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Experiment two
• Cultivation Techniques of microorganism
– Culture medium
– Inoculation and transfer techniques
• Examination of Microbial Flora
– Examination of bacteria in the air
– Examination of bacteria in the Skin
– Examination of bacteria in the Throat
Cultivation Techniques
of microorganism
Culture medium
• concept:
is the mixture of various nutrients that is
suitable for the growth of microorganisms.
Culture medium
• Classification of culture medium：
– According to basic ingredients:
• Minimal essential growth medium
• enrichment medium
• selective medium
• differential medium
– According to physical condition:
• liquid medium
• solid medium: 1.5～2.5% of agar
• semisolid medium: 0.3～0.5% of agar
Culture medium
• Classification of culture medium：
– Phenomena of bacterial growth:
• In liquid medium：surface growth pellicle,
uniformly turbid, sediment in bottom
• On solid medium ：
– Confluent growth
– Colony
• In semisolid medium
– Only grow along the line of inoculation
– Grow diffusely
Inoculation and transfer techniques
 Streak plate technique------isolation and culture
 Slant inoculation-------pure culture
 Liquid medium inoculation ------pure culture
 Semisolid medium inoculation ------pure culture
Streak Plate Method
• PURPOSE:
Isolation and culture of bacteria growing
together in a specimen
• MATERIALS:
l. Mixed broth culture of Escherichia coli
and staphylococcus aureus.
2. Nutrient agar plate.
Streak Plate Method
• PROCEDURE:
– Flame your inoculating loop until the wire
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.
Streak Plate Method
• PROCEDURE:
– Cover the petridish, invert the
plate. Sterilize the loop, label
your name, date et al.
– Incubate the plate at 37℃ for 1824 hours.
– Observe the bacterial
colonies.
Streak Plate Method
• RESULT:
observe the location and
characteristics of the bacterial
colonies:
Size
Shape: circular, irregular, spreading
Color
Density: transparent, or opaque
Elevation
Margin
Hemolysis
Surface: rough or smooth, dry or moist.
Bacillus subtilis
mucoid
Staphylococcus aureus
Streptococcus pyogenes
Agar Slope Method
• MATERIALS :
1. Agar slope
2. Colonies on agar plate
• PROCEDURE :
1. With the flame-sterilized wire inoculating loop,
transfer a small amount of bacteria from the
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.
Agar Slope Method
• RESULTS :
There are many similar wet colonies on
the surface. If there are some other
forms, it indicates culture sample is not
pure.
Liquid Medium Culture
• MATERIALS：
1. Peptone water
2. Colonies on agar plate
• PROCEDURE：
1. Flame -sterilize the wire inoculating loop.
2. Insert the wire loop containing a small
amount
of bacteria into the liquid culture robe.
3. Scratch the wall of tube over the broth in
order
Liquid Medium Culture
• PROCEDURE：
4. Flame the mouth of the tube and reinsert
the
cotton plug. Flame-sterilize the wire
loop.
5. Label the tube, incubate at 37℃ for 24
hours
6. Observe the result.
• RESULTS:
turbid, sediment, pellicle
pellicle
contrast
sediment
turbid
Semisolid medium Culture
• METHODS:
1. Flame-sterilize inoculating needle.
2. Insert the needle with a small bacteria to the
center of the culture, be care not to touch the
bottom of the tube, then draw it out in the
same way.
3. Flame the mouth of the tube and reinsert the
cotton
plug. Flame-sterilize the needle.
4. Label the tube, incubate for 24 hours at 37℃.
5. Observe the result.
Semisolid medium Culture
• 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.

Examination of
Microbial Flora
Examination of bacteria in air
• METHODS:
1. Take a nutrient agar plate, choose any place
inside,
open the plate, expose it in air for 5 minutes,
2. cover it and mark place, class and group. .
3. Observe the results after cultivation in a
incubator for
24 hours.
• RESULTS
Count colonies which grow in the agar plate.
Examination of bacteriain the Skin
• 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.
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.
• RESULTS
before the
disinfection
after the
disinfection.
Incubate plates for 18~24 hours at 37℃
Examination of bacteria in the Throat
• PROCEDURE :
1. Label a blood agar plate with the initial
2. Held a blood agar plate 15-cm distant from the
mouth
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℃.