Name: _________________________ Period: ____ Date: __________________
Skills: Bacteria Culture Techniques
Part 1: Making Yogurt – Role of Bacteria
For a dairy product to be called yogurt, it must contain two bacteria: Streptococcus thermophilus
and Lactobacillus bulgaricus. Many types of yogurt incorporate other species as well, including
Lactobacillus acidophilus and Lactobacillus casei. In many countries, yogurt must also contain live
bacteria and remain unpasteurized, with pasteurized yogurts being specially labeled.
The bacteria consume natural milk sugars and excrete lactic acid, which causes
the milk proteins to begin to curdle and create a more solid mass. At the same
time, the increased acidity of the dairy is too high for most harmful bacteria, so
the yogurt keeps itself clean. Other bacteria can be added for flavor or health
benefits, especially Lactobacillus acidophilus.
Yogurt is rich in protein and minerals and can in theory be drunk by people
suffering from lactose intolerance, because it contains an enzyme that breaks down lactose in the
intestines.
Procedure:
1. Heat 2 cups of milk almost to boiling at about 80 °C. Be sure to heat the milk in a
beaker (for edible only) provided and not in a mason jar. Mason jar will crack at high
temperature.
2. Cool to the milk to about 112 °F (= 44.4 °C) or cool enough for you to hold the jar.
3. Add ¼ cup powdered milk.
4. Add 1 T. yogurt starter culture and stir well.
5. Incubate at 105 – 122°F (= 40.6 - 50°C) for 8-12 hours (overnight).
6. Serve with desired topping. Enjoy!
Questions
1. What are two main bacteria used in most yogurt cultures?
Streptococcus thermophilus and Lactobacillus bulgaricus.
2. Explain the role of bacteria in production of yogurt.
The bacteria consume natural milk sugars and excrete lactic acid, which causes the milk
proteins to begin to curdle and create a more solid mass.
Describe the taste of the final product: Sour
3. Do research. Discover another bacteria used in our daily lives. Write its scientific name;
describe its role (in detail describe how it works), and describe or draw its appearance.
See bacteria notes paper (E. coli) and notes on video
Part 2: Bacteria & You (Macroscopic Observation of Bacteria)
Populations of microbes (such as bacteria and yeasts) inhabit the skin and
mucosa. Their role forms part of normal, healthy human physiology, however
if microbe numbers grow beyond their typical ranges (often due to a
compromised immune system) or if microbes populate atypical areas of the
body (such as through poor hygiene or injury), disease can result.
Traditionally, bacteria have been described by how they grow - what they
grow on, color of the colony, and so forth. More recently, bacteria have been described on the basis
of DNA sequencing.
It is estimated that 500 to 1000 species of bacteria live in the human gut and a roughly similar
number on the skin. Bacterial cells are much smaller than human cells, and there are at least ten
times as many bacteria as human cells in the body (approximately 1014 versus 1013).
Many of the bacteria in the digestive tract, collectively referred to as the gut
flora, are able to break down certain nutrients such as carbohydrates that
humans otherwise could not digest. The majority of these commensal
bacteria are anaerobes, meaning they survive in an environment with no
oxygen. Normal flora bacteria can act as opportunistic pathogens at times
of lowered immunity.
Prelab Question:
1. Identify and describe at least three types of bacteria found on your skin. Be sure to source
more than two references.
A. ___________________________
B. ___________________________
C. ___________________________
2. Which area of your body do you expect to find most bacteria? Why?
__________________________________________________________________________
Purpose: To identify the characteristics of a bacterium on an agar plate (colonial morphology).
Procedure:
1. Divide the LB agar plate into 3 segments and label them with
each body part that you are going to swab. Be sure to add you
initial and group number.
2. Obtain a package of sterile cotton swabs.
3. Using the technique shared in the class, swab the areas then
streak the plate.
4. Incubate overnight at 37 °C. Be sure to keep one LB plate for control.
5. On the following class period, check for bacterial growth (count colonies) and describe the
bacteria’s appearance in the table provided.
Disposal: Place the used swab in the rubbish can and place all contaminated agar plate in 10%
bleach bucket (open Petri-dish and submerge in the bleach solution).
To describe, use the following colonial characteristics/culture characteristics:
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WHOLE SHAPE or COLONY SIZE i.e. less than 1mm = punctiform (pin-point).
EDGE/MARGIN OF COLONY: magnified edge shape
CHROMOGENESIS (pigmentation): white, buff, red, purple, etc.
OPACITY OF COLONY: transparent (clear), opaque, translucent (almost clear, but distorted vision–
like looking through frosted glass), iridescent (changing colors in reflected light)
ELEVATION OF COLONY (turn the place on end to determine height)
SURFACE OF COLONY: smooth, glistening, rough, dull (opposite of glistening), rugose (wrinkled)
CONSISTENCY: butyrous (buttery), viscid (sticks to loop, hard to get off), brittle/friable (dry, breaks
apart), mucoid
EMULSIFIABILITY OF COLONY: Is it easy or difficult to emulsify? Does it form a uniform
suspension, a granular suspension, or does not emulsify at all?
ODOR: Absent or present? If it has an odor, what does it smell like?
Data:
Sample Area:
Colony shape
Colony Edge/Margin
Colony Pigmentation
Colony Opacity
Colony Elevation
Colony Surface
Colony Consistency
Colony Emulsifiability
Colony Odor
Questions:
1. Which area of your body seems to have more bacteria than others? ____________________
2. Identify one of the bacteria observed in your body. What was your determining factor?
____________________________________________________________________________
Part 3: Streaking Bacteria
Cultures ordered from a supply company or stock center will probably not consist of genetically
identical bacteria. The bacteria will all be of the same species, and available as a single strain.
However, random mutations may still exist due to the large number of bacteria present. To
obtain a source of genetically identical bacteria, streak plates are used.
Streaking a plate allows the bacteria to be spread out so that a single bacterium can be
isolated from all other bacteria. This technique is called streaking for individual colonies. Since
bacteria are so small, you will not be able to see that isolated bacterium. However, that
bacterium will reproduce itself by binary fission (typical division time is on the order of 20
minutes), resulting in bacteria which are genetically identical to the original bacterium and to
each other. These bacteria are visible as a small round colony growing where there had been
one isolated bacterium. This method allows you to use the individual colony repeatedly and
expect similar results. Moreover, using cells derived from a single colony minimizes the chance
of using a cell mass contaminated with a foreign microorganism.
There are several acceptable streak plate methods. The method described here is
called the "T'' streak and is one of the easiest.
1. Light a Bunsen burner in your bench space. To maintain sterile conditions, inoculation
should occur within 20 cm of the flame. Wait 20 seconds before opening the petri dish and
inoculating. This gives the flame time to sterilize the local air. Remember that you want to
achieve sterile conditions. Do not work with the plate close to your face. This will violate the
sterile environment.
2. Use a marker or wax pencil to draw a T on the bottom of a plate of nutrient agar. This
divides the plate into three sections (One section covers one half the plate. The other half is
divided into two quarters.
Figure 5.2: Draw a "T'' on the bottom of your petri dish as shown.
3. Sterilize the inoculating loop by holding its tip in the flame until it turns red.
4. Lift up the lid of the plate you will be inoculating and poke the inoculating loop through the
agar close to the side of the Petri dish to cool it. This prevents the heat from killing the
bacteria sample you want to use. The heat will not harm the agar. Try to lift the lid of the plate
up only as much as is necessary to put the loop inside. If you completely remove the lid, it can
become contaminated with bacteria from the environment.
5. Touch the loop to the edge of the colony growing on the plate. Then take the loop and place
the lid securely back on the plate.
6. Set the plate you will be streaking so that its bottom is sitting on the bench top and you can
see the T clearly. The largest section should be at the top. Carefully lift up the lid and touch
the inoculating loop to the upper left hand corner of the largest section of the plate. Move the
loop from left to right, back and forth, across the surface of the agar. See Figure . Since
nutrient agar is a gel with properties similar to Jell-O, do not push down with the loop or you
will gouge the agar.
Touch the inoculating loop to the upper left hand corner and then move it across the agar from left to right as shown.
7. Replace the lid of the petri dish and flame the loop again to kill any remaining bacteria on it.
Rotate the plate 90 degrees counterclockwise. Carefully lift the lid slightly and touch the loop
into the left side of the plate which contains the area you streaked in the previous step. Move
the loop across the surface of the agar until it is in the smaller section in the upper right of the
plate. Within that quarter of the plate, move the loop back and forth across the agar surface.
Touch the loop to the area previously streaked and then move the loop across the agar as shown.
8. Repeat Step 7 as shown below.
Touch the loop on the previously streaked area. Then move the loop across the agar onto the third area as shown.
9. Replace the lid of the petri dish and flame the loop again to kill any remaining bacteria on it.
10. Incubate the streak plate at 37°C until you can see individual colonies (15-20 hours).
Plates are inverted to prevent condensation that might collect on the lids from falling back on
the agar and causing colonies to run together).
Incubate the streak plate until you can see individual colonies.
11. Take time for responsible clean up.
 Segregate bacterial cultures for proper disposal.
 Wipe down lab bench with soapy water, 10% bleach solution, or disinfectant (such as
Lysol) at end of lab.
 Wash hand before leaving lab.
Questions:
1. What is the purpose for bacterial streaking? _________________________________________
a) What is the reason for the zigzag streak pattern? __________________________________
____________________________________________________________________________
b) Why is the inoculating loop resterilized between each new streak?____________________
____________________________________________________________________________
c) Why should a new streak intersect only the end of the previous one at a single point?
___________________________________________________________________________
2. Draw a step by step process in how you would go about streaking without the T chart on the
Petridish and with four streaks.
3. Describe the appearance of a single E. coli colony. Why can it be considered genetically
homogenous?
__________________________________________________________________________
Streaking Practical:
__ Pass (date: _________________)
__ Fail (retake date: _______________________)
Part 4: Gram Staining (Microscopic Observation)
In 1884, Hans Christian Gram, a Danish doctor working in Berlin, accidentally stumbled on a
method which still forms the basis for the identification of bacteria. While examining lung tissue
from patients who had died of pneumonia, he discovered that certain stains were preferentially
taken up and retained by bacterial cells. Over the course of the next few years, Gram developed a
staining procedure which divided almost all bacteria into two large groups - the Gram + (dark
purple/violet crystal violet stain is obtained) and Gram – (pink safranin counterstain obtained).
This property reveals fundamental differences in the cell envelope between the two groups. Gram
positive bacteria have many layers of peptidoglycan in their cell wall; Gram negative bacteria have
only one or two peptidoglycan layers but, additionally, they have an outer membrane. These
differences have important consequences. For example, certain antibiotics cannot penetrate the
outer membrane of Gram-negative bacteria, which are intrinsically resistant to these drugs as a
consequence.
Below is a schematic, showing how the Gram stain works:
Prelab Question:
What is the major difference between Gram + and Gram – bacteria? What do you expect to see in
the lab (describe the color and the shape of the bacteria)?
Bacteria stained: _____________________
Procedure:
Before we can stain we need to do a smear prep (fix bacteria onto a slide) to prevent the sample
from being lost during a staining procedure. A smear can be prepared from a solid or broth
medium. Below are some guidelines for preparing a smear for a Gram-Stainining.
Part 1: Smear Preparation
1. Spread the bacteria suspension on a clean glass slide using a sterilized wire loop.
2. Allow the smear to air dry
3. Heat-fix the smear by passing the slide through a flame 3 times; allow the slide to cool
before staining.
What not to do in smear preparation:
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Forget to clean the slide
Use too much material - suspension should be just barely cloudy
Use so much liquid that it takes forever to dry
Heat the smear before letting it air dry, boiling the bacteria instead of attaching them
Overheat the smear, melting cell walls and possibly breaking the slide.
Part 2: Gram Staining
1.
2.
3.
4.
5.
Flood the prepared slide with crystal violet stain and allow to stand for 1 minute.
Rinse the slide gently with deionized water.
Flood the slide with working gram iodine solution and allow to stand for 1 minute.
Rinse the slide gently with deionized water.
Rinse the slide gently with Decolorizer solution for 10 seconds; decolorization is complete
when the solution runs clear from the slide. The action 1/1 decolorizer is very rapid. The
exposure limit of the slide should be determined by the technologist.
6. Rinse the slide gently with deionizee water.
7. Flood the slide with Safranin Stain and allow to stand for 1 minute.
8. Rinse the slide gently with deionized water.
9. Blot the slide dry with absorbent paper and examine the slide under oil immersion lens.
10. Results: Gram positive bacteria are stained purple and green; and negative bacteria are
stained pink to red.
Result: Observe and raw the bacteria under the microscope. Be sure to identify the
magnification and Gram staining result.
Magnification: _______________ X
Was your bacteria Gram + or Gram -? ________________________
Notes on proper microscope usage (oil-immersion)
________________________________________________
________________________________________________
________________________________________________
________________________________________________
________________________________________________
________________________________________________
Lab Practical: Smear & Gram Staining
____ Pass (Date:
) _____ Fail (retake date:
)
Reason: ___________________________________
Disposal: Be sure to clean up the microscope lenses properly with Kimwipes. Slides will be
collected.
Part 5: Small-Scale Suspension Culture (overnight culture)
Overnight cultures are used for purification of plasmid DNA and for inoculating mid-log cultures.
1. Label and sterile 50-ml tube with your name and the date. The large
tube providers a greater surface area for good aeration culture.
2. Use a 10-ml pipette to sterilely transfer 5 ml of LB broth into the
tube.
a. Attach pipette aid or bulb to pipette. Briefly flame pipette cylinder.
b. Remove cap of LB bottle using little finger of hand holding pipette bulb, flame mouth of
LB bottle.
c. Withdraw 5 ml of LB. Reflame mouth of bottle, and replace cap.
d. Remove cap of sterile 50-ml culture tube. Briefly flame mouth of culture tube.
e. Expel sample into tube. Briefly reflame mouth of tube, and replace cap.
3. Locate well-defined colony 1-4 mm in diameter on a freshly streaked plate.
4. Sterilize inoculating loop in the Bunsen Burner flame until it glows red hot.
Then, continue to pass lower half of its handle through the flame.
5. Cool loop tip by stabbing it several times into the agar near the edge of plate.
6. Use loop to scrape up a visible cell mass from selected colony.
7. Sterilely transfer colony into culture tube:
a. Remove cap of the culture tube using little finger of hand holding
b. Briefly flame mouth of culture tube.
c. Immerse loop tip in broth, and agitate to dislodge cell mass.
d. Briefly reflame mouth of culture tube, and replace cap.
loop.
8. Reflame loop before placing it on lab bench.
9. Loosely replace cap to allow air to flow into culture. Affix a loop of tape over the cap to prevent
it from becoming dislodged during shaking.
10. Incubate for 12-24 hours at 37 °C, preferably with continuous agitation. Shaking is not
essential for a culture to be used for plasmid purification. The culture can be incubated at 37 °C,
without shaking, for 1 or more days.
11. Take time for responsible cleanup.
a. Segregate for proper disposal bacterial cultures and tubes, pipettes, and micropipettor tips
that have come into contact with the cultures.
b. Disinfect overnight culture and pipettes and tips with 10% bleach or disinfect.
c. Wipe down lab bench with soapy water, 10% bleach solution, or disinfect (such as Lysol)
d. Wash hands before leaving lab.
Questions:
1. Why is 37°C the optimum temperature for E. coli growth? ________________________
2. Give two reasons why it is ideal to provide continuous shaking for a suspension culture.
__________________________________________________________________________
3. What is the growth phase reached by a suspension of E. coli following overnight shaking
at 37°C? __________________
4. Approximately how many E. coli cells are in a 5mL suspension culture at stationary
phase? _____________________________
Part 6: Mid-long culture of E. coli
Cells in mid-log growth can generally be rendered more competent to uptake plasmid DNA than
can cells at stationary phase. Mid-log cells are used in the class transformation protocols. The
protocol begins with an overnight suspension of culture of E. coli (from part 5). Incubation with
agitation has brought the culture to stationary phase and ensures a large number of health cells
capable of further reproduction.
The object is to subculture (reculture) a
small volume of the overnight culture in
a large volume of fresh nutrient broth.
This “re-sets” the culture to zero growth,
where after a short lag phase, the cells
enter the log-growth phase. As a general
rule, 1 volume of overnight culture (the
inoculum) is added to 100 volumes of
fresh LB broth in an Erlenmeyer flask.
To provide good aeration for bacterial
growth, the flask volume should be at
least four times the total culture volume.
Prepare culture:
1. Sterilely transfer 1ml of overnight culture into 100ml of LB broth at room temperature.
2. If using a 1-ml overnight culture:
a. Remove cap from overnight culture tube, and flame mouth. Do not place cap on lab bench.
b. Remove foil cap from flask, and flame mouth. Do not place cap on lab bench.
c. Pour entire overnight culture into flask. Reflame mouth of flask, and replace foil cap.
3. Incubate at 37 °C with continuous shaking.
4. Store mid-log culture in the refrigerator until ready to use. This slows cell growth. Cells can be
stored on ice for up to 2 hours prior to use. This arrests cell growth.
Note: We will be using this culture for E. coli DNA extraction Lab.
5. Take time for responsible cleanup.
Questions:
1. What variables influence the length of time for an E. coli Culture to reach mid-log
phase?
2. What are the disadvantage of beginning a mid-log culture from a colony scraped off
a plate, as opposed to an inoculum of over night culture?
Definition: Define following words.
 Nutrient Agar:
 Ampicillin:
 Stab Culture:
 Slant Culture: