Transcript Microbial Biotechnology

Microbial Biotechnology
Reem Alsharief
Lab 3
General Methods of Isolation and
selection of Microorganism
•Microbial isolation: To separate (a pure strain)
from a mixed bacterial or fungal culture.( to
obtain a pure culture)
•A microbiological culture(or microbial culture)
:is a method of multiplying microbial organisms
by letting them reproduce in predetermined
culture media under controlled laboratory
conditions.
Purpose of Culturing
• To determine the type of organism and its
abundance in the sample being tested.
• It is one of the primary diagnostic methods of
microbiology and used as a tool to determine
the cause of infectious disease by letting the
agent multiply in a predetermined media.
• To study the properties of bacteria
• To create antigens for laboratory use
• To test for Antibiotic sensitivity
•Typing with Bacteriophages and Bacteriocins
susceptibility.
Bacterial Culture Streaking
Bacterial culture streaking allows bacteria to
reproduce on a culture medium in a controlled
environment.
The process involves spreading bacteria across
an agar plate and allowing them to incubate at
a certain temperature for a period of time.
Bacterial streaking can be used to identify and
isolate pure bacterial colonies from a mixed
population.
A Streak Plate
A single bacterial colony
Obtaining single colonies is important as it
enables
• The size,
•The shape
• Colour of the individual colonies to be
examined.
• It can also highlight the presence of
contaminating micro-organisms
1. Streak Plate Methods
A. Crossing Shape ( The quadrant streak
technique)
Tools you need:
• Gloves
• Inoculating Loop (or sterile toothpick)
• Bunsen Flame
• Agar plates
•
Culture media with microorganism
Technique:
1. Flame the inoculating loop until it is red hot
and then allow it to cool
2. Remove a small amount of bacterial growth
with a sterile inoculating loop (either from a
broth, single colony from a plate or a slant)
3. Immediately streak the inoculating loop very
gently over a quarter of the plate using back
and forth motion
4. Flame the loop again and allow it to cool.
Going back to the edge of area 1 that you just
streaked, extend the streaks into the second
quarter of the plate (area 2).
5. flame the loop again and allow it to cool.
Going back to the area that you just streaked
(area 2), extends the streaks into the third
quarter of the plate (area 3)
6. Flame the loop again and allow it to cool.
Going back to the area that you just streaked
(area 3), extends the streaks into the center
fourth of the plate (area 4)
Quadrant streak
B. Zigzag or Snake shape technique
Procedure:
1. While wearing gloves, sterilize an inoculating loop
by placing it at an angle over a flame. The loop
should turn orange before you remove it from the
flame. A sterile toothpick may be substituted for
the inoculating loop. Do not place toothpicks over
a flame.
2. Remove the lid from a culture plate containing the
desired microorganism.
3. Cool the inoculating loop by stabbing it into the
agar in a spot that does not contain a bacterial
colony.
4. Pick a colony and scrape off a little of the
bacteria using the loop. Be sure to close the
lid.
5. Using a new agar plate, lift the lid just enough
to insert the loop.
6. Streak the loop containing the bacteria at the
top end of the agar plate moving in a zig-zag
horizontal pattern until 1/3 of the plate is
covered.
7. Sterilize the loop again in the flame and cool it
at the edge of the agar away from the
bacteria in the plate that you just streaked.
8. Rotate the plate about 60 degrees and spread the bacteria
from the end of the first streak into a second area using
the same motion in step 6.
9. Sterilize the loop again using the procedure in step 7.
10. Rotate the plate about 60 degrees and spread the
bacteria from the end of the second streak into a new
area in the same pattern.
11. Sterilize the loop again.
12. Replace the lid and invert the plate. Incubate the plate
overnight at 37 degrees Celsius (98.6 F̊).
13. You should see bacterial cells growing in streaks and in
isolated areas
ZigZag Shape
2. Serial dilution for bacterial
enumeration
An easier method is to spread bacteria over a wide area (i.e.
nutrient agar plate) and count the number of colonies that
grow. If the bacteria are spread out enough, each bacterial
cell in the original sample should produce a single colony.
Bacterial cell numbers need to be reduced, which is done by
repeatedly diluting the amount of bacteria you have in your
sample. A small amount of bacteria sample is mixed with a
diluent solution (such sterile broth), and then successive
dilutions are made. A small amount of each of the diluted
bacteria samples is then spread onto an agar plate. The
numbers of bacteria colonies that grow on each plate are
counted.
Materials:
• Gloves.
• Mixed bacterial culture.
• 6 tubes of distilled water ( each tube contains
9ml of dis. Water)
• 12 Petri dishes ( 2 per 1 tube sample)
• Nutrient agar media ( cooled at 45 ̊C in a
water bath)
• Pump Pipettes.
• Bunsen flame
Procedure:
1. One milliliter from the sample was transferred to
9 ml of distilled water in tube A and mixed. This was a
1/10 serial dilution.
2. One milliliter from tube A will be transferred to 9 ml of
distilled water in tube B and mixed. This was
another 1/10 serial dilution. The total dilution up to this
point is 1/100.
3. Then 1 ml from tube B will be transferred to 9 ml of
sterile media in tube C and mixed. This is a 1/10
serial dilution. The total dilution up to this point is
1/1000.
4. Continue the serial of dilution until the total
dilution reach up to 1/1000 000 (10 ̄⁶)
Tip: change the Pipette each time you dilute the
solution.
5. Pour about 15-20 ml of the nutrient agar into each
dish and shake it slightly in a circular round.
6. Incubate the agar plates in a 37 degree incubator
and you should see the bacterial colonies in 24-48
hours.
7. Count the number of bacteria colonies that appear
on each of the plates that has between 30 and 200
colonies. Any plate which has more than 200 colonies
is designated as "too many to count" (TMTC). Plates
with fewer than 30 colonies do not have enough
individuals to be statistically acceptable.
8. To compute the estimated number of bacteria
on the surface that you tested, use the following
formula:
B = N/D
B = number of bacteria
N = number of colonies counted on a plate
D = dilution factor (either 1, 10 or 100)
Thank You