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Culture Lab
Week 2
2013
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Outline Plating Techniques
Types of culturing techniques
• Pour-plating
• Spread-plating
• Streak-plating
Used for: • Determining bacterial concentrations
• Isolating colonies & purifying cultures
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Pour-plate technique
• Main purpose: To estimate bacterial cell numbers
• A known volume of a bacterial sample is introduced into a Petri dish.
• The nutrient medium, in which the agar is kept liquid by holding it in a water bath at about 50⁰C, is poured over the sample, which is then mixed into the medium by agitation of the plate.
• When the agar solidifies, the plate is incubated.
• After incubation, count colonies on plates that have 25-250 colonies (CFUs).
• Colonies will grow within the agar as well as on the surface of the agar plate.
• Usually the concentration of bacterial cells is unknown so multiple plates are poured in a dilution gradient to achieve a statistically valid plate count.
• This procedure is usually used in food and environmental microbiology.
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Pour-plate technique
• The progenitor from which a particular pure culture is derived may be either a single cell or a group of related cells; therefore, the progenitor is termed a colony-forming unit (CFU).
• By knowing the colony count and dilution factor, the concentration
of CFU/mL can be determined.
• For example: If 1 mL produces 32 colonies at a dilution factor of 104, the concentration is 3.2 x 105 CFU/mL.
• CFU/mL is almost always smaller than # of cells/ml from a microscopic count (Why?)
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Serial dilutions and plate counts
Original
inoculum
1 ml 1 ml 1 ml 1 ml 1 ml
9 ml
broth
in each tube
Dilutions 1:10 1:100 1:1000 1:10,000 1:100,000
1 ml 1 ml 1 ml 1 ml 1 ml
1:10 1:100 1:1000 1:10,000 1:100,000 (10-1) (10-2) (10-3) (10-4) (10-5)
Plating
Calculation: Number of colonies on plate × reciprocal of dilution of sample = number of bacteria/ml
(For example, if 54 colonies are on a plate of 1:1000 dilution, then the count is 54 × 1000 = 54,000 bacteria/ml in sample.)
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Spread-plate Technique
• Main purpose:
– To estimate bacterial cell numbers
• A known volume of bacterial sample is plated onto a solid agar
• A bent glass rod (hockey stick) is used to spread the liquid over the entire surface of the agar
• Again, dilution gradients are used
• After incubation, count colonies on plates that have 25–250 colonies (CFUs)
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Streak-plate technique
• Main purpose: To achieve isolated colonies through a dilution gradient by streaking, especially from a mixed population
• In this technique, a sterile inoculating loop is used to spread an inoculum across the surface of a solid medium in Petri dishes
• The loop is used to lightly streak a set pattern that gradually dilutes (thins out) the sample to a point that CFUs are isolated from one another
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Streak-plate technique
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Isolated colonies
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Streak-plate technique cont’d
• After incubation, cells should give rise to isolated colonies
• The various types of organisms present are distinguished from one another by differences in colony characteristics
• Isolated colonies can be re-streaked on fresh plates to ensure purity and subjected to testing to identify the microbe – Biochemical, immunological, molecular, etc.
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How the Technique is Performed
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Four-way streak-plate inoculation
Cappuccino,JG.,N. Sherman , Microbiology: A Laboratory Manual. Eight edition, 2008. Pearson Benjamin Cummings Publishers.
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Types of Media Used in Our Lab
• Complex Media
• Differential Media
• Selective Media
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Lab activity
You will receive: – Broth culture of a mixture of E.coli (a gram negative bacilli) and
Staphylococcus aureus* (a gram positive cocci). – 2 petri plates (Nutrient agar and MacConkey agar).
You will: – Use the streak plate method on both plates to isolate individual colonies.
You should: – Record your observations next lab (Refer to lab manual pages 97-99). Assigned Reading: Lab manual pages 83-95 (Use of differential, selective, and enriched media, and experiment 12). Experiment 11 & 13 procedures are NOT included. * Staphylococcus aureus is used in this experiment instead of Micrococcus luteus.
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Some bacterial colonies characterizations
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Demo plates
At the back bench
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The use of blood agar as a differential medium
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Beta hemolytic colonies
Alpha hemolytic colonies
Non(gamma)-hemolytic colonies
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Non-hemolytic colonies on blood agar example Enterococcus faecalis
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Beta hemolysis on blood agar example Staphylococcus aureus
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Lactose fermenter colonies on MacConkey agar Example: E.coli
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Lactose fermenter colonies on MacConkey agar Example: Klebsiella pneumoniae
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Non-Lactose fermenter colonies on MacConkey agar Example: Pseudomonas aeruginosa
21 Arvidson, C. et al. Cultivation media for bacteria. http://www.microbelibrary.org/library/laboratory-test/2782-cultivation-media-for-bacteria
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Clicker Question
Today’s plating technique is the:
A: Pour plate
B: Streak plate
C: Spread plate
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Clicker Question
MacConkey agar is an example of a _____________ media
A: Differential
B: Selective
C: All-purpose nutrient media
D: Both A and B
E: All of the above
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Clicker Question
On MacConkey agar, lactose-fermenting bacteria appear _____________ and non-lactose fermenters appear ______________
A: red/pink; translucent
B: translucent; red/pink
C: blue; red/pink
D: red/pink; blue
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References
• Tortora G, Funke B and Case C. Microbiology: An Introduction. 11th edition. Pearson- Benjamin Cummings.
• Cappuccino,JG.,N. Sherman , Microbiology: A Laboratory Manual. Eight edition, 2008. Pearson Benjamin Cummings Publishers.
• Levinson, W. Review of Medical Microbiology and Immunology. Ninth edition, 2006. McGraw-Hill Publication.
• Bauman RW. Microbiology with diseases by body system. Second edition.2009. Pearson Benjamin Cummings Publications.
• Cappuccino,J.G. & N.Sherman. Microbiology: A Laboratory Manual. Eighth edition, 2008. Pearson Benjamin / Cummings Publishers.
• Harley, P.J. Laboratory Exercises in Microbiology. Seventh edition, 2008. Published by McGraw-Hill Higher Education.
Websites: • http://www.asm.org/ • http://www.medmaster.net/atlasofmicrobiol.html • http://www.accessmedicine.com/resourceTOC.aspx?
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