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: 1/17/15
Post lab due: 1/31/15
4.3. Obtaining a Pure Culture
 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
Growing Microorganisms on a Solid Medium
 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
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1
Sterilize
loop.
4
Sterilize
loop.
6
2 Dip loop
Into culture.
3 Streak first area.
5 Streak second area.
Starting point
Agar containing
nutrients
Sterilize
loop.
4.7. Cultivating Prokaryotes in the Laboratory
 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
• Differential media contains substance that microbes
change in identifiable way
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Colony Zone of clearing
(a)
(b)
a: © Christine Case/Visuals Unlimited; b: © L. M. Pope and D. R. Grote/Biological Photo Service
3.2. Microscopic Techniques: Dyes and Staining
 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
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10 m
© Dr Gladden Willis/Visuals Unlimited/Getty
3.2. Microscopic Techniques: Dyes and Staining
 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
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1 m
© E. Chan/Visuals Unlimited
3.2. Microscopic Techniques: Dyes and Staining
 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
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10 m
© Jack M. Bostrack/Visuals Unlimited
3.2. Microscopic Techniques: Dyes and Staining
 Gram stain most common for bacteria
 Two groups: Gram-positive, Gram-negative
• Reflects fundamental difference in cell wall structure
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Steps in Staining
1 Crystal violet
State of Bacteria
Appearance
Cells stain purple.
(primary stain)
2 Iodine
Cells remain purple.
(mordant)
3 Alcohol
(decolorizer)
4 Safranin
(counterstain)
(a)
Gram-positive cells
remain purple;
Gram-negative cells
become colorless.
Gram-positive cells
remain purple;
Gram-negative cells
appear pink.
(b)
b: © Leon J. Le Beau/Biological Photo Service
10 µm
The Gram-Positive Cell Wall
 Gram-positive cell wall has thick peptidoglycan layer
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N-acetylglucosamine
N-acetylmuramic acid
Teichoic acid
Peptidoglycan
and teichoic acids
Gel-like
material
Peptidoglycan
(cell wall)
Cytoplasmic
membrane
Gel-like
material
Gram-positive
(b)
Cytoplasmic
membrane
Peptidoglycan
Cytoplasmic
membrane
(a)
(c)
(c): © Terry Beveridge, University of Guelph
0.15 µm
The Gram-Negative Cell Wall
 Gram-negative
cell wall has
thin peptidoglycan layer
 Outside is
unique outer
membrane
 Periplasm
 LPS
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
O antigen
(varies in length and
composition)
Porin protein
Core polysaccharide
Lipid A
Lipopolysaccharide
(LPS)
(b)
Outer
membrane
(lipid bilayer)
Outer
membrane
Peptidoglycan
Lipoprotein
Periplasm
Cytoplasmic
membrane
Peptidoglycan
Periplasm
(c)
Cytoplasmic
membrane
(inner membrane;
lipid bilayer)
Outer
Cytoplasmic
Peptidoglycan membrane Periplasm membrane
(a)
(d)
(d): © Terry Beveridge, University of Guelph
0.15 µm
Cell Wall Type and the Gram Stain
 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