Tools of the Laboratory:
The Methods for Studying Microorganisms
Chapter 2
What are the challenges if you want to study microbes?
• In their nature habitats microbes are found in complex associations with other microbes.
• Microbes are small so to study them you need to isolate them and grow them under artificial conditions.
• Microbes are invisible.
• Microbes are everywhere and they often contaminate your isolated experimental microbes.
The Five I’s of Microbiology
Inoculation
Incubation
Isolation
Inspection
Identification
Major Techniques Performed by Microbiologists to Locate, Grow, Observe, and Characterize Microorganisms
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Major Techniques Performed by Microbiologists to Locate,
Grow, Observe, and Characterize Microorganisms
Specimen Collection:
Nearly any object or material can serve as a source of
microbes. Common ones are body fluids and tissues,
foods, water, or soil. Specimens are removed by some
form of sampling device: a swab, syringe, or a special
transport system that holds, maintains, and preserves
the microbes in the sample.
A GUIDE TO THE FIVE I’s: How the Sample Is Processed and Profiled
1
2
Syringe
Microscopic morphology:
shape, staining reactions
Bird
embryo
Streak plate
Subculture
1
2
Inoculation:
The sample is placed into a container of sterile medium containing appropriate nutrients
to sustain growth. Inoculation involves spreading the sample on the surface of a solid
medium or introducing the sample into a flask or tube. Selection of media with specialized
functions can improve later steps of isolation and identification. Some microbes may
require a live organism (animal, egg) as the growth medium.
4
3
Incubation:
An incubator creates the proper growth temperature and other
conditions. This promotes multiplication of the microbes over a
period of hours, days, and even weeks. Incubation produces a
culture—the visible growth of the microbe in or on the medium.
Biochemical
tests
Isolation
Incubator
Blood bottle
Isolation:
One result of inoculation and incubation is
isolation of the microbe. Isolated microbes
may take the form of separate colonies (discrete
mounds of cells) on solid media, or turbidity
(free-floating cells) in broths. Further isolation by
subculturing involves taking a bit of growth from
an isolated colony and inoculating a separate
medium. This is one way to make a pure culture
that contains only a single species of microbe.
Immunologic
tests
DNA
analysis
5
Inspection:
The colonies or broth cultures are observed
macroscopically for growth characteristics
(color, texture, size) that could be useful in
analyzing the specimen contents. Slides are
made to assess microscopic details such as
cell shape, size, and motility. Staining techniques
may be used to gather specific information on
microscopic morphology.
Identification:
A major purpose of the Five I’s is to determine
the type of microbe, usually to the level of
species. Information used in identification can
include relevant data already taken during initial
inspection and additional tests that further
describe and differentiate the microbes.
Specialized tests include biochemical tests to
determine metabolic activities specific to the
microbe, immunologic tests, and genetic analysis.
Inoculation
Culture: the propagation of microorganisms with various media
Medium (pl. media): a nutrient used to grow microorganisms outside their natural habitat
Inoculation: the implantation of microorganisms into or onto culture media Inoculation – Clinical Specimens Include
Blood
cerebrospinal fluid
Sputum
Urine
Feces
diseased tissue
Incubation
The Incubator: media containing inoculants are placed in temperature‐controlled chambers
Usual laboratory propagation temperatures fall between 20°C and 40°C
Atmospheric gases such as O2 and CO2 may be required for the growth of certain microbes
During incubation, microbes grow and multiply, producing visible growth in the media
Various Conditions of Cultures
Pure Culture
(a)
Various conditions of cultures. (a)
Three tubes containing pure
cultures of Escherichia coli (white),
Micrococcus luteus (yellow), and
Serratia marcescens (red). A pure
culture is a container of medium
that grows only a single known
species or type of microorganism.
This type of culture is most
frequently used for laboratory
study, because it allows the
systematic examination and control
of one microorganism by itself.
Mixed Culture
(b)
(b) A mixed culture is a
container that holds two
or more identified, easily
differentiated species of
microorganisms, not
unlike a garden plot
containing both carrots
and onions. Pictured
here is a mixed culture
of M. luteus (bright
yellow colonies) and E.
coli (faint white
colonies).
Contaminated Culture
(c)
(c) A contaminated culture was once
pure or mixed (and thus a known entity)
but has since had contaminants
(unwanted microbes of uncertain
identity) introduced into it, like weeds
into a garden. Contaminants get into
cultures when the lids of tubes or Petri
dishes are left off for too long, allowing
airborne microbes to
settle into the medium. They can also
enter on an incompletely sterilized
inoculating loop or on an instrument that
you have inadvertently reused or
touched to the table or your skin.
This plate of S. marcescens was
overexposed to room air, and it has
developed a large, white colony. Because
this intruder is not desirable and
not identified, the culture is now
contaminated.
Media in Different Physical Forms
Liquid
(a)
Semisolid
(b)
Media in different physical forms.
(a) Liquid media are water-based
solutions that do not solidify at
temperatures above freezing and
that tend to flow freely when the
container is tilted. Growth occurs
throughout the container and can
then present a dispersed, cloudy, or
particulate appearance. Urea broth
is used to show a biochemical
reaction in which the enzyme
urease digests urea and releases
ammonium. This raises the pH of
the solution and causes the dye to
become increasingly pink. Left:
uninoculated broth, pH 7; middle:
weak positive, pH 7.5; right: strong
positive, pH 8.0.
1
2
Solid/Reversible to Liquid
3
4
(b) Semisolid media have more
body than liquid media but less
body than solid media. They do not
flow freely and have a soft, clotlike
consistency at room temperature.
Semisolid media are used to
determine the motility of bacteria
and to localize a reaction at a
specific site. Here, sulfur indole
motility medium (SIM) is pictured.
The (1) medium is stabbed with an
inoculum and incubated. Location
of growth indicates nonmotility (2)
or motility (3). If H2S gas is
released, a black precipitate forms
(4).
(c)
(c) Media containing 1%–5% agar are
solid enough to remain in place
when containers are tilted or
inverted. They are reversibly solid
and can be liquefied with
heat, poured into a different
container, and resolidified. Solid
media provide a firm surface on
which cells can form discrete
colonies. Nutrient gelatin contains
enough gelatin (12%) to take on a
solid consistency. The top tube
shows it as a solid. The bottom tube
indicates what happens when it is
warmed or when microbial enzymes
digest the gelatin and liquefy it.
The Media
Food for Microbes in the Laboratory
(Physical states of media)
liquid
semisolid
solid (can be converted to liquid)
solid (cannot be liquefied)
The Media
Food for Microbes in the Laboratory (cont.)
Agar ‐ complex polysaccharide from the alga Gellidium
liquefies at 100°C and solidifies at 42°C and can be poured in liquid form that will not harm the microbe or the handler
flexible and moldable; can hold moisture and nutrients
not a digestible nutrient for microorganisms
Chemical Content of Media
Chemically Defined
Media where chemical composition of media’s composition are precisely defined
Contain pure organic and inorganic compounds that vary little from one source to another
Molecular content specified by an exact formula
Chemical Content of Media
Minimal media
contain nothing more than a few essential compounds such as salts and amino acids
some contain a variety of defined organic and inorganic chemicals
Chemical Content of Media
Complex media
contain at least one ingredient that is not chemically definable
extracts of animals, plants, or yeasts
blood, serum, meat extracts, or infusions
present a rich mixture of nutrients for microbes that have complex nutritional needs
Chemically Defined and Complex Media
Selective and Differential Media
Selective media
contains one or more agents that inhibit the growth of a certain microbes but not others
important in the primary isolation of a specific type of microorganism from samples containing dozens of species
Selective and Differential Media
Differential media
allow multiple types of microorganisms to grow but are designed to display differences among those microorganisms
differentiation shows as variations in colony
size or color
media color changes
formation of gas bubbles
precipitates
Selective and Differential Media
Media can be both selective and differential
Dyes are often used as pH indicators that change colors in response to the production of an acid or base
Comparison of Selective and Differential Media
Mixed
sample
Mixed
sample
General-purpose
nonselective medium
(All species grow.)
Selective medium
(One species grows.)
(a)
General-purpose
nondifferential medium
(All species have a similar
appearance.)
(b)
Differential medium
(All 3 species grow but may
show different reactions.)
Miscellaneous Media
Reducing medium
contains a substance (thioglycolic acid or cystine) that absorbs oxygen or slows the penetration of oxygen
important for growing anaerobic bacteria
Carbohydrate fermentation media
contain sugars that can be fermented
and a pH indicator that shows this reaction
can contain a Durham tube to collect gas bubbles
Miscellaneous Media
Transport media
used to maintain and preserve specimens that have to be held for a period of time before clinical analysis
sustain delicate species that die rapidly if not held under stable conditions
Isolation
Based on the concept that if an individual cell is separated from other cells on a nutrient surface, it will form a colony
Colony: a macroscopic cluster of cells appearing on a solid medium arising from the multiplication of a single cell
Requires the following
-
a medium with a firm surface
-
a Petri dish
-
inoculating tools
Methods for Isolating Bacteria
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Steps in a Streak Plate
(a)
1
2
3
4
5
Note: This method only works if the spreading tool (usually an
inoculating loop) is resterilized after each of steps 1–5.
Steps in Loop Dilution
(b)
1
2
3
1
2
3
Steps in a Spread Plate
(c)
“Hockey stick”
1
2
© Kathy Park Talaro and Harold Benson
Inspection and Identification
Microbes can be identified through
microscopic appearance
characterization of cellular metabolism
determination of products given off
during growth, presence of enzymes, and mechanisms for deriving energy
genetic and immunological characteristics
details of these techniques will be covered in chapter 15
Maintenance and Disposal of Cultures
Cultures and specimens constitute a potential hazard
Prompt disposal is required
Stock cultures represent a “living catalog”
for study and experimentation
The American Type Culture Collection (ATCC) in Manassas, VA is the largest culture collection in the U.S. Microbial Size
Macroscopic organisms can be measured in the range from meters (m) to centimeters (cm)
Microscopic organisms fall into the range from millimeters (mm) to micrometers (μm) to nanometers (nm)
viruses measure between 20 – 800 nm smallest bacteria measure around 200 nm
protozoa and algae measure 3 – 4 mm
-6
1 micron = 1 x 10 meters = 0.000001 meters
The Size of Things
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Macroscopic View
1 mm
Louse
Range of
human eye
Reproductive
structure
of bread mold
Microscopic View
100 µm
Range
of
light microscope
10 µm
Colonial alga
(Pediastrum)
Red blood cell
Most bacteria fall
between 1 and
10 µm insize
1 µm
Escherichia coli bacteria
200 nm
Mycoplasma bacteria
100 nm
Range 10 nm
of
electron
microscope
1 nm
Require special
microscopes
0.1 nm
(1 Angstrom)
AIDS virus
Polio virus
Flagellum
Large protein
Diameter of DNA
Amino acid
(small molecule)
Hydrogen atom
Principles of Light Microscopy
Magnification
objective lens: closest to the specimen, forms the initial image called the real image
ocular lens: forms the second image called the virtual image that will be received by the eye and converted to the retinal and visual image
Total magnification (see next slide)
Principles of Light Microscopy
Power of Objective
Usual power of ocular
Total magnification
4x scanning objective
10x
40x
10x low power objective
10x
100x
40x high dry objective
10x
400x
100x oil immersion objective
10x
1000x
Principles of Light Microscopy
Resolution = resolving power
the capacity of an optical system to distinguish or separate two adjacent points or objects from one another
the human eye can resolve two objects that are no closer than 0.2 mm apart
The Effect of Wavelength on Resolution
(a)
Low resolution
(b)
High resolution
Principles of Light Microscopy
Oil Immersion Lens
uses oil to capture light that would otherwise be lost to scatter
reducing scatter increases resolution
Objective lens
Air
Oil
Slide
oil immersion lens can resolve images that are at least 0.2 μm
in diameter and at least 0.2 μm
apart
Principles of Microscopy
Contrast
refractive index: a measurement of the degree of bending that light undergoes as it passes from one medium to another
the higher the difference in refractive indexes, the greater the contrast the iris diaphragm can control the amount of light entering the condenser and increase contrast
special lenses and dyes are also used to increase contrast
Comparison of Types of Microscopy
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Table 2.6 Comparison of Types of Microscopy
Visible light as source of illumination
Microscope
Bright Field The bright-field microscope is the most widely used type of light microscope.
Although we ordinarily view objects like the words on this page with light reflected off the
surface, a bright-field microscope forms its image when light is transmitted through the
specimen. The specimen, being denser and more opaque than its surroundings, absorbs some
of this light, and the rest of the light is transmitted directly up through the ocular. As a result,
the specimen will produce an image that is darker than the surrounding brightly illuminated
field. The bright-field microscope is a multipurpose instrument that can be used for both live,
unstained material and preserved, stained material.
2,000x
0.2 µm
(200 nm)
Paramecium (400x)
Dark Field A bright-field microscope can be adapted as a dark-field microscope by adding a
special disc called a stop to the condenser. The stop blocks all light from entering the objective
lens—except peripheral light that is reflected off the sides of the specimen itself. The resulting
image is a particularly striking one: brightly illuminated specimens surrounded by a dark
(black) field. The most effective use of dark-field microscopy is to visualize living cells that
would be distorted by drying or heat or that cannot be stained with the usual methods. Darkfield microscopy can outline the organism’s shape and permit rapid recognition of swimming
cells that might appear in dental and other infections, but it does not reveal fine internal details.
2,000x
0.2 µm
Paramecium (400x)
Phase-Contrast If similar objects made of clear glass, ice, cellophane, or plastic are immersed in
the same container of water, an observer would have difficulty telling them apart because they have 2,000x
similar optical properties. Internal components of a live, unstained cell also lack contrast and can
be difficult to distinguish. But cell structures do differ slightly in density, enough that they can alter
the light that passes through them in subtle ways. The phase-contrast microscope has been
constructed to take advantage of this characteristic. This microscope contains devices that
transform the subtle changes in light waves passing through the specimen into differences in light
intensity. For example, denser cell parts such as organelles alter the pathway of light more than
less dense regions (the cytoplasm). Light patterns coming from these regions will vary in contrast.
The amount of internal detail visible by this method is greater than by either bright-field or darkfield methods. The phase-contrast microscope is most useful for observing intracellular structures
such as bacterial spores, granules, and organelles, as well as the locomotor structures of
eukaryotic cells such as cilia.
Paramecium (400x)
2,000x
Differential Interference Like the phase-contrast microscope, the differential interference contrast
(DIC) microscope provides a detailed view of unstained, live specimens by
manipulating the light. But this microscope has additional refinements, including two prisms that
add contrasting colors to the image and two beams of light rather than a single one. DIC
microscopes produce extremely well-defined images that are vividly colored and appear threedimensional.
Amoeba proteus (160x)
(first): © Carolina Biological Supply, Co/Visuals Unlimited; (second–fourth): © Michael Abbey/Visuals Unlimited
0.2 µm
0.2 µm
Comparison of Types of Microscopy
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Table 2.6 (continued)
Ultraviolet rays as source of illumination
Microscope
Confocal The scanning confocal microscope overcomes the problem of cells or
Maximum Practical Magnification
Resolution
2,000x
0.2 µm
structures being too thick, a problem resulting in other microscopes being unable to
focus on all their levels. This microscope uses a laser beam of light to scan various
depths in the specimen and deliver a sharp image focusing on just a single plane. It is
thus able to capture a highly focused view at any level, ranging from the surface to the
middle of the cell. It is most often used on
fluorescently stained specimens but it can also be used to visualize live unstained
cells and tissues
Myofibroblasts, cells involved in
tissue repair (400x)
Electron beam forms image of specimen
Microscope
Scanning Electron Microscope (SEM) The scanning electon microscope
provides some of the most dramatic and realistic images in existence. This instrument
is designed to create an extremely detailed three-dimensional view of all kinds of
objects—from plaque on teeth to tapeworm heads. To produce its images, the SEM
bombards the surface of a whole metal-coated specimen with electrons while scanning
back and forth over it. A shower of electrons deflected from the surface is picked up
with great fidelity by a sophisticated detector, and the electron pattern is displayed as
an image on a television screen.
You will often see these images in vivid colors. The color is always added afterwards;
the actual microscopic image is black and white.
Maximum Practical Magnification
Resolution
100,000,000x
10 nm
Algae showing cell walls made of
calcium discs (10,000x)
(Top): Courtesy of Dr. Jeremy Allen/University of Salford, Biosciences Research Institute; (bottom): © Science Photo Library RF/Getty Images
Preparing Specimens for the Microscope
Specimens are usually prepared by mounting a sample on a suitable glass slide that sits on the stage between the condenser and the objective lens
The manner in which it is prepared depends on
the condition of the specimen, either living or preserved
the aims of the examiner: to observe overall structure, identify microorganisms, or see movement
the type of microscopy available: bright
field, dark‐field, phase‐contrast, or
fluorescence
Fresh, Living Preparations
Placed on wet mounts or in hanging drop mounts to observe as near to the natural state as possible
Cells are suspended in water, broth, or saline to maintain viability and provide space for locomotion
Wet mount
consists of a drop or two of culture placed on
a slide and overlaid with a cover slip
Hanging drop
a drop of culture is placed in a concave (depression) slide, Vaseline adhesive or sealant, and cover slip are used to suspend the sample
Short‐term mounts such as these provide a true assessment of size, shape, arrangement, color, and motility
Fixed, Stained Smears
More permanent mounts used for long‐term study
Smear technique developed by Robert Koch over 100 years ago
spread a thin film made from a liquid suspension of cells on a slide
air dry
heat fix: heat gently to kill the specimen and attach to the slide
Stains
Unstained cells in a fixed smear are difficult to see regardless of magnification and resolving power
Staining is any procedure that applies colored chemicals (dyes) to specimens
basic dyes have a positive charge
acidic dyes have a negative charge
Bacteria have numerous negatively charged substances and attract basic dyes
Acidic dyes are repelled by cells Negative vs. Positive Staining
Positive stain: dye sticks to the specimen and gives it color
Negative stain: does not stick to the specimen but settles some distance from its outer boundary, forming a silhouette
-
negatively charged cells repel the negatively charged dye and remain unstained
-
smear is not heat fixed so there is reduced distortion and shrinkage of cells
-
also used to accentuate a capsule
-
nigrosin and India ink are used
Simple vs. Differential Staining
Simple stains: only require a single dye and an incomplicated procedure
cause all the cells in the smear to appear more or less the same color, regardless of type
reveal shape, size, and arrangement
Differential stains:
use two differently colored dyes: the primary dye and the counterstain
distinguish cell types or parts
more complex and require additional chemical reagents to produce the desired reaction
Simple Stains
Simple Stains
(a) Crystal violet stain of Escherichia coli (b) Methylene blue stain of Corynebacterium
Types of Differential Stains
Gram stain ‐ developed in 1884 by Hans Christian Gram
consists of sequential applications of
crystal violet (the primary stain), iodine (the mordant), an alcohol rinse (decolorizer), and safranin (the counterstain)
different results in the Gram stain are due to differences in the structure of the cell
wall and how it reacts to the series of
reagents applied to the cells
remains the universal basis for bacterial classification and identification
a practical aid in diagnosing infection and
guiding drug treatment
Types of Differential Stains
Acid‐fast stain
-
differentiates acid‐fast bacteria (pink) from non‐acid‐fast bacteria (blue)
-
originated as a method to detect Mycobacterium tuberculosis
-
these bacteria cell walls have a particularly impervious cell wall that holds fast (tightly or tenaciously) to the dye (carbol fuschin) when washed with an acid alcohol decolorizer
-
also used for other medically important bacteria, fungi, and protozoa
Types of Differential Stains
Endospore stain
similar to the acid fast stain in that a
dye is forced by heat into resistant
bodies called spores or endospores
stain distinguishes between spores
and vegetative cells
significant in identifying gram‐
positive, spore‐forming members of
the genus Bacillus and Clostridium
Differential Stains
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Differential Stains
(a) Gram stain. Purple cells are
gram-positive. Pink cells are
gram-negative.
(b) Acid-fast stain. Red cells are
acid-fast. Blue cells are non-acidfast.
(c) Spore stain, showing endospores
(red) and vegetative cells (blue)
a,b: © Jack Bostrack/Visuals Unlimited; c: © Manfred Kage/Peter Arnold/Photolibrary
Special Stains
Used to emphasize cell parts that are not revealed by conventional staining methods
Capsule staining
used to observe the microbial capsule
an unstructured protective layer surrounding the
cells of some bacteria and fungi
negatively stained with India ink
Flagellar staining
used to reveal tiny, slender filaments used by bacteria for locomotion
flagella are enlarged by depositing a coating on
the outside of the filament and then staining it