Experiment two Cultivation Techniques of microorganism
Cultivation Techniques of microorganism Culture medium Inoculation
and transfer techniques Examination of Microbial Flora Examination
of Microbial Flora Examination of bacteria in the air Examination
of bacteria in the Skin Examination of bacteria in the Throat
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Cultivation Techniques of microorganism
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Culture medium concept: concept: is the mixture of various
nutrients that is suitable for the growth of microorganisms. is the
mixture of various nutrients that is suitable for the growth of
microorganisms.
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Culture medium Classification of culture medium Classification
of culture medium According to basic ingredients: Minimal essential
growth medium Minimal essential growth medium enrichment medium
enrichment medium selective medium selective medium differential
medium differential medium According to physical condition: liquid
medium liquid medium solid medium : 1.5 2.5% of agar solid medium :
1.5 2.5% of agar semisolid medium : 0.3 0.5% of agar semisolid
medium : 0.3 0.5% of agar
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Culture medium Classification of culture medium Classification
of culture medium Phenomena of bacterial growth: In liquid medium
surface growth pellicle, uniformly turbid, sediment in bottom In
liquid medium surface growth pellicle, uniformly turbid, sediment
in bottom On solid medium On solid medium Confluent growth Colony
In semisolid medium In semisolid medium Only grow along the line of
inoculation Grow diffusely
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Inoculation and transfer techniques Streak plate
technique------isolation and culture Slant inoculation-------pure
culture Liquid medium inoculation ------pure culture Semisolid
medium inoculation ------pure culture
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Streak Plate Method PURPOSE: PURPOSE: Isolation and culture of
bacteria growing together in a specimen Isolation and culture of
bacteria growing together in a specimen MATERIALS: MATERIALS: l.
Mixed broth culture of Escherichia coli l. Mixed broth culture of
Escherichia coli and staphylococcus aureus. and staphylococcus
aureus. 2. Nutrient agar plate. 2. Nutrient agar plate.
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Streak Plate Method PROCEDURE: PROCEDURE: Flame your
inoculating loop until the wire glows red. glows red. Allow the
loop to cool and get a loopful of the suspension of sample. Pick up
your plate and streak the surface, Flame the loop before streaking
next section. When streaking, be care not to cut into the agar and
not to be far away from flame.
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Streak Plate Method PROCEDURE: PROCEDURE: Cover the petridish,
invert the plate. Sterilize the loop, label your name, date et al.
Incubate the plate at 37 for 18- 24 hours. Observe the bacterial
colonies. colonies.
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Streak Plate Method RESULT: RESULT: observe the location and
characteristics of the bacterial colonies: observe the location and
characteristics of the bacterial colonies: Size Size Shape:
circular, irregular, spreading Shape: circular, irregular,
spreading Color Color Density: transparent, or opaque Density:
transparent, or opaque Elevation Elevation Margin Margin Hemolysis
Hemolysis Surface: rough or smooth, dry or moist. Surface: rough or
smooth, dry or moist. Bacillus subtilis Proteus vulgaris
Agar Slope Method MATERIALS : MATERIALS : 1. Agar slope 1. Agar
slope 2. Colonies on agar plate 2. Colonies on agar plate PROCEDURE
: PROCEDURE : 1. With the flame-sterilized wire inoculating loop,
1. With the flame-sterilized wire inoculating loop, transfer a
small amount of bacteria from the transfer a small amount of
bacteria from the colony on agar plate. Then streak on the agar
slope. colony on agar plate. Then streak on the agar slope. 2.
Sterile the mouth of tubes, replug the test tubes and flame the
loop. 3. Label and incubate at 37 for 18-24 hours 4. Observe your
result.
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Agar Slope Method RESULTS : RESULTS : There are many similar
wet colonies on the surface. If there are some other forms, it
indicates culture sample is not pure. There are many similar wet
colonies on the surface. If there are some other forms, it
indicates culture sample is not pure.
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Liquid Medium Culture MATERIALS MATERIALS 1. Peptone water 1.
Peptone water 2. Colonies on agar plate 2. Colonies on agar plate
PROCEDURE PROCEDURE 1. Flame -sterilize the wire inoculating loop.
1. Flame -sterilize the wire inoculating loop. 2. Insert the wire
loop containing a small amount 2. Insert the wire loop containing a
small amount of bacteria into the liquid culture robe. of bacteria
into the liquid culture robe. 3. Scratch the wall of tube over the
broth in order 3. Scratch the wall of tube over the broth in order
to let bacteria drop into the liquid. to let bacteria drop into the
liquid.
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Liquid Medium Culture PROCEDURE PROCEDURE 4. Flame the mouth of
the tube and reinsert the 4. Flame the mouth of the tube and
reinsert the cotton plug. Flame-sterilize the wire loop. cotton
plug. Flame-sterilize the wire loop. 5. Label the tube, incubate at
37 for 24 hours 5. Label the tube, incubate at 37 for 24 hours 6.
Observe the result. 6. Observe the result. RESULTS: RESULTS:
turbid, sediment, pellicle turbid, sediment, pellicle
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pellicle sediment turbid contrast
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Semisolid medium Culture METHODS: METHODS: 1. Flame-sterilize
inoculating needle. 1. Flame-sterilize inoculating needle. 2.
Insert the needle with a small bacteria to the 2. Insert the needle
with a small bacteria to the center of the culture, be care not to
touch the center of the culture, be care not to touch the bottom of
the tube, then draw it out in the same way. bottom of the tube,
then draw it out in the same way. 3. Flame the mouth of the tube
and reinsert the cotton 3. Flame the mouth of the tube and reinsert
the cotton plug. Flame-sterilize the needle. plug. Flame-sterilize
the needle. 4. Label the tube, incubate for 24 hours at 37 . 4.
Label the tube, incubate for 24 hours at 37 . 5. Observe the
result. 5. Observe the result.
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Semisolid medium Culture RESULTS: RESULTS: Motile bacteria will
migrate from the line of inoculation to form a diffuse turbidity in
the surrounding medium; nonmobile bacteria will grow only along the
line of inoculation.
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Examination of Microbial Flora
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Examination of bacteria in air METHODS: METHODS: 1. Take a
nutrient agar plate, choose any place inside, 1. Take a nutrient
agar plate, choose any place inside, open the plate, expose it in
air for 5 minutes, open the plate, expose it in air for 5 minutes,
2. cover it and mark place, class and group.. 2. cover it and mark
place, class and group.. 3. Observe the results after cultivation
in a incubator for 3. Observe the results after cultivation in a
incubator for 24 hours. 24 hours. RESULTS RESULTS Count colonies
which grow in the agar plate. Count colonies which grow in the agar
plate.
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Examination of bacteriain the Skin PROCEDURE : PROCEDURE : 1.
Soak sterile cotton swab in sterile saline for a moment, scrub
finger or skin of forearm with the swab for several times, then
streak on the half surface of a agar plate. 1. Soak sterile cotton
swab in sterile saline for a moment, scrub finger or skin of
forearm with the swab for several times, then streak on the half
surface of a agar plate. 2. Disinfect your skin by 2.5% tincture of
iodine and 75% alcohol, repeat the former step by streaking the
cotton swab on the other half surface of the agar plate. 2.
Disinfect your skin by 2.5% tincture of iodine and 75% alcohol,
repeat the former step by streaking the cotton swab on the other
half surface of the agar plate. 3.Incubate plates for 18~24 hours
at 37 observe the result. 3.Incubate plates for 18~24 hours at 37
observe the result. RESULTS RESULTS Compare the distribution of
bacteria before and after the disinfection. Compare the
distribution of bacteria before and after the disinfection.
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before the disinfection after the disinfection. Incubate plates
for 18~24 hours at 37
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Examination of bacteria in the Throat PROCEDURE : PROCEDURE :
1. Label a blood agar plate with the initial 1. Label a blood agar
plate with the initial 2. Held a blood agar plate 15-cm distant
from the mouth 2. Held a blood agar plate 15-cm distant from the
mouth with the dominant hand. with the dominant hand. 3. Rapidly
remove the cover of the plate with another hand, cough heavily to
the exposed blood agar 3 and 4 times to ensure mucous in the throat
will drop to the agar surface, and then immediately close the
plate. 4. Incubate the plate in an inverted position for 18~24
hours at 37 . RESULTS RESULTS Observe the growth of various kinds
of bacteria Observe the growth of various kinds of bacteria