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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

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Streptococcus pyogenes Staphylococcus aureus mucoid

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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