can be counted and represents a single cell in the original
solution. If a milliliter of solution is mixed in the agar then the number of colonies
represents the number of organisms per milliliter of solution. Usually a portion of a
milliliter is mixed in the agar so the number of colonies counted must be multiplied by
the dilution factor to determine the number of organisms in a milliliter of solution. When
counting colonies in agar it is difficult to accurately count more than 300 colonies on a
plate. Less than 30 colonies on a plate are considered statistically insignificant. When
evaluating a solution for bacteria a series of dilutions is usually made and cultured. The
plate with 30-300 colonies is counted and the number multiplied by the dilution factor for
that plate to determine the number of bacteria per milliliter in the original solution. This
method is used to evaluate the number of organisms in milk, drinking water, and even
the water at the beach. While the cells grow and are isolated from each other in a pour
plate, they will not develop typical colonial morphology and are not easily accessible for
further testing.

26
Procedure

Inoculation of a Broth Culture

Materials
Mixed Culture in broth
Inoculating loop
Bacticinerator
Incubator
Sterile nutrient broth

Students work individually.

1. Label the sterile nutrient broth with the source of the culture and your initials.

2. Sterilize the loop.

3. Using appropriate aseptic technique, remove a loopful of broth from the mixed
culture tube.

4. Insert the loop into the sterile broth tube and swirl gently. Sterilize the loop.

5. Incubate the broth at 37o C for 24-48 hours.

6. Observe broth for turbidity. Record results in table at end of the procedure
section.

Inoculating an Agar Slant

Materials
Mixed Culture in broth
Inoculating loop
Bacticinerator
Incubator
Sterile nutrient agar slant

Students work individually.

1. Label the sterile nutrient agar slant with the source of the culture and your
initials.

2. Sterilize the loop.

3. Using appropriate aseptic technique, remove a loopful of broth from the


mixed culture tube.
27
4. Insert the loop into the sterile agar slant tube and starting at the base of
the slant, draw the loop up the slant. Do not penetrate the agar. Sterilize
the loop.

5. Incubate the slant at 37o C for 24-48 hours.

6. Observe the slant for growth. Record results in table at end of the
procedure section.

Streak Plate

Materials
Mixed Culture in broth
Inoculating loop
Bacticinerator
Incubator
Sterile nutrient agar plate

Students work individually.

1. Label the sterile nutrient agar plate with the source of the culture and your
initials.

2. Sterilize the loop.

3. Using appropriate aseptic technique, remove a loopful of broth from the


mixed culture tube.

4. Lift the agar plate from the lid and streak about half of the plate. The loop
should be parallel to the agar surface to prevent digging into or gouging
the agar.

28
5. Return the plate to the lid. Sterilize the loop. Lift the agar plate and make
one streak into the inoculated portion of the plate. Finish by streaking about one-
fourth of the uninoculated plate.

6. Return the plate to the lid. Sterilize the loop. Lift the agar plate and make
one streak into the second inoculated portion of the plate. Finish by
streaking the remaining one-fourth of the uninoculated plate. Sterilize the
loop.

7. Place the plate in a 37o C incubator for 24-48 hours. Observe for growth
and record your results in the table provided at the end of the procedure section.

29
Pour Plate
Materials
Mixed Culture in broth
Inoculating loop
Bacticinerator
Incubator
Nutrient agar deep, liquefied
Sterile petri dish
Sterile pipette

Students work in pairs.

1. Label the bottom of the sterile petri plate with the source of the culture and your
initials. Turn the plate so the lid is facing up.

2. Obtain two tubes of liquefied nutrient agar, one for each student in the pair. The
nutrient agar was boiled (100o C) to melt the agar. Agar at that temperature
would kill bacteria, so the agar has been cooled to 60o C and held in a water bath
to maintain that temperature. This should not kill the bacteria when they are
introduced to the liquid agar and will also reduce the amount of condensation that
will collect on the lid of the petri dish.

3. Work quickly. The agar will solidify at 42o C. One student of the pair should
aseptically transfer two drops of the mixed culture broth to one of the agar tubes.
The other student should aseptically transfer one drop of the mixed culture broth
the second agar tube. Mix the tubes by rolling the tubes between your hands,
then pour the inoculated liquid agar into a labeled sterile petri dish. Gently move
the dish in a figure eight to completely cover the bottom of the dish with agar.

4. Allow the agar to solidify. Add to the labeling the amount of mixed culture used
in the agar. A milliliter contains approximately 20 drops. Two drops would be
approximately 0.1 ml and 1 drop would be approximately 0.05 ml.

5. Incubate the plates at 37o C for 24-48 hours. Examine the plates for growth and
record the results in the table below.

Culture Growth Isolation


Broth
Slant
Streak Plate
Pour Plate 0.1 ml
Pour Plate 0.05 ml

30
Review Questions

How can you tell growth has occurred in a broth culture?

What is the purpose of agar?

At what temperature does agar liquefy?

At what temperature does agar solidify?

Why is liquefied agar cooled to 60o C before adding organisms?

List two methods for obtaining isolated colonies.

What is the primary purpose of the streak plate?

How many colony types did you observe on your streak plate?

Describe each colony type observed using standard terms.

What is the primary purpose of the pour plate?

31
How many colonies did you observe on the 0.1 ml pour plate?

How many colonies did you observe on the 0.05 ml pour plate?

If possible determine the number of organisms in the original mixed culture broth.
The dilution factor for 0.1 ml is 10 and for 0.05 ml the dilution factor is 20.

What is the general formula for determining the number of organisms in a solution using
a pour plate?

Serial dilutions are made of milk. The following information is collected.

Dilution Number of Colonies


1:100 312
1:1000 262
1:10000 22

How many organisms are in a milliliter of the milk tested?

32
Identification of Gram Positive Cocci
Staphylococcus

Introduction

The genus Staphylococcus contains both pathogenic and non-pathogenic


organisms. They do not produce endospores but are highly resistant to drying,
especially when associated with organic matter such as blood, pus, and other tissue
fluids. Most staphylococci are found routinely on the surface of the skin. Breaks in skin
and mucous membranes allow entrance of these organisms into the body where they
may cause disease.

The three major species include Staphylococcus aureus, Staphylococcus epidermidis,


and Staphylococcus saprophyticus. The latter two are rarely implicated in disease, but
have been isolated in cases of endocarditis and urinary tract infections under certain
circumstances. Staph. aureus is considered the pathogenic strain, causing abscesses,
boils, carbuncles, acne and impetigo. Less commonly, pneumonia, osteomyelitis,
endocarditis, cystitis, pyelonephritis, and food poisoning have been attributed to this
organism. These three strains of staphylococci can be distinguished from each other by
a number of biochemical tests.

Principle

The identification of organisms is based on cellular, cultural and biochemical


characteristics. All species of Staphylococcus are Gram positive cocci. On nutrient
agar they tend to be white, circular, entire, convex colonies. On blood agar
Staphylococcus aureus may show hemolysis of the agar in the area around the colony.

Additional biochemical tests that are useful in separating the Staphylococcus


species include catalase, coagulase, growth and fermentation of mannitol salt, and
resistance or susceptibility to the antibiotic novobiocin.

The catalase test determines if the organism produces the enzyme catalase that
breaks down hydrogen peroxide to water and oxygen.
__catalase__
2 H2O2 > 2 H2 O + O2

This enzyme allows organisms to breakdown harmful metabolites of aerobic respiration


and may be seen in aerobic and facultatively anaerobic organisms. There are other
enzymes that some organisms produce to handle toxic endproducts of metabolism so
not all aerobes or facultative anaerobes produce catalase.

Pathogenic organisms require mechanisms to help them overcome host defense


mechanisms. One mechanism involves coating the bacterial cells in a body substance,
such as fibrin, to fool the immune system. The coating of a natural body substance will
33
not trigger an immune response. The enzyme coagulase causes fibrin to be deposited
on bacterial cells.

Some organisms can not tolerate a high osmotic pressure. Media containing
higher than normal salt concentrations may inhibit the growth of these non-tolerant
organisms. Mannitol salt agar contains a high salt concentration so only salt tolerant
organisms will grow on it. Additionally, mannitol salt agar contains the sugar mannitol.
Some organisms can utilize mannitol as a food source and will produce acid
endproducts from this metabolism. Since this process is invisible an indicator is added
to the media to detect changes in pH. Phenol red is the indicator used in mannitol salt
agar. It is red at a neutral pH but turns yellow if conditions in the media become acidic.

Antibiotic susceptibility is another test that can be used to identify organisms. A


filter paper disc is impregnated with an antibiotic, in this case novobiocin. When the
disc is placed on agar, the antibiotic diffuses through the agar. An organism susceptible
to the antibiotic will be unable to grow on the media containing the antibiotic. A zone of
inhibition (no growth) will be seen around the disc. The size of the zone indicates the
resistance or susceptibility of the organism to the antibiotic.

Procedure

Catalase

1. Place a drop of 3% H2 O2 on a glass slide.

2. Touch a sterile loop to a culture of the organism to be tested and pick up a visible
mass of cells.

3. Mix the organism in the drop of hydrogen peroxide.

4. Observe for immediate and vigorous bubbling.

5. Dispose of slide in the contaminated slide container.

Interpretation: Bubbling indicates a positive (+) test and scant or no bubbling indicates a
negative (-) test.

Coagulase

1. Dispense 1 drop of Test Latex onto one of the circles on the reaction card and 1
drop of Control Latex onto another circle.

2. Touch a sterile loop to a culture of the organism to be tested and pick up a visible
mass of cells. Mix the cells in the drop of Test Latex.

3. Repeat Step 2 for the Control Latex.


34
4. Pick up and hand rock the card for up to 20 seconds and observe for
agglutination or clumping of the latex particles.

5. Dispose of the reaction card in the biohazard container.

Interpretation: Agglutination of the Test Latex with no agglutination of the Control Latex
is considered a positive (+) test for coagulase. No agglutination in either the Text Latex
or Control Latex is considered negative (-) for coagulase. All reactions occurring after
20 seconds should be ignored. If agglutination occurs in the Control Latex the
agglutination is due to some factor other than the enzyme coagulase and the test
results are invalid.

Mannitol Salt Agar

1. Label a tube of mannitol salt agar with the organism to be tested and your initials.

2. Using a sterile loop transfer the organism to be tested to the surface of the
mannitol salt agar slant.

3. Incubate the tube at 35o C. for a minimum of 18 hours.

4. Examine the tube for evidence of growth on the slant and for a color change from
red to yellow.

6. Remove the markings from the tube using Gram’s decolorizer on a paper towel
and place the tube in the designated area for disposal.

Interpretation: Two different characteristics of the organism are determined with this
agar. The first is the organism’s ability to tolerate a high salt environment. Evidence of
growth on the slant indicates the organism can grow in a high salt environment.
Organisms that can ferment the sugar mannitol produce an acid end product that
changes the red pH indicator in the media to yellow. Any yellow in the media is
considered a positive test for mannitol fermentation. It is possible for organisms to grow
on the media and not ferment mannitol.

Novobiocin Susceptibility

1. Divide a nutrient agar plate into three sections.

2. Label a section with the name of the organism to be tested.

3. Using a sterile loop transfer the test organism to the plate and streak the section
for confluent growth.

4. Aseptically transfer a novobiocin antibiotic disc to the center of each streaked


area. Gently press the disc to the surface of the agar.
35
5. Invert the plate and place in the incubator for a minimum of 18 hours.

6. Examine the plate for a zone of inhibition of growth around the antibiotic disc.

7. Using a metric ruler, measure the diameter of the zone of i nhibition and record
the measurement in millimeters (mm).

8. Discard the plate in the biohazard container.

Interpretation: A zone of growth inhibition 17 mm or less in diameter indicates


resistance (R) to novobiocin. If the zone is greater than 17 mm the organism is
susceptible (S) to novobiocin.

LABORATORY INSTRUCTIONS

Cultures provided: Staphylococcus aureus


Staphylococcus epidermidis
Staphylococcus saprophyticus

Students work individually unless otherwise noted.

1. Make a smear of one of the organisms provided. (See page 18) Complete the
remainder of the laboratory work before heat fixing, staining and examining the
smear.

2. Perform a catalase test on all organisms.

3. Select one of the three organisms and perform a coagulase test. Allow the other
members of your group to observe your results. Observe the results of the other
2 organisms.

4. Select one of the three organisms and inoculate a mannitol salt agar slant. As in
step 3, observe the results of all three organisms

5. Test each organism for novobiocin susceptibility. Each person should test all
three organisms.

6. Record all results on the Laboratory Record Sheet. (Page 37)

7. As time permits, Gram stain the smear prepared in Step 1 (Page 22).

36
MCB 1000L
Identification of Staphylococcus

Test Staph. aureus Staph. epidermidis Staph.


saprophyticus
Gram Stain
Catalase

Coagulase
Growth on mannitol
salt
Mannitol
fermentation

Novobiocin
susceptibility

All species of Staphylococcus are Gram ___________________

____________________ and positive for the ___________________________

test. Also, all Staphylococcus species tolerate ___________________________

as indicated by their growth on mannitol salt agar.

Which test differentiates Staph. aureus from the other species of Staphylococcus?

How can you differentiate Staph. epidermidis from Staph. saprophyticus?

37
Identification of Gram Positive Cocci
Streptococcus

Introduction

Members of the genus Streptococcus are responsible for disease as well as


being part of the normal flora of humans. Among the diseases caused are bacterial
pneumonia, meningitis, tonsillitis, endocarditis, scarlet fever, erysipelas, and urinary
tract infections. Streptococcus species are also found normally in the mouth and on the
skin surface.

The streptococci are classified by two major methods: hemolytic activity and
serologic classification of Lancefield.

Classification Based on Hemolytic Activity


When grown on sheep blood agar, streptococci display one of three types of
hemolysis of the red blood cells in the agar.

Alpha hemolysis --The red blood cells in the media are partially digested
producing a greening of the agar.

Beta hemolysis--The red blood cells in the media are completely digested
producing a clearing of the agar.

Gamma hemolysis--No change is noted in the agar. The red blood cells are not
affected by the organism.

Expected alpha beta gamma


Hemolysis
never always never
Streptococcus pyogenes
never usually sometimes
Streptococcus agalactiae
sometimes sometimes usually
Streptococcus bovis
always never never
Streptococcus pneumoniae
sometimes sometimes usually
Enterococcus faecalis

38
Classification Based on Lancefield Proteins

Rebecca Lancefield, working with various streptococcal species, discovered


proteins in the cell wall that were unique to certain organisms. These proteins were
labeled Group A, Group B, Group C, and so on through Group M. Currently three
Lancefield Groups are of medical importance: Group A, Group B, a nd Group D. Of the
organisms used in this lab the following correlations apply:

Group A Strep--Streptococcus pyogenes


Group B Strep--Streptococcus agalactiae
Group D Strep--Streptococcus bovis, Enterococcus (Streptococcus) faecalis

Streptococcus pneumoniae does not possess Lancefield proteins and is not classified in
one of the Lancefield groups. Viridans streptococci is the term applied to alpha
hemolytic Streptococcus species that lack Lancefield proteins.

Principle

All Streptococcus species are Gram positive cocci. Some will only grow on an
enriched agar, such as 5% sheep blood agar. On sheep blood agar the colonies are
usually gray, punctiform, convex, and entire. Various species display alpha, beta or
gamma hemolysis. Important biochemical tests include catalase, bacitracin
susceptibility, optochin susceptibility, growth in high salt broth, hemolysis patterns seen
with the CAMP test, and the ability to hydrolyze esculin.

The bacitracin and optochin susceptibility tests are similar to the no vobiocin
susceptibility test used for the identification of Staphylococcus species. Filter paper
discs impregnated with the appropriate chemical are placed on an agar surface. The
chemical diffuses through the agar. Organisms that are susceptible to the chemical will
not grow on the agar containing the chemical. The size of the zone of growth inhibition
determines the organisms susceptibility to the chemical.

CAMP factor is a diffusable protein produced by certain species of


Streptococcus. This factor will react with the beta toxin produces by Staphylococcus
aureus to rapidly lyse sheep red blood cells. When a CAMP producing Streptococcus is
grown near a beta toxin producing strain of Staphylococcus aureus a definite hemolytic
pattern is produced.

Only a few organisms can tolerate a salt concentration of 6.5% NaCl. Those that
can will grow in high salt broth.

Bile esculin agar contains bile that inhibits the growth of many organisms. Some
organisms can hydrolyze esculin to esculetin and dextrose. Esculetin will react with
ferric citrate in the media to produce a black-brown product.

39
Procedures

Bacitracin Susceptibility
1. Divide a sheep blood agar plate into four quadrants.

2. Label a quadrant with the name of the organism to be tested.

3. Using a sterile loop aseptically transfer the test organism to the plate and streak
the quadrant for confluent growth.

4. Aseptically transfer a bacitracin disc (A disc) to the center of the quadrant.


Forceps may be used to position the disc. Gently press the disc to the surface of
the agar but do not embed the disc in the agar.

5. Invert the plate and place in the incubator for a minimum of 18 hours.

6. Examine the plate for a zone of inhibition of growth around the disc. When
finished discard the plate in the biohazard container.

Interpretation: Any zone of inhibition of growth is considered positive (+) for this test. If
a red ring can be seen around the disc this is considered a positive test. This test
should be done only on organisms that display beta hemolysis .

Optochin Susceptibility

1. Divide a sheep blood agar plate into four quadrants.

2. Label a quadrant with the name of the organism to be tested.

3. Using a sterile loop aseptically transfer the test organism to the plate and streak
the quadrant for confluent growth.

4. Aseptically transfer an optochin disc (P disc) to the center of the quadrant.


Forceps may be used to position the disc. Gently press the disc to the surface of
the agar but do not embed the disc in the agar.

5. Invert the plate and place in the incubator for a minimum of 18 hours.

6. Examine the plate for a zone of inhibition of growth around the disc. Using a
metric ruler, measure the diameter of the zone of inhibition and record the
measurement in millimeters (mm). When finished discard the plate in the
biohazard container.

Interpretation: A growth inhibition zone of 15-30 mm is considered a positive (+) test.


Zone sizes of less than 15 mm are considered negative (-) for this test. This test should
be done only on organisms that display alpha hemolysis.
40
CAMP Test

1. Obtain a sheep blood agar plate that has been prepared for a CAMP test by
having Staphylococcus aureus streaked in a single line down the center of the
plate.

2. Lines have been drawn on the plate perpendicular to the Staph. streak. These
will act as guidelines for inoculating the plate. Label one of the lines on the
CAMP plate with the organism to be tested.

3. Using a sterile loop obtain a sample of the test organism. Using a single streak
and moving from the outer edge of the CAMP plate toward the Staph. steak,
inoculate the CAMP plate with the test organism. Do not allow the test organism
to directly touch the Staph. streak or streak across the Staph. streak. The test
organism should be streaked using one of the perpendicular lines as a guide.

4. Invert the plate and place it in the incubator for a minimum of 18 hours.

5. Observe the plate for the development of a distinct arrowhead pattern of


hemolysis where the test organism and the Staph. almost touch.

6. Discard the plate in the biohazard container.

Interpretation: The arrowhead hemolysis pattern is considered positive (+) for this test.
No hemolysis or indistinct hemolysis patterns are considered negative (-) for this test.
This test should be done only on organisms that display beta or gamma hemolysis .

Bile Esculin

1. Label a bile esculin slant with the organism to be tested and your initials.

2. Using a sterile loop transfer the organism to be tested to the surface of the bile
esculin slant.

3. Incubate the tube for a minimum of 18 hours.

4. Examine the tube for a definite blackening of the agar.

5. Remove the markings from the tube using Gram’s decolorizer on a paper towel
and place the tube in the designated area for disposal.

Interpretation: Blackening of the agar is considered positive (+) for this test. No change
in the color of the agar is considered negative (-) for this test. This test should be done
on all suspected streptococci.

41
High Salt

1. Label a high salt broth tube with the organism to be tested and your initials.

2. Using a sterile loop transfer the organism to be tested to the broth.

3. Incubate the tube for a minimum of 18 hours.

4. Examine the tube for evidence of growth (turbidity). It may be helpful to compare
the tube to an uninoculated tube. Do not agitate the tubes before you examine
them.

5. Remove the markings from the tube using Gram’s decolorizer on a paper towel
and place the tube in the designated area for disposal.

Interpretation: Organisms that can tolerate a high salt environment (6.5% NaCl) will
grow in this broth causing the broth to become cloudy or turbid. Turbidity is considered
positive (+) for this test. Organisms that can not tolerate the high salt environment will
not grow and the broth will remain clear. Clear broth is considered negative (-) for this
test. This test should be done on all suspected streptococci.

LABORATORY INSTRUCTIONS

Cultures provided: Streptococcus pyogenes


Streptococcus agalactiae
Streptococcus pneumoniae
Enterococcus (Streptococcus) faecalis
Streptococcus bovis

Students work individually unless otherwise noted.

1. Make a smear of one of the organisms provided (See page 18). Complete the
remainder of the laboratory work before heat fixing, staining and examining the
smear.

2. Perform a catalase test (Page 34) on all organisms and record your results on
the Laboratory Worksheet (Page 44).

3. Examine all cultures for hemolysis and record your observations on the
Laboratory Worksheet (Page 44).

4. Refer to your Laboratory Worksheet (Page 44) and on all beta hemolytic
organisms set up a Bacitracin Susceptibility test.

42
5. Refer to your Laboratory Worksheet (Page 44) and on all alpha hemolytic
organisms set up an Optochin Susceptibility test.

6. Refer to your Laboratory Worksheet (Page 44) and on all beta and gamma
hemolytic organisms set up a CAMP Test. Work in lab groups to get all required
organisms tested, but be sure each member of the group sets up one test.
Organisms may be used more than once in your group if necessary.

7. Working in groups, set up a bile esculin slant on all organisms. Each member of
the group must set up at least one test.

8. Working in groups, set up a high salt broth on all organisms. Each member of
the group must set up at least one test.

9. After appropriate incubation, examine all tests and record results on the
Laboratory Worksheet (Page 44).

10. As time permits, Gram stain the smear prepared in Step 1 (Page 22).

43
MCB 1000L
Identification of Streptococcus

Test* Strep. Strep. Strep. Enterococcus Strep.


pyogenes agalactiae pneumoniae faecalis bovis
Gram Stain
Catalase

Hemolysis
Bacitracin

Optochin
CAMP Test
Bile Esculin

High Salt
*If a test is not done on an organism because it is an inappropriate test for that
organism, mark the results box with a large X.

What characteristic do Staphylococcus and Streptococcus share?

What test would distinguish Staphylococcus from Streptococcus?

An organism is GPC, catalase negative, and alpha hemolytic. List all appropriate
tests for identification of this organism.

An organism is GPC, catalase negative, and beta hemolytic. List all appropriate
tests for identification of this organism.

An organism is GPC, catalase negative, and gamma hemolytic. List all


appropriate tests for identification of this organism.

Once you know an organism is GPC, what test should you do next?

44
Throat Culture
Introduction

The human mouth has numerous and varied organisms as part of its
normal flora. Both aerobes and anaerobes flourish is this warm, moist
environment. Virtually every type of microorganism can be found in the mouth.
The most prevalent are the viridans streptococci. These alpha hemolytic
organisms account for most of the organisms that grow aerobically in a throat
culture. In addition to these Gram positive cocci, numerous species of
Staphylococcus may also be found. Neisseria, Branhamella and the anaerobic
Veillonella comprise the majority of the Gram negative cocci found in the mouth.
Various Gram negative bacilli, such as Haemophilus species and Klebsiella
pneumoniae, are also present. The nonpathogenic Corynebacterium, or
diphtheroids, are also alpha hemolytic. Diphtheroids are pleomorphic Gram
positive bacilli. Spirochetes, a few yeasts and occasional protozoa round out the
normal mouth flora. These organisms are commensals that probably protect us
from other organisms that may enter our mouths. The presence of our normal
flora prevents other organisms from finding space or nutrients to support their
growth.

While our normal flora potentially protects us from certain diseases, they
do contribute to one. The organisms of the mouth contribute to the development
of dental caries. Certain organisms adhere to the teeth forming a network for
others to adhere. These organisms produce the plaque found on your teeth.
Some of the organisms involved in plaque metabolize sugars found in the mouth
to acids that etch the tooth enamel and weaken it. If the tooth enamel is
damaged, organisms can penetrate to the pulp of the tooth damaging it. Regular
removal of these organisms and plaque helps prevent tooth decay.

Principal

The one organism responsible for disease in the throat is Streptococcus


pyogenes or Group A Strep. This organism is beta hemolytic and not part of the
normal throat flora. Sheep blood agar provides the enrichment necessary for
growing many of the Streptococcus species and also acts as a differential media.
The hemolysis produced on sheep blood agar he lps separate the normal alpha
hemolytic organisms from the pathogenic, beta-hemolytic Streptococcus
pyogenes.

Organisms that grow in the throat also need special atmospheric


conditions to grow. These organisms are exposed to the higher carbon dioxide
content in exhaled breath. To successfully grow these organisms this carbon
dioxide rich atmosphere must be reproduced. Organisms that require less
oxygen are known as micoraerophiles. In the laboratory this atmosphere may be
produced by placing the plates in a large jar, lighting a candle in the jar and

45
replacing the lid. As the candle burns, some of the oxygen in the jar is converted
to carbon dioxide.

Typically pharyngitis would cause redness and possibly pockets of pus on


the back of the throat. When culturing a throat these areas indicating
inflammation should be swabbed to provide the specimen. Usually a swab in a
protective plastic sleeve (Culturette) is used to take a throat culture. Once the
specimen has been taken the swab is returned to its protective sleeve and an
ampule of transport media is broken in the bottom of the sleeve. Transport
media is a special purpose media that contains balanced salts to protect the
specimen from pH changes and keeps the swab moist while in transit to the
laboratory for culturing. Nutrients are not provided so growth does not occur but
the organisms can survive for several hours in the transport media, particularly if
refrigerated.

Procedure

1. Obtain a sheep blood agar plate, sterile swab and tongue depressor.

2. Label the agar plate with your “patient’s” name.

3. Using the tongue depressor, flatten the patient’s tongue. Having the
patient say “Ahhhh” helps flatten the tongue. Being careful not to touch
any other parts of the mouth, use the sterile swab to firmly swab the back
of the patient’s throat. Use care. Some people have a very strong gag
response and this may induce vomiting.

4. Gently roll the swab across the surface of the blood agar plate. Using a
sterile loop, streak the plate for isolation. First streak through the area
where you rolled the swab and cover approximately half of the plate.
Sterilize the loop and streak one quarter of the plate streaking into the
original streaking only once. Repeat the procedure for the remaining
quarter of the plate streaking into the second streaking only once. Discard
the swab and tongue depressor in the biohazard container.

5. Place the plate in a candle jar. The jar will be incubated at 35-37o C for a
minimum of 18 hours.

6. Following incubation, examine the plate for the presence of beta hemolytic
colonies. A predominance of beta hemolytic colonies would indicate a
possible throat infection with Streptococcus pyogenes.

7. When finished examining the plate, discard in the biohazard container.

46
Review Questions

1. List 3 organisms that are considered normal throat flora.

2. Why is sheep blood agar used for throat cultures?

3. What organism is pathogenic in the throat?

4. What incubation conditions are required for throat cultures?

5. What is the purpose of the candle jar?

6. Define microaerophile.

7. What are viridans streptococci?

8. What are the most predominant aerobic organisms in the throat?

9. Complete the table below.

Colony Colonial Gram Stain


Morphology Results
Colony 1

Colony 2

47
Identification of Gram Negative Bacilli—Oxidase
Introduction

The oxidase test can be used in the identification of GNB to distinguish


non-fermenters (oxidase positive) from fermenters (oxidase negative).

Principle

The oxidase test checks for the presence of the enzyme indophenol
oxidase. Tetramethyl-para-phenylenediamine (oxidase reagent) will be oxidized
in the presence of atmospheric oxygen by indophenol oxidase causing the
formation of a dark-purple compound known as indophenol.

Procedure

Organisms used
Pseudomonas aeruginosa
E. coli
Proteus vulgaris

1. Students work in groups to complete this lab.

2. Obtain a sterile swab. Touch the swab to the organism being tested.

3. Place one drop of oxidase reagent on the organism on the swab. Using
more than one drop of reagent may dilute the color reaction and result in a
false negative.

4. Observe the swab for 10-30 seconds for the development of a dark-purple
color around the edge of the organism. This is interpreted as a positive
test. No color change or a color change after 30 seconds is interpreted as
a negative test.

5. Share your results with the other members of your group.

6. Record all results in the chart below.

7. Dispose of the swabs in the biohazard container. The reagent droppers


may be discarded is the regular trash can.

Results Pseudomonas E. coli Proteus


aeruginosa vulgaris
Oxidase

48
Review Questions

1. Based on your results, which organism(s) could be classified as a non-


fermenter?

2. E. coli and Proteus vulgaris are members of the family Enterobacte riaceae
so their reactions are representative of the entire family (all members of
the family behave the same way). Klebsiella pneumonia is also a member
of the family Enterobacteriaceae. What would its oxidase test result be?
Is it a fermenter or a non-fermenter?

49
Identification of Gram Negative Bacilli—Urea
Introduction

The urea test can be used in the identification of GNB, particularly those in
the family Enterobacteriaceae.

Principle

If an organism produces the enzyme urease it will break down urea to


ammonia and carbon dioxide.
urease
urea > ammonia + carbon dioxide

Ammonia will increase the pH of the media to 8.0 or higher. The media
contains phenol red as a pH indicator. At a pH 8.0 or higher the indicator is a
bright pink color. If urea is split to ammonia and carbon dioxide the pH change
will cause the media to turn bright pink and the test will be considered positive for
urease.

Procedure

Organisms used
Pseudomonas aeruginosa
E. coli
Proteus vulgaris

1. Work in groups to complete this lab.

2. Urea media may be either a broth or a slant. Obtain urea media and
inoculate tubes with the three organisms listed above. Use appropriate
aseptic technique when inoculating the tubes.

3. Incubate the tubes for a minimum of 18 hours at 35-37o C.

4. Examine the tubes for a color change. Tubes that are bright pink are
considered positive for the test. Any other color change is considered
negative. Remove labels from the tubes and discard in the designated
area.

5. Record all results in the table below.

Result Pseudomonas E. coli Proteus


aeruginosa vulgaris
Urea

50
Review questions

1. What enzyme is produced by organisms that can split urea?

2. What is the indicator used in urea media?

3. Why does the media turn pink when the test is positive?

51
Identification of Gram Negative Bacilli—TSI
Introduction

The TSI (Triple Sugar Iron) agar provides information concerning glucose
fermentation, utilization of the sugars lactose and sucrose, and the anaerobic
respiratory process that uses sulfur as the final electron acceptor to produce
hydrogen sulfide. This information is useful in the identification of Gram negative
bacilli.

Principle

TSI Agar contains three sugars: glucose (0.1%), lactose (1.0%), and
sucrose (1.0%). It also contains phenol red to indicate a change in pH and
ferrous sulfate to demonstrate H2S production.

Sugar Fermentation

Fermentation is an anaerobic process. When sugar is fermented an acid


endproduct is produced and sometimes gas. Phenol red turns yellow under acid
conditions and red under alkaline conditions.

Yellow agaràacid productionàsugar fermentation

The enzymes for glucose fermentation are constitutive enzymes so


glucose is the first choice of an organism for fermentation. The acid produced
will turn the agar in the tube yellow. Some organisms also produce gas from
glucose fermentation. This gas may be trapped in the agar pushing the agar up
in the tube or causing cracks or bubbles in the tube. It is possible for the gas to
escape around the agar and not be detected.

If only glucose can be used the organism quickly uses the available
glucose in the tube. In order to survive the organism begins using the protein in
the agar as a carbon source. The first step of protein utilization is deamination
(forming ammonia—alkaline). Deamination is an aerobic process and only
occurs on the slant. Those organisms that can ferment only glucose deaminate
proteins to continue to survive and the slant reverts to red due to the alkaline
conditions produced.

Organisms that can use either sucrose or lactose (or both) will begin to
ferment these sugars once the glucose has been consumed. The enzymes
required for utilization of these sugars are inducible so the presence of sucrose
and lactose in the media will activate the necessary operons. Acid will continue
to be produced as a result of the metabolism of the lactose and/or sucrose and
the tube will remain yellow. There is a sufficient quantity of either sugar in the
media to support the organism for at least 2-3 days.

52
Hydrogen Sulfide (H 2S) Production

Anaerobic respiration does not require oxygen since an inorganic salt acts
as the final electron acceptor instead of oxygen. Sulfur is one of the anaerobic
electron acceptors used by some facultative and obligate anaerobes. Sulfur is
readily available in the environment and in media in both organic (amino acids)
and inorganic (sulfates) molecules.

Hydrogen sulfide is a colorless, volatile liquid. In order to detect its


presence in the media, ferrous sulfate is used as an indicator.

H2S + ferrous sulfate à ferrous sulfide (Black precipitate)

H2S production is an anaerobic process so the black precipitate will


appear only in the butt of the TSI tube.

Reporting Results and Interpretation

Black butt—H2S + Yellow agar—acid—A


No black in butt—H2S – Red agar—alkaline—K
Gas produced—G
Report slant/butt

Report Interpretation

A/A Glucose and sucrose and/or lactose fermented

A/AG Glucose fermented with gas production,


sucrose and/or lactose fermented
K/A Glucose only fermented

K/AG Glucose only fermented with gas produced

H2S + Hydrogen sulfide produced

H2S -- Hydrogen sulfide negative

K/K No sugars fermented

K/H2S Lactose and sucrose not fermented


H2/S production (black butt) is all that can be
seen

A/ H2S Lactose and/or sucrose fermented


H2/S production (black butt) is all that can be
seen

53
Procedure

Organisms used
Pseudomonas aeruginosa
E. coli
Proteus vulgaris

1. Students work in groups to complete this exercise

2. Label a TSI slant with one of the organisms to be tested.

3. All tests in this media rely on anaerobic conditions. To provide this the
organism must be introduced into the media, not on the surface. Using a
sterile inoculating needle , touch the organism to be tested and stab the
TSI media penetrating to the bottom of the tube. When removing the
needle, streak the slant.

4. Incubate all tubes for at least 18 hours at 35-37o C.

5. After incubation examine the tubes for color changes. Record all results in
the table below.

6. When finished examining the tubes, remove all labels and markings and
place in the designated contaminated area.

Organism Fermentation H2S Production


Pseudomonas
aeruginosa
E. coli

Proteus vulgaris

54
Review Questions

Why is the media stabbed when inoculating it?

Why does the slant turn red if only glucose is fermented?

Which organism(s) produced gas? How could you tell?

Which organism(s) produced H2S? How could you tell?

What is the interpretation of the results for Pseudomonas aeruginosa?

What is the interpretation of the results for E. coli?

What is the interpretation of the results for Proteus vulgaris?

Do the TSI fermentation results match the oxidase results?

55
Identification of Gram Negative Bacilli—Motility
Introduction

Procaryotic cells (bacteria) have a single strand of protein for a flagellum.


The flagellum is the only organelle for motility in the procaryotic cell. Eucaryotic
cells may move by using flagella, cilia, or pseudopods. Motility in bacteria
indicates that the organism has flagella.

Principle

Bacterial flagella may be stained using a special flagellar stain to


demonstrate their presence. This procedure is somewhat tedious. An alternate
way to show bacteria have flagella is to demonstrate their ability to move. Since
the only organelle for motility in bacteria is the flagellum, movement indicates the
presence of flagella. Media that contains half the agar content as usual is semi-
solid. It does not pour but is too soft to produce a slant. This consistency will
allow bacteria to swim through the agar from the initial inoculation point if they
posses flagella. Cells will be distributed along the migration route and will cause
the media to become cloudy so the trail will be visible. If a tetrazolium salt
(triphenyltetrazolium chloride or TTC) is added to the medium the bacterial
presence in the media will be much easier to see. TTC is colorless and soluble
in the oxidized form but becomes insoluble and turns red when reduced. Since
any metabolic process involves the oxidation of molecules to produce energy, the
dye is readily reduced by microbial growth and other microbial activities, such as
motility. When TTC is present in the media the cloudy trail left by bacteria
swimming through the media is red. The semi-solid agar technique is the most
common test for motility in the clinical laboratory.
Motility may also be demonstrated using the hanging drop method. A
drop containing bacteria is suspended from a cover slip using a depression slide.
The slide is then examined to see if the organisms are moving directionally for a
distance of 2-3 cell lengths. This is considered true motility. Due to the size of
the bacterial cell it is possible to see Brownian movement if the cells are non-
motile. This movement is the result of molecular bombardment of the cells and is
not due to the presence of flagella.

56
Procedure

Organisms used
E. coli
Klebsiella pneumoniae
Enterobacter cloacae

1. Students work in groups to complete this exercise.

2. Obtain a tube of motility media. Using a sterile inoculating needle


inoculate the motility media by stabbing halfway into the agar.

3. Incubate for a minimum of 18 hours at 35o C.

4. After incubation, examine the initial stab line. If the line is sharp the
organisms did not move and there is no motility. If the line is fuzzy, shows
a cloud of growth around it, or is indistinct in any way the organism was
able to move away from the initial stab line and is motile.

5. Record all results in the table below. Remove all marks from the tube and
discard in the designated area.

6. OPTIONAL: If available, observe the demonstration of the hanging drop


technique.

Organism E. coli Klebsiella Enterobacter


pneumoniae cloacae

Results

Review Questions

What is Brownian movement? Is it motility?

What organelle(s) for motility do bacteria posses?

List three methods that may be used to demonstrate flagella in bacteria.

57
Identification of Gram Negative Bacilli—IMViC
Introduction

IMViC is a mnemonic to remember the four biochemical tests being used:


Indole, Methyl red, Voges-Proskauer, and Citrate. These four tests help divide
the Enterobacteriaceae into two major groups—the E. coli group and the
Enterobacter-Klebsiella group.

Principle

Indole

Organisms that posses the enzyme tryptophanase can break down the
amino acid tryptophan to indole. When indole reacts with para-dimethyl-
aminobenzaldehye (Kovac’s reagent) a pink -colored complex is produced.
Tryptophan is plentiful in most media, but growth on blood agar or chocolate agar
produces the best effects.

Methyl Red

Some organisms produce acid from the metabolism of glucose in a


sufficient quantity to produce a pH of 4.4 in the media. These acids are not
further metabolized and are said to be stable acids. At a pH of 4.4 or less the pH
indicator methyl red is a bright cherry red.

Voges-Proskauer

Some organisms initially produce acid from glucose metabolism but


further metabolize the acid produced to neutral end products, such as acetoin,
and 2,3-butanediol. Initially the pH may drop to 4.4 but the neutral end products
raise the pH so the methyl red test will be negative. Acetoin and 2,3 -butanediol
under alkaline conditions will react with alpha-naphthol (1-naphthol) to produce a
mahogany red color.

Citrate

Citrate contains carbon. If an organism can use citrate as its only source
of carbon the citrate in the media will be metabolized. Bromthymol blue is
incorporated into the media as an indicator. Under alkaline conditions this
indicator turns from green to blue. The utilization of citrate in the media releases
alkaline bicarbonate ions that cause the media pH to increase above 7.4.

58
Procedure
Organisms used
E. coli
Klebsiella pneumoniae
Enterobacter cloacae

1. Students work in groups to complete this exercise.

2. Obtain a DrySlide indole test card. Using a sterile loop transfer cells from
an agar plate or slant to the test area on the card. Observe for the
development of a pink color within 30 seconds. Record your results in the
table provided (Page 60) and discard the test card in the biohazard
container.

3. Obtain an MRVP broth and using aseptic technique inoculate the tube. It
is important to inoculate this test heavily. Incubate for at least 24 hours at
35o C.

4. Obtain a citrate slant. Aseptically inoculate the slant and incubate for at
least 24 hours at 35o C.

5. After incubation of the MRVP broth obtain a spot plate and a sterile
dropper. Observe the MRVP for turbidity. If turbidity is not noted the test
results are not reliable. The tube may be re-incubated until growth is
evident. Place 3 drops of turbid broth into two of the wells on the spot
plate.
Methyl Red Test—To one well add 1-2 drops of methyl red reagent.
Observe for an immediate cherry red color that indicates a positive test.
Orange or yellow is considered negative. Record your results in the table
provided (Page 60).
Voges-Proskauer Test—To the remaining well add 2 drops of alpha-
naphthol and 1 drop of potassium hydroxide (KOH). Observe for the
development of a mahogany red color. The color development takes 20
minutes or longer. Be extremely careful with the KOH. It is caustic and
may cause burns if it gets on your skin. The mahogany red color is
considered positive. Record your results in the table provided (Page 60).
Remove all marks from the MRVP tube and discard in the designated
area. Clean the spot plate by covering the surface with disinfectant. Allow
the disinfectant to sit for a few minutes, then rinse with water, wash and
dry. Return the spot plates to their original location.

6. Observe the citrate for a change from green to blue. Blue is considered
positive for this test. Record your results in the table provided (Page 60).
Remove all marks from the tube and discard in the designated area.

59
Organism Indole Methyl Red Voges- Citrate
Proskauer
E. coli

Enterobacter
cloacae
Klebsiella
pneumoniae

Review Questions

What is the IMViC pattern for the E. coli group?

What is the IMViC pattern for the Enterobacter-Klebsiella group?

What is the difference between a reagent and an indicator?

Complete the following table.

Test Substrate Reagent Indicator Positive


Result
Indole
Methyl Red
Voges-
Proskauer
Citrate

Is it possible for an organism to be Methyl red and Voges-Proskauer positive?


Explain your answer.

What might happen if the MRVP tests are read too soon?

60
Disc Diffusion Susceptibility Methods
Introduction

When a filter paper disc impregnated with a chemical is placed on agar


the chemical will diffuse from the disc into the agar. This diffusion will place the
chemical in the agar only around the disc. The solubility of the chemical and its
molecular size will determine the size of the area of chemical infiltration around
the disc. If an organism is placed on the agar it will not grow in the area around
the disc if it is susceptible to the chemical. This area of no growth around the
disc is known as a “zone of inhibition”.

Principle

Antiseptics, disinfectants and antibiotics are used in different ways to


combat microbial growth. Antiseptics are used on living tissue to remove
pathogens. Disinfectants are similar in use but are used on inanimate objects.
Antibiotics are substances produced by living organisms, such as Penicillium or
Bacillus, that kill or inhibit the growth of other organisms, primarily bacteria.
Many antibiotics are chemically altered to reduce toxicity, increase solubility, or
give them some other desirable characteristic that they lack in their natural form.
Other substances have been developed from plants or dyes and are used like
antibiotics. A better term for these substances is antimicrobials, but the term
antibiotic is widely used to mean all types of antimicrobial chemotherapy.

Many conditions can affect a disc diffusion susceptibility test. When


performing these tests certain things are held constant so only the size of the
zone of inhibition is variable. Conditions that must be constant from test to test
include the agar used, the amount of organism used, the concentration of
chemical used, and incubation conditions (time, temperature, and atmosphere).
The amount of organism used is standardized using a turbidity standard. This
may be a visual approximation using a McFarland standard 0.5 or turbidity may
be determined by using a spectrophotometer (optical density of 1.0 at 600 nm).
For antibiotic susceptibility testing the antibiotic concentrations are
predetermined and commercially available. Each test method has a prescribed
media to be used and incubation is to be at 35-37o C in ambient air for 18-24
hours.

The disc diffusion method for antibiotic susceptibility testing is the Kirby-
Bauer method. The agar used is Meuller-Hinton agar that is rigorously tested for
composition and pH. Further the depth of the agar in the plate is a factor to be
considered in the disc diffusion method. This method is well documented and
standard zones of inhibition have been determined for susceptible and resistant
values. There is also a zone of intermediate resistance indicating that some
inhibition occurs using this antimicrobial but it may not be sufficient inhibition to
eradicate the organism from the body.

61
The standardized methods for antiseptic and disinfectant testing are more
rigorous and more difficult to reproduce in a student laboratory. Two common
tests are the Phenol Coefficient Test (a comparison of the effect of the chemical
and phenol on several organisms) and the Use Dilution Test (testing the
chemical under actual conditions of use). A disc diffusion test can be used to
approximate the Use Dilution Test. The chemical under consideration is used to
saturate a filter paper disc. This disc is then used to introduce the chemical to
the agar for testing. The actual zo ne sizes have not been standardized as in the
Kirby-Bauer method, but a comparison of zone sizes for the same chemical
among organisms will provide an approximate effectiveness of the chemical.

Procedure

Kirby-Bauer Antimicrobial Susceptibility Test

Organisms to be tested:
Staphylococcus aureus
E. coli

Procedure
1. Students will work independently in the laboratory exercise.

2. Obtain a plate culture of one of the organisms to be tested.

3. Using a sterile loop, emulsify a colony from the plate in the sterile saline
solution. Mix thoroughly making sure that no solid material from the colony is
visible.

4. Repeat this procedure until the turbidity of the saline solution matches that of
the standard available for your class.

5. Dip the swab into the broth culture of the organism. Gently squeeze the swab
against the inside of the tube to remove excess fluid. Use the swab to streak
a Mueller-Hinton agar plate or a nutrient agar plate for a lawn of growth. This
is best accomplished by streaking the plate in one direction, then streaking at
right angles to the first streaking, and finally streaking diagonally. End by
using the swab to streak the outside diameter of the agar.

6. Allow the plates to dry for about 5 minutes.

62
7. Antibiotic disks can be placed on the surface of the agar using a dispenser
that dispenses multiple disks at the correct distance apart, or by obtaining
individual disks and placing them on the surface of the agar using flame
sterilized forceps.

a. Dispenser method:
1. Obtain the dispenser containing the correct antibiotic disks for the
organism you are using.

2. Place the dispenser over the surface of the plate and using the
lever/plunger dispense the disks.

3. Using sterile forceps or a loop, gently press the disks onto the
surface of the agar, taking care not to press them into the agar.

b. Dispensing individual disks:


1. Obtain 6 of the appropriate individual disk dispensers.

2. Using the levers, dispense the disks at equal distances apart on the
surface of the agar.

3. Using flame sterilize forceps or a sterile loop gently press the disks
onto the surface of the agar.

4. 6 disks may also be individually placed onto the surface of the agar
using sterile forceps.

8. Invert the plates and incubate for 24 hours at 37° C.

9. Using a metric ruler measure the diameter of the zone of inhibition (if present)
for each antibiotic used.

10. Compare the measurement obtained from the individual antibiotics to the
table of standards to determine if the bacterial species tested is resistant or
sensitive to the antibiotic.

11. Use the data you collected and that of the rest of the class to fill in the table
below. Discard the plates in the biohazard container.

Antibiotic
Staph.
aureus
E. coli

63
Zone Diameter (mm) Interpretation Chart
Antibiotic Resistant Intermediate Susceptible
Tetracycline = 14 15-18 = 19
Ciprofloxacin = 15 16-20 = 21
Enoxacin = 14 15-17 = 18
Erythromycin = 13 14-22 = 23
Penicillin
Staphylococci = 28 = 29
Oxacillin
Staphylococci = 10 11-12 = 13
Tobramycin = 12 13-14 = 15
Ceftriaxone = 13 14-20 = 21
Kanamycin = 13 14-17 = 18
Clindamycin = 14 15-20 = 21
Piperacillin
Gram negatives = 17 18-20 = 21
Ampicillin
Gram negative enterics = 13 14-16 = 17
Staphylococci = 28 = 29

64
Antiseptic/Disinfectant Susceptibility Test

Organisms used
Staphylococcus aureus
E. coli
Bacillus cereus
Pseudomonas aeruginosa

1. Students work individually on this laboratory exercise.

2. Obtain one of the organisms to be tested, 5 nutrient agar plates, and a


sterile swab.

3. Dip the swab into the broth culture of the organism. Gently squeeze the
swab against the inside of the tube to remove excess fluid. Use the swab
to streak a nutrient agar plate for a lawn of growth. This is best
accomplished by streaking the plate in one direction, then streaking at
right angles to the first streaking, and finally streaking diagonally. End by
using the swab to streak the outside diameter of the agar. Repeat this
procedure for the remaining plates.

4. Place a disc soaked in an antiseptic or disinfectant in the center of each


plate. Be sure to label the plates with the organism and chemical used.

5. Incubate the plates in the standard upside down position until the next lab
period.

6. Measure the diameter of the zone of inhibition for each chemical. The
class will share data so you can fill in the table provided.

7. Discard the plates in the biohazard container.

Chemical

Staph.
aureus
E. coli

Bacillus
cereus
Ps.
aeruginosa

65
Review Questions

What conditions must be held constant when doing disc diffusion procedures?

Define

Antiseptic

Disinfectant

Antibiotic

Zone of inhibition

According to your results, which chemical is the most effective? On what do you
base this conclusion?

What are the standard tests used for determining the e ffectiveness of antiseptics
and disinfectants?

What is the standard method used for antimicrobial susceptibility testing?

66
Guidelines for the Identification of Unknown Organisms

Unknown organisms will be grown on 5% Sheep blood agar or Nutrient agar and
will be distributed as streak plates with isolated colonies. Each plate will
represent a pure culture.

Each student is responsible for doing their own work but may ask other students
for opinions, advice, or instructions on what to do.

The Lab Instruc tor may provide assistance on all but the last two unknowns.
(Unknown 3 and Unknown 4)

On any of the unknowns, students are allowed to refer to class notes, texts, and
any other source of information they may have developed during the course.

An Unknown Report Form will be provided for each unknown. Students may
have only one form for each unknown. The form must be filled out neatly and
completely using blue or black ink. Spelling must be correct and all test notations
must be appropriate. This report should be treated as though it were a part of a
patient chart. Erasures, liquid paper, and any obliteration of information recorded
on the form are not allowed (loss of points). Should errors occur the student
must make a single mark through the error, initial, date, and report the corrected
result.

+ fd m/d/y ?

The steps for identifying an unknown are


• Gram stain
• Colonial morphology
• Biochemical testing

Only the biochemical tests necessary for identification of the organism are
appropriate. Unnecessary tests will result in a deduction of points from the final
grade. Tests are to be ordered from the lab instructor using the appropriate form.
This form must also be filled out completely. A separate order form is used for
each unknown. More than one order form may be used for one unknown. If a
test is ordered the results must appear on the report form or a brief explanation
as to why the test was not done should be made. Failure to do so will result in a
loss of points from the final grade.

Gram staining and initial spot testing will give the student a general idea
concerning the identity of the unknown. Only tests useful for identifying the
suspected organism should be performed. A sufficient number of tests should be
performed so that the organism may be identified at the next laboratory session.

67
Test results must be reported appropriately. Failure to do so may result in
a loss of points from the final grade. Most results can be reported with a + or -.
That is sufficient. A detailed description of the results is inappropriate.

The identity of the unknown should be written appropriately. The genus


name should be capitalized but not the species name. If you are in the habit of
printing in all upper case letters, be sure to differentiate the first letter of the
genus name as larger than the others. Failure to do so will lead to a loss of
points on the final grade.

Below is an example of the test request form. The top should be


completely filled out and the appropriate tests checked. This will be retained by
the Laboratory Instructor and attached to your completed Unknown Report Form.
It is considered in grading your unknown.

MCB 1000L
Media/Test Request Form

Name___________________________________Date___________________Unknown Number________

Requested Media/Test Materials

Coagulase_______________ TSI_________________

Mannitol Salt____________ Urea________________

Novobiocin_______________ Indole_______________

Optochin________________ MRVP______________

Bacitracin_______________ Citrate______________

CAMP__________________ Motility_____________

High Salt________________

Bile Esculin______________

68
Name____________________________

Date Started_______________________

MCB 1000L
Unknown Report Form

Unknown Number______________

Gram Stain Results______________ Date________________

Colony Morphology (include media used) Date________________

Test Performed Results Date

Identity of Unknown___________________________________________

Date completed__________________

69
References

Howard, Barbara J. (ed.) 1987. Clinical and pathogenic microbiology,


2nd ed. Mosby, Baltimore.

Finegold, Sidney M., William J. Martin, and Elvyn G. Scott. 1978.


Diagnostic microbiology, 5 th ed. Mosby, Baltimore.

BBL TM DrySlideTM Indole. 1998. Technical Insert, Becton Dickinson


Microbiology Systems, Sparks, Maryland.

BBL ® Oxidase. 1995. Technical Insert, Becton Dickinson


Microbiology Systems, Sparks, Maryland.

70