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.)

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

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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11

Four-way streak-plate inoculation

Cappuccino,JG.,N. Sherman , Microbiology: A Laboratory Manual. Eight edition, 2008. Pearson Benjamin Cummings Publishers.

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

Non-hemolytic colonies on blood agar example Enterococcus faecalis

Beta hemolysis on blood agar example Staphylococcus aureus

Lactose fermenter colonies on MacConkey agar Example: E.coli

Lactose fermenter colonies on MacConkey agar Example: Klebsiella pneumoniae

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

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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