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Basic Bacterial Culture and Identification
Mary T. Johnson, Ph.D.
Culture of Normal Flora Organisms
MATERIALS:
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Sterile cotton swab
Bacteriological loop
Nutrient Agar plates
Biohazard discard container
STUDENT DIRECTIONS:
1. Carefully swab the selected skin surface site in an area approximately 4
cm in diameter. Swab thoroughly, rotating the swab to ensure that a good
inoculum has been obtained.
2. Transfer the bacteria to the agar culture dish by touching the swab to the
agar surface in a single spot. Once you feel confident that a good
inoculum has been transferred, the swab can be discarded in the
biohazard container.
3. With the inoculating loop, proceed to streak in a second and third streak
pattern (see illustration). Briefly, isolate bacterial colonies by pulling
several streaks out from the original swab inoculum site. Use the method
shown above to avoid tearing the agar.
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4. Incubate the plate at 37oC for 24-48 hours.
5. Test the colonies that have grown by Gram staining; identify
Staphylococcus aureus colonies by testing Gram-positive cocci using a
specific latex agglutination reagent that binds to Staphylococcus aureus.
Pure Culture Techniques
Microbiologists have developed special techniques and equipment to isolate and
grow pure cultures of microorganisms that are free from contaminating forms.
This exercise is designed to give the student an introduction to these special
techniques. It is important that techniques be practiced until some degree of skill
is developed. Pure cultures are essential when identifying unknowns. During the
course of the semester, each student will be expected to develop independently
a patient-oriented case to correlate with each of 4 separate bacterial isolates. It is
important, therefore, to maintain unique isolates in pure culture to be submitted
with each case summary. Further instruction will be given during later labs.
THE STREAK PLATE TECHNIQUE
The procedure of streaking a plate with an inoculating loop is used to spread
millions of cells over the surface of a solid medium so that some individual cells
are deposited at a distance from all others. These cells grow and reproduce,
forming an isolated colony. One or more colonies will be well separated from all
others and represent a source of a pure culture. The procedure is similar to the
one used for streaking from the collection swab.
MATERIALS:
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Streak plates from the previous lab session
Nutrient agar plates (NA)
Bunsen burner
Bacteriological loop
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STUDENT DIRECTIONS:
Examine the streak plates prepared during the previous lab period and locate a
number of well-isolated colonies. Now you must transfer a portion of each colony
to a separate agar slant. To "pick" a colony you will be using an inoculating loop.
Sterilize the loop in the burner flame, let cool 3-5 seconds then touch the end of
the loop to the isolated colony, picking up the microorganisms from the colony.
Now re-cover the streak plate and pick up one NA plate. You will now be holding
the inoculating loop in your right hand and the fresh NA plate in your left (lefties
reversed).
Remove the lid from the plate, place the inoculating loop at one edge of the plate
and with a sweeping stroke, inoculate the agar using the same tri-streak method
as used for the initial isolation. Replace the lid. Flame the loop and proceed to
inoculate another plate from different colonies. Try to use colonies that are visibly
different in morphology. Incubate the plates in the 37oC incubator until the next
class period.
There is nothing difficult about picking colonies and inoculating slants, but you
must avoid contamination.
TESTS FOR SUSCEPTIBILITY TO ANTIMICROBIAL AGENTS
Bacteria generally can be grouped with respect to antibiotic susceptibility. Gram
positive bacteria are more permeable to many of the most common antibiotics,
probably because they lack the outer membrane of the Gram negatives. This
makes them more susceptible as a group to the actions of antimicrobial agents.
However, generalizations concerning susceptibility and resistance are difficult to
make because of the vast differences in bacterial species. In addition, the
emergence of new antibiotic resistant strains in particular environments makes it
increasingly important to adequately monitor resistant strains. On a global scale,
outbreaks of Shigella, E. coli and Salmonella carrying drug-resistance plasmids
have emphasized the need to monitor drug resistance within populations and to
make this information widely available.
KIRBY-BAUER DISC METHOD
One simple and commonly used assay for microbial susceptibility is the disc
diffusion or Kirby-Bauer method, which will be demonstrated. Antibioticimpregnated discs release antibiotic into the surrounding medium when placed
on the surface of solid agar. Untreated agar plates are inoculated with test
microorganisms and antibiotic discs are placed on the agar surface. Disc
diffusion is a qualitative method based on an approximation of the effect of
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antibiotic on bacterial growth on a solid medium. A zone of growth inhibition
around the antibiotic disc will occur if the organism is susceptible to the antibiotic.
As the distance from the disc increases, there is a logarithmic decrease in
antibiotic concentration. Standardized regression curves have been developed
that correlate inhibition zone size to the minimum inhibitory concentration of the
antibiotic. However, even though it is the most common test for antimicrobial
effectiveness, not all bacteria can be tested using disc diffusion.
MATERIALS:
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Bacterial cultures
Sterile loops
Antibiotic discs
Agar plates
Bunsen burner
Striker
STUDENT DIRECTIONS:
1. Light the bunsen burner and let it burn for five minutes to create a clean
work environment.
2. With an inoculating loop, pick one colony from the plate you inoculated
during the previous class. You will use it to grow a lawn of tiny bacterial
colonies.
3. Use the loop to distribute the bacteria completely over the surface of the
agar. Where you previously needed to streak for isolated colonies, now
you are streaking for maximum distribution over the surface of the agar.
Turn the plate 90o and streak the entire surface again.
4. Repeat step #3 until you are satisfied with the amount of bacteria you
have deposited on the agar.
5. Use the antibiotic disc dispenser to release several discs onto the agar
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surface. Press down with another bacteriological loop to secure.
6. Observe the growth pattern after at least 24 - 48 hours of growth (standard
read time is 48 hours).
MINIMUM INHIBITORY CONCENTRATION (MIC)
and MINIMUM BACTERICIDAL CONCENTRATION
The minimum inhibitory concentration (MIC) method uses doubling dilutions of
antibiotics solubilized in bacteriological medium to assess the susceptibility of
specific bacteria to the antimicrobial agents selected. The MIC test measures the
lowest concentration of an antibiotic that inhibits the visible growth of test
microorganisms. In practice, microtiter plates containing prediluted antibiotics in
lyophilized form are reconstituted and incubated along with test bacteria.
Once an MIC has been performed a minimum bactericidal concentration (MBC)
can subsequently be determined. The MBC is set up with subcultures made from
each MIC well that appears visually clear. The MBC is the concentration of
antibiotic included in the first tube from which colonies calculated as less than
99.9% growth is recovered.
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Comparison of Kirby-Bauer and MIC test results:
Bacterial Staining
THE GRAM STAIN
The Gram stain exploits the basic differences in the outer layers of bacteria so
that certain bacterial groups retain the initial stain while others readily lose this
dye during the decolorization process. Bacteria that decolorize will be stained the
color of the counterstain. The first two clues to the identification of any bacterium
is the shape of the organism and the Gram staining characteristic. In fact, in
some situations need no further information is needed (at least at the beginning)
and you will come across microbiologists referring toan isolated organism as a
"Gram-negative rod" or a "Gram-positive coccus". With more experience in
microbiology, this information actually tells you a great deal about the genus to
which the organism belongs.
MATERIALS:
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Gram stain solutions
Microscope slides
Agar plate cultures of Escherichia coli and Staphylococcus aureus
STUDENT DIRECTIONS:
To perform the Gram stain, a good smear is absolutely essential. Too thick a
smear will produce a slide giving false positive reactions. Too thin a smear will
require lots of time seeking organisms on the slide.
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1. Prepare a good smear
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Place one drop of water on a microscope slide.
With an inoculating loop, pick one colony; place colony in the drop of
water and make a milky suspension.
Allow the smear to air dry.
Heat-fix the smear by passing the slide over the burner flame several
times. Take care not to overheat (overheating will lyse the cells). Test the
temperature by placing the slide on the back of your hand. If it’s too hot for
your hand, it’s too hot for the bacteria.
2. Flood the slide with crystal violet stain and leave for 1 minute.
3. Pour off the crystal violet stain, wash with water, and flood the slide with
Gram’s iodine solution. Allow a contact time of 1 min.
4. Gently rinse the slide with water from a squirt bottle.
5. Blot the slide dry to remove excess water with a Kimwipe.
6. Holding the slide on a slant, flood the slide with the combination 95% acetonealcohol/safrinin solution, watching for the dye to continue washing from the cells.
As soon as most of the crystal violet dye stops leaching from the cells, gently
wash the slide in water to stop the decolorization process. Be careful not to
overdo this step.
7. Blot the slide dry or allow to air dry.
The critical step is #6, the decolorization process. In all probability it will take
some practice to train your eye to detect when the dye has stopped leaching
from the cells. Too thick a smear will protect cells lying beneath the top cells from
becoming decolorized. In this case a Gram negative organism will appear Gram
positive. Many Gram-positive cultures will appear Gram-negative or Gramvariable if the cells are over-decolorized. Is it very likely that Gram-negative cocci
would be cultured from a normal skin swab?
You should start this exercise by preparing a slide with 2 smears, one from a
Gram positive coccus (off your normal flora plates) and the Gram negative rod
(Escherichia coli). The presence of the two smears adjacent to one another will
not affect the Gram stain, but it will let you see the difference in decolorization
between the two organisms. After staining one slide, examine the results under
the microscope using the oil immersion objective. If you fail to see the red-pink
rod-shaped Escherichia or if the cocci are not a deep purple, try the stain again
with another slide--this time correcting the decolorization step (longer or shorter).
A Candida albicans culture is also interesting to examine in comparison with
Gram-positive bacteria.
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Other Bacterial Staining Methods
THE ACID-FAST STAIN
The property of acid-fastness, which is detected by the acid- fast stain, is
primarily of clinical application to detect members of the genus Mycobacterium.
M. tuberculosis, the etiologic agent of tuberculosis, is the most common
pathogen of this group. Other microorganisms, particularly the Nocardia, can be
identified by their acid-fast characteristic. The term acid-fast is derived from the
resistance displayed by acid-fast bacteria to decolorization by acid once they
have been stained by another dye.
Cultures provided:
MATERIALS:
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Mycobacterium phlei culture
Prepared Kinyoun carbol fuchsin
stain
3.0% acid-alcohol (70% ethanol
containing
0.5% hydrochloric acid)
Methylene blue counterstain
STUDENT DIRECTIONS:
1. Prepare a bacterial smear in the conventional manner (as for Gram-staining),
air-dried and heat fixed. Flood the smear with Kinyoun carbol fuchsin reagent
and allow to remain in contact for at least 5 min. Do not allow the stain to dry out
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- add more if needed.
2. Rinse the excess stain off with deionized water from the squirt bottle. Tilt the
slide to drain water.
3. Decolorize the smear with acid alcohol until the color no longer runs from the
smear. It is difficult to overdecolorize acid-fast bacteria.
4. Wash the smear with deionized water (use the squirt bottle).
5. Counterstain for 30 sec -- 1 min. with 1% methylene blue.
6. Rinse thoroughly, blot dry, and examine under oil immersion.
7. Study the stained preparation using the light microscope. A blue-stained mass
can usually be found on low-power. Examine the mass with the oil-immersion
lens. On high power, acid-fast bacteria will appear an intense, crimson red
(retaining the carbol fuchsin), while the remaining material will be blue from the
counterstain.
THE METHYLENE BLUE STAIN
The Methylene Blue Stain is considered to be a simple stain, in contrast with the
Gram and Acid-Fast Stains, which require a counterstaining step. Methylene
Blue Stain is especially useful for observing metachromasia in bacterial species
such as Corynebacteria.
MATERIALS:
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1%
Methylene blue
stain solution
• Glass
microscope
slides
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normal
flora cultures
STUDENT DIRECTIONS:Prepare a bacterial smear, as above.
1. Flood with methylene blue solution.
2. Leave stain in contact with the smear for 30 sec -- 1 min.
3. Rinse thoroughly, blot dry, and examine under oil immersion.
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Basic Bacterial Culture and Identification