Culturing and Isolation Techniques

1
Culturing and Isolation Techniques

• Bacteria require a constant nutrient supply to
  survive and grow.
• Acquire nutrients from their surroundings
  (free-living) or from a host (parasites).
• Artificial media is used to grow bacteria in a
  lab (in vitro).
• Agar is extracted from marine algae.
• A carbohydrate that cross-links to form a
  semi-solid mesh.
• Melts at 100 C, solidifies at 42 C, but will
  remain a liquid at 60 C.
• Most pathogenic bacteria have an optimum growth
  temperature of
• 37 C (human body temp.)
• Organisms grown in broth cultures are apparent
  through the turbidity that the large numbers of
  cells produce in the broth.
• On agar, a solid medium, the bacterial cells form
  masses called colonies after about 18 24 hours
  of growth.
• Colonies represent one viable cell or Colony
  Forming Unit (CFU) that came to rest on the agar
  surface. This cell or CFU divides many times to
  form visible colonies on the agar.
• Isolated colonies, meaning those not touching
  other colonies, represent clones of the original
  cell or CFU since all the cells in the colony
  were derived from one cell or CFU and are
  genetically identical.
• Isolated colonies are considered to be pure
  cultures of a particular bacterial species and
  strain.

2
Colony Morphology
3
Inoculation of a Broth Culture

• Label the sterile nutrient broth with the source
  of the culture, your initials and the date.
• Sterilize a loop in the Bacticinerator.
• Using appropriate aseptic technique, remove a
  loop-ful of broth from the mixed culture tube.
• Insert the loop into the sterile broth and gently
  swirl. Retract the loop and sterilize it in the
  Bacticinerator.
• Incubate the broth at 37 C for 24 48 hours.
• Observe the broth culture for turbidity. Record
  the results in the Table on page 30 in the lab
  book.

Compare your inoculated broth tube to
the un-inoculated control tube to determine
the amount of turbidity. The more turbid the
broth The higher the bacterial count per mL of
broth.
4
Inoculating an Agar Slant

• Label the sterile nutrient agar slant with the
  source of the culture, your initials, and the
  date.
• Sterilize the loop using the bacticinerator.
• Using appropriate aseptic technique, remove a
  loopful of broth from the mixed culture tube.
• Insert the loop into the sterile agar slant tube
  and starting at the base of the slant (closest to
  the bottom of the tube), very lightly draw the
  loop in a zig-zag motion up the slant. Do not dig
  into the agar. Sterilize the loop in the
  bacticinerator.
• Incubate the slant at 37 C for 24 48 hours.
• In the following lab observe the slant for
  growth. Record the results in the table on page
  30 in the lab book.

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Streak Plates

• Allow for the growth of isolated colonies on the
  surface of the agar.
• Used to isolate clones of a particular bacterial
  species/strain.
• An isolated colony, one that is not touching any
  other colonies, is assumed to be a pure culture.
• May observe colony morphology that can be used to
  help identify the bacterial species.
• Colonies of the same organism may grow
  differently on different media, e.g. the shape,
  color, growth pattern of the colony may differ on
  other types of media.
• Colony Morphology Characteristics
• Colony color
• Type of hemolysis (if grown on Sheep Blood Agar)
• Form
• Elevation
• Margin

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Streak Plates
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Pour Plating for Colony Counts

• One of the most common methods of determining
  cell number is the viable plate count.
• A sample to be counted is diluted in a solution
  that will not harm the microbe.
• In most cases a volume of liquid from the sample
  is first diluted 10-fold.
• In most cases, a 0.1-1.0 ml portion of this first
  dilution is then diluted a further 10-fold,
  giving a total dilution of 100-fold.
• This process is repeated until a concentration
  that is estimated to be about 1000 cells per ml
  is reached.
• One method for counting viable bacteria is the
  pour plate technique, which consists of mixing a
  portion of the dilution with molten agar and
  pouring the mixture into a sterile plate.
• The sample serially diluted and individual cells
  are deposited in the molten agar and these give
  rise to colonies.
• By counting each colony, the total number of
  colony forming units (CFUs) on the plate is
  determined.
• By multiplying this count by the total dilution
  of the solution (dilution factor), it is possible
  to find the total number of CFUs in the original
  sample.

8
Calculating CFU from dilution plating results

• How does a count on a plates get converted to
  CFUs per gram or ml of sample? Let's illustrate
  the procedure with an example. Imagine that we
  perform the following experiment
• Five ml of milk are added to 45 ml of sterile
  broth. From this suspension, two serial, 1/100
  dilutions are made, and 0.1 ml is plated onto
  Plate Count Agar from the last dilution. After
  incubation, 137 colonies are counted on the
  plate.
• This problem may be illustrated as follows

The initial dilution is calculated As follows
So the initial dilution is 1/10 or 0.1 or 10-1.
9

• Remember, there are many ways to make 1/10 and
  1/100 dilutions.
• A 0.1 ml to 0.9 ml dilution is the same as a 1 ml
  to 9 ml dilution and a 13 ml to 117 ml dilution.
• Next, 1 ml of the first dilution is added to 99
  ml to make the second dilution, that is a 1/100
  dilution.
• This is repeated with third dilution giving
  another 1/100 dilution.
• Then 0.1 ml of the third dilution is plated out
  on a sterile plate.
• To obtain the concentration of bacteria in the
  original sample the dilution factor must be
  determined and then multiplied by the plate
  colony count.

The total dilution may be calculated
mathematically as follows
10

• The dilution factor is the inverse of the total
  dilution

Therefore, the Dilution factor for our example
is 106 and the

• Total Colony Forming Units (CFUs) for this sample
  is