Lab Exercises Week 2:

#2 Pure Culture#7 Defined and Undefined#22 Normal Skin Biota#6 Differential and Special Stains (Gram- stain)

Pre lab due: 1/17/15Post lab due: 1/31/15

Pure culture defined as population of cells derived from a single cell• Allows study of single species

Pure culture obtained using aseptic technique• Minimizes potential contamination

Cells grown on culture medium• Contains nutrients dissolved in water• Can be broth (liquid) or solid gel

4.3. Obtaining a Pure Culture

Need culture medium, container, aseptic conditions, method to separate individual cells• With correct conditions, single cell will multiply• Form visible colony (~1 million cells easily visible)• Agar used to solidify

• Not destroyed by high temperatures

• Liquifies above 95°C

• Solidifies below 45°C

• Few microbes can degrade

• Growth in Petri dish• Excludes contaminants

• Agar plate

Growing Microorganisms on a Solid Medium

Streak-plate method• Simplest, most commonly used method for isolating• Spreads out cells to separate

• Obtain single cells so that individual colonies can form

Growing Microorganisms on a Solid Medium

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Sterilizeloop.

Dip loopInto culture.

Streak first area.

Agar containingnutrients

Starting point

Streak second area.

Sterilizeloop.

Sterilizeloop.

General categories of culture media• Complex media contains variety of ingredients

• Exact composition highly variable

• Often a digest of proteins

• Chemically defined media composed of exact amounts of pure chemicals

• Used for specific research experiments

• Usually buffered

4.7. Cultivating Prokaryotes in the Laboratory

Hundreds of types of media available• Regardless, some

medically important microbes, and most environmental ones, have not yet been grown in laboratory

4.7. Cultivating Prokaryotes in the Laboratory

Special types of culture media• Useful for isolating and identifying

a specific species• Selective media inhibits growth of

certain species

4.7. Cultivating Prokaryotes in the Laboratory

• Differential media contains substance that microbes change in identifiable way

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(a) (b)

Zone of clearingColony

a: © Christine Case/Visuals Unlimited; b: © L. M. Pope and D. R. Grote/Biological Photo Service

Simple staining involves one dye Differential staining used to distinguish different

types of bacteria

3.2. Microscopic Techniques: Dyes and Staining

Acid-fast staining used to detect Mycobacterium• Includes causative agents of tuberculosis and

Hansen’s disease (leprosy)• Cell wall contains high concentrations of mycolic acid

• Waxy fatty acid that prevents uptake of dyes

• Harsher methods needed

• Used to presumptively identify clinical specimens

3.2. Microscopic Techniques: Dyes and Staining

Capsule stain Some microbes surrounded by gel-like layer

• Stains poorly, so negative stain often used• India ink added to wet mount is common method

3.2. Microscopic Techniques: Dyes and Staining

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10 m

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Flagella stain Flagella commonly used for prokaryotic motility

• Too thin to be seen with light microscope• Flagella stain coats flagella to thicken and make visible• Presence and distribution can help in identification

3.2. Microscopic Techniques: Dyes and Staining

1 m

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Endospore stain Members of genera including Bacillus, Clostridium

form resistant, dormant endospore• Resists Gram stain, often appears as clear object• Endospore stain uses heat to facilitate uptake of primary

dye (usually malachite green) by endospore• Counterstain (usually safranin) used to visualize other

cells

3.2. Microscopic Techniques: Dyes and Staining

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Gram stain most common for bacteria Two groups: Gram-positive, Gram-negative

• Reflects fundamental difference in cell wall structure

3.2. Microscopic Techniques: Dyes and Staining

Crystal violet(primary stain)

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Cells stain purple.

Cells remain purple.Iodine(mordant)

Safranin(counterstain)

Gram-positive cellsremain purple;Gram-negative cellsappear pink.

Alcohol(decolorizer)

Gram-positive cellsremain purple;Gram-negative cellsbecome colorless.

State of Bacteria AppearanceSteps in Staining

(a) (b) 10 µm

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b: © Leon J. Le Beau/Biological Photo Service

Gram-positive cell wall has thick peptidoglycan layer

The Gram-Positive Cell Wall

(a)

(c)

CytoplasmicmembranePeptidoglycan

Gram-positive

(b)

Gel-likematerial

Peptidoglycanand teichoic acids

Cytoplasmicmembrane

Cytoplasmicmembrane

Peptidoglycan(cell wall)

Gel-likematerial

N-acetylglucosamine N-acetylmuramic acid Teichoic acid

0.15 µm

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(c): © Terry Beveridge, University of Guelph

Gram-negative cell wall has thin peptido-glycan layer

Outside is unique outer membrane

Periplasm LPS

The Gram-Negative Cell Wall

Lipoprotein

Peptidoglycan

(a)

Peptidoglycan

Cytoplasmicmembrane

(d)

Lipopolysaccharide(LPS)

Porin protein

Outermembrane(lipid bilayer)

Periplasm

Cytoplasmicmembrane(inner membrane;lipid bilayer)

PeptidoglycanOuter

membranePeriplasm

Cytoplasmicmembrane

(c)

Periplasm

Outermembrane

Lipid A

Core polysaccharide

O antigen(varies in length andcomposition)

0.15 µm

(b)

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(d): © Terry Beveridge, University of Guelph

Crystal violet stains inside of cell, not cell wall• Gram-positive cell wall prevents crystal violet–iodine

complex from being washed out• Decolorizing agent thought to dehydrate thick layer of

peptidoglycan; desiccated state acts as barrier

• Solvent action of decolorizing agent damages outer membrane of Gram-negatives

• Thin layer of peptidoglycan cannot retain dye complex

Cell Wall Type and the Gram Stain