Name:
Section:
Introductory Microbiology
Prelab Assignment
Preparing Unknown Cultures: Morphological Characteristics
1. (1pt.) What is the purpose of preparing a reserve stock?
2. (1pt.) When will you use your working stock?
3. (1pt.) Where will the working stock and reserve stock be stored between lab sessions?
4. (1pt.) List two examples of morphological characteristics of bacteria.
5. (1pt.) Write the appropriate color for each cell type after the following reagents are applied.
Reagent
Gram positive
Gram negative
Smears before treatment
Crystal violet
Gram's iodine
Ethyl alcohol
Safranin
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Lab Exercise
Preparing Unknown Cultures: Morphological Characteristics
Unknown number
10/13/14
Today you will begin to accumulate information that will lead to the identification of an unknown
bacterial species. Identifying unknown samples is an essential endeavor in clinical laboratories in order to
determine an appropriate method of treatment or eradication. In order to identify the scientific name of your
unknown, various tests will be performed over the next two weeks. First the morphological traits will be examined.
Morphological characteristics include most of the cell's external traits: staining characteristics, motility, cultural
characteristics, and cell shape. Next, several of the physiological abilities of the unknown will be determined.
Physiological characteristics refer to the chemical reactions a certain species possibly can perform.
I. Unknown Cultures.
A slant culture will be provided for you and your partner. You must not contaminate your unknown
cultures! Your expertise in aseptic technique is part of your grade in that if your cultures become contaminated,
you may have to proceed with testing one of the species in your culture. You may not choose the correct species.
Therefore, your fundamental concern is that the cultures do not become contaminated.
You will require two cultures, a reserve stock and a working stock. Both are cultured on agar slants.
All tests and inoculations are performed from the working stock, the original slant given to you. Between
experiments, the working stock is stored in the refrigerator to prevent overgrowth. The first step is to subculture
from the working stock into a sterile slant that will become your reserve stock. After incubation, the reserve stock
also is stored in the refrigerator, but is not used for tests. If for some reason your working stock becomes unusable,
there is a backup culture. If this occurs, the reserve stock becomes your working stock, and you must prepare a
new reserve stock.
II. Determining the scientific name of your unknown
Each student must research the characteristics of several species of bacteria to determine the identity of
the unknown. Previously, it was very difficult to classify, and, therefore, identify, bacteria because of the diversity
of bacterial characteristics and the lack of available fossil information. Today, however, several resources are
available to determine the correct species. Bergey's Manual of Systematic Bacteriology is the most
comprehensive source of information concerning the characteristics, phylogeny (evolution), and identification of
bacteria. This manual is divided into several volumes. The first edition of the Bergey’s Manual grouped bacteria
into Sections based primarily on general characteristics. The second edition of Bergey's Manual (2001) consists of
several volumes arranged by Domain (Archaea or Bacteria), then phyla, classes, orders, families, genera and
species. This hierarchy is based on genetic analysis. A smaller version, called Bergey’s Manual of Determinative
Bacteriology also is found in the lab. Some groups may find it informative.
The first step is to determine the unknown’s genus by skimming through Bergey's table of contents. Find
all of the possible unknowns. As you accumulate your unknown’s traits through the following tests, begin to
narrow down your choices. Remember, your results may not be identical to the characteristics found in the manual.
Often, the manual itself will list characteristics as occurring "usually", "generally", etc. It is primarily a process of
eliminating those species most different from yours.
Another helpful resource is the Difco Manual also on reserve in the HCC library. The Difco Manual
details materials and procedures for producing media and reagents utilized in microbiology. Of greater interest,
however, the manual explains the purpose of the medium and test results for various genera and species. Refer to
the index for each type of medium and reagent.
Finally, students may use online sites and the microbiology textbooks and manuals located in the lab or
the library.
NEVER WRITE IN ANY BOOK IN THE LIBRARY OR LAB!!!!!!!!
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III. Possible Unknowns
Possible Unknown
Bacillus megaterium
Sporolactobacillus inulinus
Mycobacterium smegmatis
Staphylococcus aureus
Staphylococcus epidermidis
Streptococcus sanguis
Micrococcus luteus
Escherichia coli
Enterobacter aerogenes
Proteus vulgaris
Pseudomonas aeruginosa
Bergey’s, Edition One
Vol. 2, Section 13
Vol. 2, Section 13
Vol. 2, Section 16
Vol. 2, Section 12
Vol. 2, Section 12
Vol. 2, Section 12
Vol. 2, Section 12
Vol. 1, Section 5
Vol. 1, Section 5
Vol. 1, Section 5
Vol. 1, Section 4
Bergey’s, Edition Two
Vol. 3
Vol. 3
Vol. 4
Vol. 3
Vol. 3
Vol. 3
Vol. 4
Vol. 2
Vol. 2
Vol. 2
Vol. 2
IV. Assignment
In this exercise several of the morphological characteristics of the unknown are determined (overview of the
procedures, p. 4). The most important morphological characteristics to determine are the Gram stain and cell shape
since they will establish the specific physiological tests to perform in the next lab.
After completing each test, record your results in the lab report and the Unknown Characteristics Chart.
Next, look up all of the possible unknowns listed above in Bergey’s Manual of Determinative Bacteriology. In the
Unknown Characteristics Chart, record the morphological characteristics of ALL of the possible unknowns. In this
way, you can begin eliminating those that do not resemble your unknown.
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Procedure Overview
Original Working Stock.
All inoculations will be performed
from this tube.
2.
1.
TSA Plate.
Reserve Stock.
Subculture on the TSA
plate using a quadrant
streak. Incubate 48 hr.
Using an inoculating needle,
subculture into this tube by
making a single, straight
inoculation from the bottom of
the slant to the top. Incubate 48
hr.
3.
Perform the spore test. Boil a
heavy inoculation in broth, then
plate on a TSA plate. Incubate 48
hr.
6.
On Day 2, make observations of
cultural characteristics on the
A) Reserve Stock tube and B) TSA
quadrant streak plate.
5.
4.
Perform a Gram stain using the
instructions on the following
pages.
Using an inoculating needle, perform the
motility test using semisolid motility media.
Incubate 48 hr.
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Materials (per group of 2)
Safety glasses, aprons, gloves
Unknown working stock
1 trypticase soy agar slant
2 trypticase soy agar plates
1 tube of nutrient broth
1 tube of semisolid motility media
Inoculating loop
Inoculating needle
Bunsen burner and striker
Additional Gram Stain materials:
Distilled water
Gram staining kit: crystal violet, Gram's iodine, ethyl alcohol, safranin, wash-bottle with distilled water
Clothespin
Kimwipes
Microscope slide(s)
Coverslips
Additional Spore Test materials:
500 ml beaker
Hot plate
Test tube tongs
Bactispreader
95% ethyl alcohol in glass screw top jars
Step 1: Prepare the reserve stock as instructed on the previous page (overview). Label tube with your initials and
unknown number using masking tape. Incubate for 48 hr.
Step 2: Quadrant streak the TSA plate. Label the plate with your initials, the unknown number, and ‘quadrant
streak’. Incubate for 48 hr.
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Step 3: Spore test
Endospores resist harmful environmental conditions such as high temperatures, chemicals, and
desiccation. Bacteria that form endospores include the genera Bacillus, Sporolactobacillus, and Clostridium. The
Gram stain will not penetrate the endospore. Therefore, after Gram staining cells containing endospores, a clear
area inside of the cell may be seen. Survival in the presence of high temperatures also may be an indication of the
presence of endospores.
Materials:
500 ml beaker
hot plate
test tube tong
thermometer
1. Fill the beaker with 250 ml of tap water.
2. Bring the water between 70-80C.
3. Heavily inoculate a nutrient broth tube with your unknown.
4. Place the tube in the boiling water and boil for 10 minutes. Allow to cool.
5. Pipet 1.0 ml to the center of the TSA plate and spread evenly with the bactispreader.
6. Incubate for 48 hours and observe for growth.
-Spore-former = growth present
-Non-spore-former = no growth
Positive control: Bacillus subtilis
Step 4: Perform a motility test.
Materials:
Semisolid motility media
Straight inoculating needle.
Motile bacteria contain one or more flagella that rotate to move the cell. The type of motility test you will
perform is extremely valuable when working with pathogens because there is less contact with the organisms than
with slide techniques. Motility agar is a semisolid so if the bacteria have flagella, the cells are able to move
through the medium, forming a cloud of growth away from the site of inoculation.
Procedure:
First be sure that the needle is VERY STRAIGHT. IT MUST NOT BE BENT.
1. Gently touch your working stock cells with a flamed and cooled needle. Stab the needle into the semisolid
media, in the center, approximately 3/4 of the way down. Pull the needle out in the same hole you went in. DO
NOT MOVE THE NEEDLE AROUND IN THE AGAR! YOU NEED A STRAIGHT STAB TO OBTAIN
GOOD RESULTS!
2. Incubate at 37C for 48 hours.
3. Observe results on Day 2 after incubation. Compare your results with the positive control, Proteus vulgaris.
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Step 5: Perform a Gram stain from your working stock.
Gram staining kit: crystal violet, Gram's iodine, ethyl alcohol, safranin, wash-bottle with distilled water
When staining, do not touch the slide or the bacteria with the dropper. Wear an apron.
1. Smear preparation: Follow the procedure in the Widespread Distribution of Bacteria Lab Exercise. The
smear must not be too thick.
2. While holding the slide over the sink, cover the smear with crystal violet. Leave the crystal violet on
the smear for 60 seconds.
3. Utilizing the wash-bottle of distilled water, wash off the stain for 7 seconds. Pour the water on one
end of the slide, and let it flow over the specimen. Do not pour the water directly on the specimen. Pour
off excess water.
4. Cover the smear with Gram's iodine. Leave the Gram's iodine on the smear for one minute.
5. Utilizing the wash-bottle of distilled water, wash off the stain for 10 seconds. Pour the water on one
end of the slide, and let it flow over the specimen. Do not pour the water directly on the specimen. Pour
off excess water.
6. Pour 95% ethyl alcohol (the decolorizer) over the smear until decolorization has occurred (i.e., when
the solution flowing off of the slide is colorless; ~10 seconds).
7. Utilizing the wash bottle of distilled water, wash off the smear for 2 seconds. Pour the water on one
end of the slide, and let it flow over the specimen. Do not pour the water directly on the specimen. Pour off
excess water.
8. Counter-stain with safranin. Leave the safranin on the smear for 50 seconds.
9. Wash with distilled water for 5 seconds.
10. Gently blot dry with Kimwipes, and dry at room temperature. Place a coverslip on the specimen, and
view the cells under the microscope.
*In addition to cell shape and color after Gram staining, look for clear areas inside of the cells.
Since the stains may not penetrate an endospore, clear areas are an indication of the presence of
endospores.
WHEN YOU HAVE FINISHED USING THE MICROSCOPE, COMPLETE THE
STUDENT MICROSCOPE CHECKLIST. COMPLETE ALL STEPS.
TURN THE CHECKLIST IN TO YOUR INSTRUCTOR.
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Color of Gram positive and Gram negative cells at each stage:
Gram positive
Gram negative
After smear preparation
clear
clear
After application of crystal
violet
purple
purple
After application of Gram's
iodine
purple
purple
After application of ethyl
alcohol
purple
clear
After application of safranin
purple
red/pink
Function of each reagent:
Reagent
Function
Crystal violet
Primary stain (first stain used); both cell types stained purple
Gram's iodine
Mordant: combines with crystal violet. This combination is insoluble in Gram positive cell walls (i.e.,
it cannot be decolorized with ethyl alcohol). It is soluble in Gram negative cell walls (i.e., it can be
decolorized with ethyl alcohol).
Ethyl alcohol
Decolorizer: removes purple crystal violet from Gram negative cell walls but not Gram positive.
Safranin
Counterstain (second stain used): adds color to Gram negative bacteria. The color of Gram positive
bacteria remains purple with crystal violet.
Gram positive bacteria retain the purple color after application of the decolorizing agent (ethyl
alcohol) primarily because of the many peptidoglycan layers in the cell wall. Once the Gram's
iodine and the crystal violet combine, they cannot be removed easily from the peptidoglycan in
the cell wall. Gram negative bacteria have relatively few layers of peptidoglycan so they readily
can be decolorized.
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Step 6: After incubation, use a magnifying lens to observe the cultural characteristics on the A) Reserve Stock
slant and B) TSA quadrant streak.
Macroscopic traits such a colony shape, size and color also are utilized to identify bacterial species.
Several types of media are commonly used. You will restrict your observations to colony characteristics on an agar
slant (your reserve stock) and an agar plate. Since both the slant and the plate are inoculated and incubated on Day
1 of the lab, observations will be made on Day 2.
See page 11 for illustrations of each characteristic.
A. Reserve stock slant
Day 2 Procedure:
1. Use your reserve stock after incubation in order to make colony characteristic observations on a TSA slant.
2. DO NOT TAKE THE CAP OFF OF THE TEST TUBE. You may want to wipe the outer surfaces of the tube
with a Kimwipe.
3. Determine the following characteristics (see illustrations).
1) Abundance of growth: none, slight, moderate, or large
2) Pigmentation: color of growth
3) Optical characteristics:
a. Transparent: completely clear, full transmission of light through the growth
b. Translucent: partial transmission of light through the growth
c. Opaque: no transmission of light through the growth, solid
4) Form: appearance of the single-line streak of growth
a. Filiform: continuous, threadlike growth with smooth edges
b. Echinulate: continuous, threadlike growth with irregular edges
c. Beaded: noncontinuous to semicontinuous colonies
d. Effuse: thin, spreading growth
e. Arborescent: treelike growth
f. Rhizoid: rootlike growth
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B. TSA plate
Procedure:
1. After incubation (Day 2), determine the following colony characteristics (see illustrations page 11).
1) Size of colonies: pinpoint, small, moderate, or large
2) Pigmentation: color of colonies
Most colonies are white, off-white, yellow, red, gray.
3) Form: the overall shape of the colonies (see handout)
a. Punctiform: small round with an unbroken peripheral edge
b. Circular: almost perfectly round, unbroken peripheral edge
c. Irregular: Indented peripheral edge
d. Spindle: elongated oval shape
e. Rhizoid: root-like spreading growth
f. Filamentous: root-like spreading growth, often has more and thinner filaments than a rhizoid
colony
4) Margin: the appearance of the outer edge of the colonies
a. Entire: smooth, even edge
b. Lobate: highly indented irregular edges
c. Erose: irregular indented edges, smaller indents than lobate
d. Undulate: regular wavy indentations
e. Serrate: tooth-like edges
f. Filamentous: threadlike, spreading edges
g. Curled: wavy edges with a layered appearance
5) Elevation: the degree to which the colonies are raised on the agar surface. Hold the plate at eye level to
make these observations.
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Name:
Section:
Unknown number:
Introductory Microbiology
LAB REPORT
Preparing Unknown Cultures:
Morphological Characteristics
1. Your results.
Gram stain
Cell shape
Motility
Spore formation
Cultural characteristics
TSA slant:
1) Abundance of growth
2) Pigmentation
3) Optical characteristics
4) Form
TSA plate:
1) Form:
2) Margins:
3) Elevation:
4) Size of colonies:
5) Pigmentation:
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2. (1pt.) Write the function of each reagent.
Reagent
Function
crystal violet
Gram's iodine
ethyl alcohol
safranin
3. (1pt.) What is the function of the endospore?
4. (1pt.) Why does ethyl alcohol decolorize Gram negative cells but will not decolorize Gram positive cells?
5. (1pt.) List another indication of the presence of endospores besides the boiling spore test.
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6. (1pt.) List the Gram stain and shape of the following possible unknowns. Write the Gram stain and shape for
each. Cross off all that do not match the Gram stain and shape of your unknown.
Bacillus megaterium
Sporolactobacillus inulinus
Mycobacterium smegmatis
Staphylococcus aureus
Staphylococcus epidermidis
Streptococcus sanguis
Micrococcus luteus
Escherichia coli
Enterobacter aerogenes
Proteus vulgaris
Pseudomonas aeruginosa
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Student Microscope Checklist
When finished using the microscope, complete the following.
Check off each step when completed.
Name:
Date:
Lab Section:
Microscope Number: HCC
Bring the stage to its lowest level.
Click the 4X objective into place.
Remove the slide and dispose of appropriately.
Clean objective lenses with swabs, and, if using oil, liquid lens cleaner.
Clean eyepieces with dry swabs.
Clean condenser lens with dry swabs.
If necessary, clean the stage with a damp Kimwipe.
Turn the light switch OFF. Do not move the microscope for a few minutes
before putting it away.
Rotate the head so that the eyepieces are facing forward (away from the arm).
Replace the dust cover.
Return the microscope to the appropriate location in the cabinet.
Check to be sure all waste is removed from the sink and floor.
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