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EXPERIMENT

Aseptic Technique and


Culturing Microbes
Hands-On Labs, Inc.
Version 42-0239-00-03
Review the safety materials and wear goggles when
working with chemicals. Read the entire exercise
before you begin. Take time to organize the materials
you will need and set aside a safe work space in
which to complete the exercise.
Experiment Summary:
You will outline aseptic techniques and describe
safe handling of microbes. You will identify plating
techniques and media for various types of bacterial
cultures. You will aseptically transfer microbes
between various media forms. You will observe
microbial growth on solid and liquid media. You will
incubate and store pure cultures for use in future
experiments.

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Experiment Aseptic Technique and Culturing Microbes

Objectives
Upon completion of this laboratory, you will be able to:
●● Outline aseptic techniques and describe safe handling of microbes.
●● Identify plating techniques and media for microbial cultures.
●● Describe the biology of Escherichia coli, Saccharomyces cerevisiae, and Staphylococcus
epidermidis.
●● Apply aseptic technique to transfer microbes between media forms.
●● Examine microbial growth on solid and liquid media.
●● Create pure cultures of bacteria and yeast and isolate individual colonies.

Time Allocation: 4 hours + 4 day incubation period

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Materials
Student Supplied Materials

Quantity Item Description


1 Bottle of bleach
1 Box of matches or lighter
1 Camera, digital or smartphone
1 Coffee cup
1 Hand soap
1 Isopropyl (rubbing) alcohol (C3H8O)
1 Large cooking pot (at least 8” deep)
1 Oven mitt or kitchen towel
1 Roll of paper towels
1 Source of bright light for observing culture broths
1 Source of tap water
1 Stove
HOL Supplied Materials

Quantity Item Description


1 Apron
1 E. coli tablet in vial
1 Face mask with ear loops
3 Inoculation loop
6 Nutrient broth-5mL tube
1 Pair of gloves
1 Pair of safety goggles
1 Permanent marker
3 Petri dish, 60 mm
2 Plastic cup, 9 oz
1 S. epidermidis tablet in vial
1 Small graduated pipet, 5 mL
2 Tea candle
1 Test tube clamp
1 Test tube rack, 6 x 21 mm
1 Thermometer
2 Tryptic soy agar (TSA), 18 mL tube*
1 Yeast packet

*Agar in kit may be labeled as “Nutrient Agar.”

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Experiment Aseptic Technique and Culturing Microbes

Note: To fully and accurately complete all lab exercises, you will need access to:
1. A computer to upload digital camera images.
2. Basic photo editing software, such as Microsoft Word® or PowerPoint®, to add labels, leader
lines, or text to digital photos.
3. Subject-specific textbook or appropriate reference resources from lecture content or other
suggested resources.

Note: The packaging and/or materials in this LabPaq kit may differ slightly from that which is listed
above. For an exact listing of materials, refer to the Contents List included in your LabPaq kit.

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Experiment Aseptic Technique and Culturing Microbes

Background
Aseptic Techniques
When working with microorganisms, the goal is to transfer the bacterium of interest to various
media without introducing other unwanted microbes into the culture. In addition, aseptic
technique provides a safe working environment. Aseptic techniques are the techniques needed
to prevent the accidental or inadvertent spread of microorganisms beyond the intended
working environment. Aseptic techniques are vital when isolating a pure culture from a mixed or
contaminated culture.
1. Hands must be washed, surfaces must be cleared of unnecessary items, and work surfaces
must be sterilized with a 10% bleach solution or a bleach-based cleaner before beginning the
Experimentation. See Figure 1.

Figure 1. Washing hands. Proper hand-washing before and after an experiment can greatly
reduce the risk of contaminating both you and your results. © Andrew Bassett

2. The equipment used to isolate cultures has to be sterilized. Either an open flame or
isopropyl alcohol may be used to eliminate contamination that is naturally picked up from
the environment. In campus laboratories, equipment is often sterilized with an autoclave or
micro-incinerator.
3. When opening cultures or sterile media:
●● Work quickly and efficiently.
●● Do not talk or breath over the culture.

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●● When tubes and vials are open, keep them tilted away from the mouth and nose.
●● Never lay caps or lids on a bench or work area, as these areas most likely have contaminants.
●● When working with a culture, ensure the instruments used are sterile. This will include
inoculation loops and swabs. Sterile instruments are provided by Hands-On Labs, Inc.
(HOL) You must work to maintain the instruments’ sterility.
4. Some general precautions include the following:
●● Avoid producing aerosols (anything that can be introduced into the air). This includes
avoiding splashing when pipetting media and inoculating cultures.
●● Keep the lid to the culture closed whenever possible.
●● Incubate plates inverted (agar side up). Agar contains a lot of water. When plates are
incubated with the lid side up, water will condense on the lids; this condensation can drip
back onto the agar plate and possibly contaminate and/or smear the results.
●● Always write on the bottoms of the plates, not the lids. This will prevent unlabeled plates
if lids are misplaced.
●● When not in use, tubes and plates should be properly stored away from children and pets.
●● All equipment and work surfaces should be cleaned with a 10% bleach solution or bleach
based cleaning product at the end of the experiment.

Media and Its Uses


HOL provides various forms of media that grow cultures of microbes. In general, media exist in
two major forms: liquid and solid. One liquid type of media is broth, which is depicted in Figure 2.
A microbiologist uses a broth primarily to increase the number of microorganisms.

Figure 2. Broth, a type of liquid media.

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Solid media is prepared as either slants or plates. See Figures 3 and 4. The most common solidifying
agent in both slants and plates is agar. Agar does not melt until it reaches a temperature of about
80°C (176°F). Conversely, once it has melted, it can be cooled to about 45°C (113°F) before it
solidifies. In addition, agar has the ability to grow microorganisms over a large temperature range.

Figure 3. Slant, a type of solid media.

Slants are primarily used for storage and transport of microorganisms. Organisms stabbed into
the media are protected from desiccation, allowing them to survive much longer. Slants can be
stored for weeks, even months, without significant death to the organisms that they house. The
screw cap and the small size of the vial allow a scientist to easily transport a culture from one
place to another.

Figure 4. Plate containing agar media.

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Experiment Aseptic Technique and Culturing Microbes

Plates provide a larger surface area for culturing microbes than slants. See Figure 4. Plates are
commonly used for isolating microbes. Culture samples taken from individuals or the environment
contain many types of microbes. These mixed cultures must be separated into individual colonies
of only one type of microbe to be successfully analyzed. A pure culture contains only one
species of microorganism. Proper use of aseptic techniques prevents pure cultures from being
contaminated. A contaminated culture occurs when unknown microorganisms are inadvertently
introduced often via poor technique. In addition, bacterial colony morphology (the shape of a
group of bacteria) can help distinguish among different species when using a plate.
Secondary descriptive terms are often used when describing various media. Some media are
described as a general purpose media. These media contain a rich variety of nutrients that will
facilitate the growth of a wide range or microorganisms and is therefore ideal for generalized
growth. One example of general purpose media is a Nutrient Agar plate or NA plate, which will be
used during this laboratory. In future laboratories, more specialized media that select for specific
types of microorganisms will be introduced which can aid in microbe species identification.

Over the last 100 years, thousands


of different media formulations have
been created for growing a huge variety
of microorganisms. With the advent of DNA
technology, it is estimated that we have
only grown and discovered a mere one
percent of the microorganisms living on
this planet.

Isolation Techniques
The streak plate is the primary mechanism for isolating bacteria. This technique utilizes a four
quadrant dilution that systematically reduces bacterial numbers until they are diluted enough to
form isolated colonies. The procedure is shown in Figure 5 and can be separated into four defined
steps. Carefully read the following steps as you will be streaking a number of plates during this
experiment.

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Figure 5. Bacterial isolation with a streak plate. A. A collected bacteria sample is spread over
one quadrant of the agar plate. B. A sterilized inoculation loop is used to move a small portion
of sample from the first quadrant to a second quadrant. C. A small portion of the second
quadrant is moved to the third quadrant. D. A small portion from the third quadrant is moved
to the fourth quadrant.

1. A bacterial sample must be collected. The bacterial sample may be collected by rubbing a
sterile cotton swab over a test area, such as an elevator button or a place on the human body.
Alternately, the bacterial sample may be obtained from a previous culture. Using aseptic
methods, the sample is spread over a small portion of the plate, usually about ¼ of the plate
size. This first inoculation may be referred to as the “first quadrant” and is shown in Figure 5A.
2. A small portion of the bacteria from the first quadrant is then spread to a second quadrant of
the plate so as to isolate and “dilute” a portion of the microorganisms. An instrument called
an inoculation loop is sterilized and then placed flat in the center edge of the first quadrant.
The inoculation loop is used to spread a small portion of the organism from the first quadrant
into the second quadrant, as shown in Figure 5B. Notice in the figure that it is only necessary
to move into the first quadrant a few times. The second quadrant is created with a small
amount of sample that is pulled out from the first quadrant. It is recommended that you work
from the middle of the plate each time to maximize the amount of surface area used. Using
the complete plate will optimize the dilution effect.

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3. The microorganisms are again spread using the inoculation loop. After sterilizing, the loop is
used again to spread a portion of the sample from quadrant two into a new, third quadrant
of the plate. Again, it is recommended that you work from the middle of the plate to cover as
much surface as possible. See Figure 5C.
4. The microorganisms are spread into the final quadrant using the sterilized inoculation loop.
Care must be taken to avoid accidently coming into contact with the other previously formed
quadrants. If the loop accidentally comes in contact with the first and second quadrants, the
dilution is lost.
Once the process of dilution is complete, bacteria are allowed to develop and reproduce. Figure
6 shows bacterial colonies on a streak plate. Notice that the bacterial colonies have grown the
most abundant in the first quadrant and become less abundant in each subsequent quadrant. The
fourth quadrant hosts isolated colonies as compared to smeared bacteria in the other quadrants.
Individual colonies that develop on a plate are called colony forming units (CFUs) because each
colony develops from an isolated or genetically identical clump of microorganisms.

Figure 6. Dilution of bacteria on a plate. © Alina Cardiae

Morphology
Morphology  is the size, shape, and other physical characteristics that can be used to identify
microorganisms. Even without a microscope, information about microbes can be acquired
through observation on a plate. As shown in Figure 7, different microorganisms can have different
sizes, shapes, and colors that can be seen on the plate. Additional morphological characteristics
include whether the edge of the colony is smooth or rough and whether the centers of the colony
are raised or indented. A certain species will always show this same morphology on a particular
media. Attention to detail allows the creation of pure cultures and eventual identification of a
microorganism. As you perform the Experimentation, pay very close attention and try to note the
differences you see in the isolated colonies you create.

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Figure 7. Examples of various morphologies. © Michal Kowalsky

Microbes for Cultures


In this laboratory and others you will be culturing three microbe species: Escherichia coli,
Saccharomyces cerevisiae, and Staphylococcus epidermidis. Each of these microbes is significant
to the history and study of microbiology and are used as model organisms in microbiology labs
around the world.
Escherichia coli is a rod-shaped bacteria commonly found in the lower intestine of warm-blooded
organisms. See Figure 8. Most E. coli strains, including the one provided from HOL, are harmless
and part of the normal flora of the gut. E. coli was one of the first organisms to have its genome
sequenced and is a model organism for studies involving bacterial conjugation, genetics, and
evolution.

Figure 8. Escherichia coli. © Michelangelus

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Saccharomyces cerevisiae, also known as baker’s yeast, is an ovoid single-celled fungi found on
ripe fruits in nature. See Figure 9. S. cerevisiae is harmless to most humans, but has been shown
to irritate the digestive system of individuals suffering from Crohn’s disease and ulcerative colitis.
S. cerevisiae is one of the most intensively studied eukaryotes and serves as a model organism for
studies involving fermentation, proteins, genetics, and aging.

Figure 9. Saccharomyces cerevisiae. © Krnorre

Staphylococcus epidermidis is a coccus-shaped bacteria commonly found on the skin of humans.


See Figure 10. S. epidermidis is harmless to most individuals and considered part of the normal
skin flora. S. epidermidis has been linked to infections in individuals with catheters or other
surgical implants. S. epidermidis is a model organism for study of osmotic pressure regulation,
antibiotic resistance, and biofilms.

Figure 10. Staphylococcus epidermidis. © Sebastian Kaulitzki

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Experiment Aseptic Technique and Culturing Microbes

Exercise 1: Culturing Microbes in Broth


In this exercise, you will inoculate nutrient broths with the inactive microbe samples from your
HOL kit using aseptic technique. The broths will be incubated and examined for signs of microbial
growth.
Procedure
1. Gather three nutrient broth tubes, the graduated pipet, the alcohol, disposable cups, the E.
coli and S. epidermidis vials, yeast packet, candle, a match or lighter, permanent marker, paper
towels, 10% bleach solution, safety gloves, face mask, apron, and goggles.
2. Wash your hands thoroughly with soap and warm water.
3. Put on the safety gloves, face mask, apron, and goggles.
4. Disinfect the work surface by wiping the area with a 10% bleach solution.
5. Carefully pour some undiluted bleach into the bucket for waste disposal. Anything that is
exposed to microorganisms needs to be treated with undiluted bleach before disposal.
6. Use the permanent marker to label one of the small plastic cups as “alcohol.” Fill the plastic
cup half full with the alcohol and place it on the work surface.
7. Place the pipet in a cup of alcohol making sure the alcohol enters the stem of the pipet.
8. Place a candle on the work surface and ignite the wick using a match or lighter.
9. Using the permanent marker, label a nutrient broth tube E. coli.
10. Place the E. coli vial and labeled nutrient broth on the work surface near the candle.
11. Remove the pipet from the cup and expel the alcohol inside the pipet back into the cup by
squeezing the bulb.
12. Shake the pipet while continuing to squeeze the bulb until no alcohol remains inside the
pipet. The pipet is now sterile.
13. While holding the pipet, pick up the labeled nutrient broth and gently remove the cap.
14. Pass the lip of the broth tube through the flame of the candle to sterilize the surface. See
Figure 11.

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Figure 11. Sterilizing broth tube opening. Note the culture vial also near candle.

15. Place the uncapped broth tube on the work surface and repeat steps 13-14 for the E. coli
culture vial. See figure 12.

Figure 12. Sterilizing culture vial lip.

16. Pipet .25 mL of broth from the nutrient tube into the culture vial. Be careful not to touch the
sterilized rim of either container with the tip of the pipet or your gloves. See Figure 13.

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Figure 13. Pipetting the broth into the culture vial.

17. Replace the lid on the culture vial and shake vigorously until the tablet dissolves. See Figure
14.

Figure 14. Dissolved pellet in the culture vial.

18. Pipet the dissolved tablet solution from the culture vial into the broth tube being careful not
to touch the rim of the broth tube with the tip of the pipet or your gloves. See Figure 15.

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Figure 15. Transferring tablet solution to broth tube.

19. After transferring all of the solution from the culture vial, flame the lip of the broth tube to
sterilize before screwing on the cap. See figure 16.

Figure 16. Sterilizing broth tube rim before screwing on cap.

20. Set the inoculated broth aside and place the pipet into the disposable cup containing the
alcohol. Make sure to draw alcohol into the stem of the pipet.
21. Repeat steps 9-20 for the S. epidermidis culture vial using a new broth tube labeled S.
epidermidis.
22. Open the yeast packet and carefully place approximately ½ teaspoon of the powdered contents
into an empty disposable cup. Retain the remainder of the packet for future experiments.

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23. Add approximately ¼ cup of warm tap water to the yeast and swirl until dissolved.
24. Allow the cup to sit for 10 minutes until it begins to froth. See Figure 17.

Figure 17. Yeast mixture ready for transfer to broth.

25. Label a new broth tube S. cerevisiae.


26. Repeat steps 11-14 for the S. cerevisiae broth tube.
27. Pipet .25 mL of the yeast mixture from the cup into the broth tube being careful not to touch
the rim of the tube with the pipet or your gloves.
28. After transferring the yeast solution, flame the lip of the broth tube to sterilize before screwing
on the cap. See Figure 16.
29. Extinguish the candle.
30. Sterilize the pipet in alcohol and store with the candle, safety goggles, mask, and apron for
use in Exercise 2.
31. Pour the remaining alcohol from the cup into a sink. You may rinse, dry, and reuse this cup for
Exercise 2.
32. Place the two empty culture vials with lids and the disposable cup containing the yeast
solution in a bucket of undiluted bleach for 30 minutes. The items may then be removed and
disposed of in the garbage.
33. Wipe down the work area with a 10% bleach solution.

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34. Identify a location in your home where the broth tubes can incubate, upright and untouched,
for approximately 48 hours. The location should be room temperature (21°C-25°C), away from
heating or air-conditioning vents, out of direct sunlight, and secure from children and pets. An
empty cabinet works well. If a counter top is used, place cultures in a box, such as your empty
HOL box. Use the thermometer to determine whether the location meets the requirement of
21°C-25°C, if not, identify a new location.
35. Incubate the nutrient broth cultures for 48 hours.
36. After 48 hours, inspect cultures for microbe growth by holding the nutrient tubes near a light
source.
37. Developed cultures will either appear turbid (cloudy), see Figure 18, or exhibit flocculent
growth at the bottom of the tube, see Figure 19.

Figure 18. Turbid broth from microbial growth. Compare color and clarity to Figure 19.

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Figure 19. Flocculent microbial growth in bottom of nutrient broth tube.

38. If cultures show no signs of growth after 48 hours, incubate for an additional 24 hours.
39. Take a digital photograph of each of your three developed broths.
40. Resize the images and upload into Data Table 1 of your Laboratory Report Assistant. Refer to
the appendix entitled “Resizing an Image” for guidance with resizing an image.
41. Describe each broth related to turbidity and flocculent growth in the space provided in Data
Table 1.
42. The developed broths will be used in Exercise 2 of this experiment.

Note: If Exercise 2 is not to begin immediately, return the culture tubes to the incubation location for
up to 2 additional days. If Exercise 2 will be conducted more than 2 days after Exercise 1 has been
completed, store the tubes in the refrigerator to preserve the cultures.

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Questions
A. What is the purpose of wiping down your work area with bleach before and after an
experiment?
B. List five safety precautions you used to protect yourself while culturing microbes.
C. What is the purpose of a broth in a microbiology laboratory?
D. Did each of your cultures develop at the same rate? Why might your incubation environment
favor one microbe over another?

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Exercise 2: Isolating Individual Colonies


In this exercise, you will inoculate agar plates with the broths cultured in Exercise 1. The plates
will be incubated and examined for signs of microbial growth. Then you will compare growth of
microbes in a nutrient broth versus on an agar plate.
Procedure
1. Approximately 1 hour before you begin this exercise, pour 4 agar plates. Refer to the appendix
entitled “Pouring Agar Plates” for guidance with this procedure.

Note: Plates may be poured in advance, stored in an airtight bag, and refrigerated for future use.

Note: 3 plates will be used for this lesson, reserve the 4th agar plate for future lessons.
2. Gather the developed cultures of E. coli, S. cerevisiae, and S. epidermidis, three plastic
inoculation loops, candle and lighter, three poured nutrient agar plates, a disposable cup,
alcohol, paper towels, bleach, a permanent marker, gloves, mask, apron, and goggles.
3. Wash your hands thoroughly with soap and warm water.
4. Put on the gloves, goggles, apron, and face mask.
5. Wipe down your work surface with a 10% bleach solution.
6. Fill the plastic cup labeled as “alcohol” from Exercise 1 half full with the alcohol and place it
on the work surface. Put the three inoculation loops into the cup.
7. Label the agar side of each plate with the permanent marker for the microbe it will contain: E.
coli, S. cerevisiae, or S. epidermidis. Lay each plate on the work surface.
8. Put the corresponding cultured nutrient broth near each labeled agar plate.
9. Light the candle and place it on the work surface.
10. Remove one inoculation loop from the alcohol and shake briskly to dry.
11. While holding the sterile loop, remove the cap from the E. coli broth and pass the lip through
the flame to sterilize. See Figure 20.

Figure 20. Sterilizing tip of broth tube. Note proximity of agar plate.

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12. Insert the sterile loop into the broth being careful not to touch the rim with the loop or your
gloves. See Figure 21.

Figure 21. Inserting inoculation loop into broth.

13. Submerge the loop into the broth.


14. Immediately transfer the broth adhering to the loop into one quadrant of the agar plate using
a zigzag motion. See Figure 22 and Figure 23.

Figure 22. Inoculating agar plate with loop.

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15. Place the lid on the agar plate.


16. Put the inoculation loop into the cup of alcohol.
17. Flame the lip of the broth tube and replace the cap on the tube.
18. Remove the inoculation loop from the alcohol and shake to dry.
19. Open the lid of the agar plate and touch the loop to the edge of the inoculated quadrant to
transfer microbes to the adjacent quadrant of the plate. See Figure 24.
20. Return the lid to the agar plate.
21. Put the inoculation loop into the cup of alcohol.
22. After 20 seconds, remove the inoculation loop from the alcohol and shake to dry.
23. Repeat steps 20-22 so that all four quadrants have been streaked. See Figure 23.

Figure 23. Bacterial isolation with a streak plate. A. A collected bacteria sample is spread over
one quadrant of the agar plate. B. A sterilized inoculation loop is used to move a small portion
of sample from the first quadrant to a second quadrant. C. A small portion of the second
quadrant is moved to the third quadrant. D. A small portion from the third quadrant is moved
to the fourth quadrant.

24. Place the used inoculation loop in undiluted bleach for 2 hours. It may then be disposed of in
the garbage.
25. Select a new inoculation loop from the cup of alcohol.

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26. Gather the S. cerevisiae broth and labeled agar plate and repeat steps 11-26.
27. Gather the S. epidermidis broth and labeled agar plate and repeat steps 11-26.
28. Extinguish and store the candle, safety goggles, mask, and apron for use in future experiments.
29. Pour the remaining alcohol from the cup into a sink and dispose of the cup.
30. Wipe down your work area with a 10% bleach solution.
31. Identify a location in your home where the inoculated agar plates can incubate agar-side up
(inverted) for approximately 48 hours. The location should be room temperature (21°C-25°C),
away from heating or air-conditioning vents, out of direct sunlight, and secure from children
and pets. An empty cabinet works well. If a counter top is used, place cultures in a box such,
as your empty HOL box. Use the thermometer to determine whether the location meets the
requirement of 21°C-25°C, if not, identify a new location.
32. Place the broth tubes containing live cultures into the refrigerator to preserve for future
experiments.
33. Incubate the agar plates for 48 hours.
34. After 48 hours, inspect the plates for microbial growth. See Figure 24 for an example of a
developed plate.

Note: Do not remove the lids or flip the plates when observing for growth. You will be able to see the
microbe colonies through the agar.

Figure 24. Developed colonies on agar plate. © Alina Cardiae

35. If plates do not exhibit growth after 48 hours, incubate for an additional 24 hours.
36. Take a photo of each developed plate and resize and insert into Data Table 2 of your Laboratory
Report Assistant.
37. Include the incubation time and observations for shape and color into Data Table 2. See Figure
25 for describing the shape of colonies.

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Figure 25. Colony morphology. As an example, a colony may be described as filamentous in


shape with curled margins, a flat elevation, and punctiform in size. © Julie Oliver

38. Place the developed plates into the refrigerator to preserve the microbes for future
experiments.
39. When you are finished uploading photos and data into your Laboratory Report Assistant,
save your file correctly and zip the file so that it can be sent to your instructor as a smaller file.
Refer to the appendix entitled “Saving Correctly” and the appendix entitled “Zipping Files” for
guidance with saving the Laboratory Report Assistant correctly and zipping the file.
40. Soak reusable equipment, such as test tube clamps and racks, in a 10% bleach solution for 2
hours after contact with active cultures. Reusable materials should then be rinsed with tap
water and allowed to dry before returning to the lab kit.
41. Soak all disposable equipment, such as pipets and inoculation loops, in a pure bleach solution
for 2 hours before wrapping with paper towels, sealing in a plastic bag, and placing in the
garbage. Secure all disposed items out of reach of children and pets.

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Questions
A. What is a colony and how does a colony relate to a bacterial cell? Why are colonies important
in the study of microbiology?
B. Are bacteria easier to observe on a plate or in a broth? How do the bacteria look on the plate
as compared to the broth?
C. How could you tell if one of the colonies on your pure plate was a contaminant from the
environment and did not come from your original culture?
D. Was the streak plate method effective at diluting the population size in all 4 plates? How could
your methods be improved in the future?

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