Lab 11: Isolation of Bacteria by Dilution Techniques – Flashcards
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pure culture
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single microbe; study growth, pathogenicity, metabolism, antibiotic susceptibility, or other characteristics
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Joseph Lister
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attempted to obtain pure cultures by performing serial dilutions until each of his container theoretically had one bacterium
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contamination
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presence of unwanted microorganisms
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isolating bacteria
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three dilution methods commonly used for isolating bacteria: streak plate technique, spread plate technique, and pour plate technique
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streak plate technique
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loop is used to streak the mixed sample many times over the surface of a solid culture medium in a Petri dish. Streaking is suppose to cause the bacteria to fall off the loop and ultimately to be distributed over the agar's surface; most common isolation technique used today to form colonies
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spread plate technique
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used to determine the number of bacteria in a sample; sample must be diluted; a small amount of a previously diluted specimen is spread over the surface of a solid medium using a spreading rod; after incubation bacteria is visible as colonies in and on agar plate
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pour plate technique
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used to determine the number of bacteria in a sample; a small amount of diluted sample (diluted from mixed broth culture 10x10, 10x10x10x10, and 10x10x10x10x10x10x10x10) is mixed with melted agar (45 °C) and poured into empty, sterile Petri dishes; after incubation bacteria is visible as colonies in and on agar plate
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What will a streak plate with two species of bacteria look like?
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Bacteria will be in a straight line, the will be separate from one another.
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Where will colonies be located in the pour plate?
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Throughout the agar
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Where will colonies be the largest?
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On the surface because they have more room to grow.
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mixed culture colony description:
1st quadrant
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diameter: 5mm
whole colony appearance: irregular
margin: lobate
elevation: umbonate
pigment: opaque
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mixed culture colony description:
2nd quadrant
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diameter: 1 cm
whole colony appearance: irregular
margin: undulate
elevation: flat, slight raise
pigment: opaque, slightly darker on raised area
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mixed culture colony description:
3rd quadrant
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diameter: 3 mm
whole colony appearance: circular
margin: entire
elevation: convex
pigment: yellowish
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mixed culture colony description:
4th quadrant
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diameter: 1 cm
whole colony appearance: filamentous
margin: undulate
elevation: flat
pigment: opaque
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Calculate the number of colony-forming units per milliliter in the mixed culture. Using 1:8000 dilution
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number of colonies divided by 1 x 1/8000 (flip: 8000/1)= number of colonies x 8000
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colony forming units per ml
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11,5200,000 or 11.52x10 to sixth power
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How can you tell if there is more than one bacteria in a mixed culture?
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the colony morphology
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How do colonies on the surface of a pour plate differ from those suspended in the agar?
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If they're on the surface they are obligate aerobes- microaerophiles. If in liquid, they are obligate anaerobes -aerotolerant anaerobes.
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How would you determine whether a colony was a contaminant on a streak plate?
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A contaminant would be growing in an area you didn't streak.
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How would you determine whether a colony was a contaminant on a pour plate?
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By looking at the appearance of the colonies to see if there is one that looks different
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What would happen if the plates were incubated a week longer? A month?
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A week: There would be non isolated colonies
A month: Each streak would show ore colonies and they would not be isolated
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What is a disadvantage of the streak plate technique? Of the pour plate technique?
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Streak plate can't accurately count the colonies
In pour plate, the bacteria can clump together and appear to be one colony
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Will isolated colonies always be in the fourth sector on the streak plate?
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No, sometimes you might get them in the 3rd, but usually not the fourth because of clumping colonies.
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Control has 1:400 dilution, 1 ml amount plated, and 160 colonies. What is the CFU/ml?
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160x400= 64,000
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Experimental has 1:200 dilution, 0.1 ml amount plated, and 32 colonies. What is the CFU/ml?
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3200
