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Applied Microbiology
Laboratory Manual
By
Frances Duncan
With contributions from
Valerie Walker and Neil Clark
Fourth Edition 2005
Table of Contents
Safety 3
Aseptic Technique 8
Microscopy 11
Smears 17
Staining 20
Staphylococcus 33
Streptococcus 38
Throat Culture 45
Oxidase 48
Urea 50
Motility 56
IMViC 58
References 70
2
Safety
Introduction
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.
3
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
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.
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.
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_________________
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
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.
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.
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_________________
5
Safety Review Questions
6
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
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.
8
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.
9
Conclusions
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.
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
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
11
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.
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.
12
Procedure for Focusing
1. Obtain a slide.
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.
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.
13
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.
14
Review Questions
Define:
Resolving power
Parfocal
Field
Working distance
Iris diaphragm
16
Smears
Introduction
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
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.
17
Materials/Equipment
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.
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.
8. Heat fix the slide by passing it 10 times over the top of the Bacticinerator.
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.
18
Smear Review Questions
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.
8. When removing a lid from a lid or a bottle using aseptic technique, what do you
do with the lid?
19
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.
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.
20
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.
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
2. Place the slide on the staining rack and flood the slide with stain for 1 minute.
21
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
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.
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.
22
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
23
Review Questions
What are the differences between a simple stain and a differential stain?
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?
24
Culturing and Isolation Techniques
Introduction
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
ELEVATION
________________________________________________________
Flat Convex Umbonate
MARGIN
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
Materials
Mixed Culture in broth
Inoculating loop
Bacticinerator
Incubator
Sterile nutrient broth
1. Label the sterile nutrient broth with the source of the culture and your initials.
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.
6. Observe broth for turbidity. Record results in table at end of the procedure
section.
Materials
Mixed Culture in broth
Inoculating loop
Bacticinerator
Incubator
Sterile nutrient agar slant
1. Label the sterile nutrient agar slant with the source of the culture and your
initials.
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
1. Label the sterile nutrient agar plate with the source of the culture and your
initials.
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
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.
30
Review Questions
How many colony types did you observe on your streak 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?
32
Identification of Gram Positive Cocci
Staphylococcus
Introduction
Principle
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
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.
Procedure
Catalase
2. Touch a sterile loop to a culture of the organism to be tested and pick up a visible
mass of cells.
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.
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.
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.
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
3. Using a sterile loop transfer the test organism to the plate and streak the section
for confluent growth.
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).
LABORATORY INSTRUCTIONS
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.
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.
7. As time permits, Gram stain the smear prepared in Step 1 (Page 22).
36
MCB 1000L
Identification of Staphylococcus
Coagulase
Growth on mannitol
salt
Mannitol
fermentation
Novobiocin
susceptibility
Which test differentiates Staph. aureus from the other species of Staphylococcus?
37
Identification of Gram Positive Cocci
Streptococcus
Introduction
The streptococci are classified by two major methods: hemolytic activity and
serologic classification of Lancefield.
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.
38
Classification Based on Lancefield Proteins
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.
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.
3. Using a sterile loop aseptically transfer the test organism to the plate and streak
the quadrant for confluent growth.
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
3. Using a sterile loop aseptically transfer the test organism to the plate and streak
the quadrant for confluent growth.
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.
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.
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.
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.
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
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
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.
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.
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
45
replacing the lid. As the candle burns, some of the oxygen in the jar is converted
to carbon dioxide.
Procedure
1. Obtain a sheep blood agar plate, sterile swab and tongue depressor.
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.
46
Review Questions
6. Define microaerophile.
Colony 2
47
Identification of Gram Negative Bacilli—Oxidase
Introduction
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
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.
48
Review Questions
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
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
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.
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.
50
Review questions
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
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.
Report Interpretation
53
Procedure
Organisms used
Pseudomonas aeruginosa
E. coli
Proteus vulgaris
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.
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.
Proteus vulgaris
54
Review Questions
55
Identification of Gram Negative Bacilli—Motility
Introduction
Principle
56
Procedure
Organisms used
E. coli
Klebsiella pneumoniae
Enterobacter cloacae
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.
Results
Review Questions
57
Identification of Gram Negative Bacilli—IMViC
Introduction
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
Voges-Proskauer
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
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 might happen if the MRVP tests are read too soon?
60
Disc Diffusion Susceptibility Methods
Introduction
Principle
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
Organisms to be tested:
Staphylococcus aureus
E. coli
Procedure
1. Students will work independently in the laboratory exercise.
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.
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.
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.
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
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.
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.
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?
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 ?
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.
MCB 1000L
Media/Test Request Form
Name___________________________________Date___________________Unknown Number________
Coagulase_______________ TSI_________________
Novobiocin_______________ Indole_______________
Optochin________________ MRVP______________
Bacitracin_______________ Citrate______________
CAMP__________________ Motility_____________
High Salt________________
Bile Esculin______________
68
Name____________________________
Date Started_______________________
MCB 1000L
Unknown Report Form
Unknown Number______________
Identity of Unknown___________________________________________
Date completed__________________
69
References
70