The
Five I’s: |
|
•Inoculation
–Addition
of microorganism to supporting medium
•Incubation
–Proper
growth temperature and time
•Isolation
–Creating
pure culture from a mixed population
•Inspection
–Macroscopic
and microscopic observations
•Identification
|
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How do you culture a microorganism? |
|
•To
identify the agent that causes a specific disease requires that microorganisms
be isolated and cultivated or cultured
|
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Step 1 to cultivate microorganism |
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–Inoculum (sample to be cultured)•Environmental specimens (water, air, soil)•Clinical specimens (obtained from patient)•Stored specimens (previously cultured) |
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Step 2 to cultivate microorganism |
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–Medium(collection of nutrients allowing growth of inoculum)•Liquid broth •Solid media |
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Step 3 to cultivate microorganism |
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–Incubation (placing inoculated media at proper temperature for specific period of time)•Observable growth appears in or on medium after incubation is known as a culture |
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Clinical
Specimens: |
|
•A
clinical specimen is a sample of human material to be examined or tested for
the presence of a pathogen |
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How do you properly handle a clinical specimen? (3 Steps) |
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•Must
take care not to contaminate the sample with organisms from the environment or
other region’s of the patients body
•Must
be labeled properly
•Must
be transported quickly to the lab to avoid death of microbe and minimize growth
of normal microbiota
|
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•Obtaining Pure Cultures: |
|
–Technique
for isolating the suspected pathogen from normal microbiota in a culture
|
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Pure
culture : |
|
–refers
to a cultures composed
of cells arising from a single progenitor (parent or original cell)
•Progenitor
is termed a CFU
(colony forming unit)
|
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Aseptic
technique: |
|
refers
to a set of instructions used to prevent further contamination by microbes |
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Two
common pure culture
isolation techniques: |
|
•Streak
plates
•Pour
plates |
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•Culture Media: |
|
–Used to support the
growth of microorganism or as a means of identification |
| |
–3 Physical States of Media |
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•Liquid
•Semi solid
•Solid
|
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6 types of general culture media: |
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•Defined media
•Complex media
•Selective media
•Differential media
•Anaerobic media
•Transport media
|
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Physical
States of Media
•Liquid: |
|
–Water-based
solution
–Growth
appears as cloudiness or turbidity |
| |
Physical States of Media Semisolid: |
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–Contains
agar that thickens, but does not form a solid
Used to determine
motility |
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Physical States of Media Solid: |
|
–Agar: complex polysaccharide derived from algae
–Will
allow culture media to form solid surfaces |
| |
•Defined Media: |
|
–Exact
chemical composition is known
–Used
for organisms with strict need for specific growth factors
–Not
used for most clinical cultures
|
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•Complex
media: |
|
–Exact
composition is not quite known
–Yeast,
meat, soy or protein extracts provide the nutrients
–Trypticase soy agar (TSA),
nutrient broth or nutrient agar are commonly used
|
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Selective Media: |
|
•Contains
substances that
favor the growth of
particular organisms or inhibit the growth of
unwanted ones
|
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Differential Media: |
|
•Presence
of visible changes in the media or differences in the appearance of colonies help
differentiate amongst different kinds of bacteria |
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Differential Media Blood agar- |
|
differentiates
between the ability of bacteria to digest, partially digest or inability to
digest red blood cells |
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Differential Media Carbohydrate
tubes: |
|
differentiates
between the ability to ferment types of sugars and the resulting products (acid
or gas or both) |
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Differential Media MacConkey agar- |
|
–selective
for Gram – bacteria, differentiates between non-lactose and lactose fermenting
Gram - bacteria |
| |
Anaerobic
Media: |
|
•Obligate
anaerobes need to be cultured in the absence of oxygen
–Agar
Stab
–Reducing
media that chemically combine with free oxygen and remove from media
–Anaerboic culture system (Gas
Pak machine) |
| |
Transport
Media: |
|
•Special
media to allow for transfer of clinical specimens
–Prevent
contamination of other people
–Prevent
contamination of sample
–Maintain
proper growth conditions for survival |
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Special Culture Techniques
Animal and cell culture:
|
|
Not
all organisms will grow in media, need live cells or organism |
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Special Culture Techniques
•Enrichment
culture:
|
|
–Selective
media or technique to increase the growth of bacteria found in very small
numbers |
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•Preserving
Cultures (3 steps): |
|
–Refrigeration
•Stores
for short periods of time
–Deep-freezing
(-50°C to -95°C)
•Stores
for years
–Lyophilization (removal of water
from a frozen culture)
•Stores
for decades
|
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Microscopy: |
|
Refers
to the use of light or electrons to magnify organisms not visible with the
unaided eye. |
| |
General Principles of Microscopy (4): |
|
–Wavelength
of radiation
–Magnification
–Resolution
–Contrast |
| |
General Principles of Microscopy (4) Wavelength
of Radiation: |
|
•Using
radiation of smaller wavelengths results
in enhanced microscopy
|
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General Principles of Microscopy (4) Magnification: |
|
•Increase in the size of an object (number
with X, read times)
•Determined
by multiplying magnification power of ocular and objective lenses
•Results
from the passing of radiation (light or electrons) through a lens or series of
lenses
•Magnification
is limited by resolution |
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General Principles of Microscopy (4) Resolution: |
|
•Resolution
or resolving power is the ability to distinguish
between objects that
are close together
•Dependent
on the wavelength of radiation and numerical aperture (na)of lens
–Ability
of lens to gather light
|
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General Principles of Microscopy (4) Contrast: |
|
•Refers
to the differences in the intensity between two objects
or between an object and the background
–Important
in determining resolution
–Staining
increases contrast |
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Types
of Microscopes (3)
Light Microscopes:
|
|
–Bright-field
•Simple
•Compound
–Dark-field
–Phase
•Phase
contrast
•Differential
interference contrast
–Fluorescent
Confocal |
| |
Types of Microscopes (3)
Electron Microscopes: |
|
–Transmission
(TEM)
–Scanning
(SEM) |
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Types of Microscopes (3)
Probe Microscopes: |
|
–Scanning
Tunneling (STM)
–Atomic
Force (AFM) |
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Light Microscopy
–Bright-field
microscopes•Simple: |
|
–Contain
a single magnifying lens
–Similar
to magnifying glass
–Capable
of 300X magnification
–Leeuwenhoek
used simple microscope to observe microorganisms |
| |
Light Microscopy
–Bright-field
microscopes
•Compound |
|
–Series
of lenses for magnification
–Light
passes through specimen into objective lens
–Have
one or two ocular
lenses
–Most
have condenser lens (direct light through specimen)
–Capable
of 2,000X magnification
–Background
bright, specimen darker
|
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Light Microscopy
–Dark-field microscopes: |
|
•Best
for observing pale objects
•Only
light rays scattered by specimen enter objective lens
•Increases
contrast and enables observation of more details
•Specimen
appears light against
dark background
|
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Light Microscopy
Phase microscopes: |
|
•Used to examine
living organisms or specimens that would be damage by attaching them to slides or staining
•Light rays in phase
produce brighter image, while light rays out of phase produce darker image
•Contrast is created
because light waves are out of phase |
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Phase microscopes (2 types)
Phase-contrast microscope: |
|
»Simplest
|
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Phase microscopes (2 types)
Differential
interference contrast microscope
(Normarski): |
|
Involves prisms to split light into different colors, increasing contrast, 3Dappearance |
| |
Light Microscopy
Fluorescent
microscopes: |
|
•Direct
a UV light source at specimen
•Specimen
emits visible light when bombarded by short UV rays
•Some
cells are naturally
fluorescent; others must be stained
•Used
in immunofluorescence to identify
pathogens or to make visible a
variety of proteins |
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Light Microscopy
–Fluorescent microscopy
•Confocal microscopes: |
|
Use florescent dyes or antibobies- Uses UV lazer-Creates 3D image |
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Electron Microscopy: |
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–Electron microscopes have
greater resolving power and magnification (10,000X to 100,000X)
–Detailed views of
bacteria, viruses, internal cellular structures, molecules, and large atoms
–Lenses are magnets, not glass
–Energy beam is in
form of electrons, not visible light
–Requires a vacuum, therefore only dead
specimens can be observed
|
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Two
Types of Electron Microscopes
Transmission (TEM): |
|
–Specimen
must be very thin
–Electrons
are sent through specimen, similar to
that of light microscopes
–Electron
energy is captured by a fluorescent screen, changing some energy to visible
light
–More
dense the specimen, the more electrons blocked, seen as darker on the final
image
–Finest
detailed structures of cells can be
observed |
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Two Types of Electron Microscopes Scanning (SEM): |
|
–Specimen
is coated with a metal
–Electrons
are sent across the surface of the
specimen
–Primary
electrons excite metal to release secondary electrons
–Secondary
electrons are captured by detector and photomultiplier
–Final
image is three dimensional, but of surface of
specimen only
|
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Probe Microscopy: |
|
–Magnifies
more than 100,000,000 times
–Detects
individual molecules and atoms
–Uses
a probe that has been sharpened to end in a single atom
|
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2 Types Probe Microscopes Scanning tunneling microscopes: |
|
•Probe
is passed back and
forth and slightly above specimen
•Measures
the flow of voltage
back and forth from the probe to
specimen,
translates to an image
|
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2 Types Probe MicroscopesAtomic
force microscopes: |
|
•Probe
is passed along the
surface of the specimen
•Tip
moves up and down
with contour of surface, movement is translated to image
|
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Staining, what does it do? |
|
•Increases
contrast and resolution by
coloring specimens with stains/dyes |
| |
Types of stains (4): |
|
–Simple stains
–Differential stains
•Gram stain
•Acid-fast stain
•Endospore stain
–Special stains
•Negative (capsule)
stain
•Flagellar stain
–Electron microscope
stains |
| |
Wet mount: |
|
–Cells
are suspended in liquid, one or two drops are added to slide, slide is overlaid
with coverslip
|
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Hanging
drop: |
|
–Best
for observing motility
–Suspended
cells are added to coverslip, coverslip is inverted onto a
depression slide, seal with Vaseline to prevent drying
|
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How to prepare
a specimen for staining: 2 Steps |
|
•Create
a smear by spreading a thin sample of specimen
on a slide and air drying
•Fixation process adheres
sample to slide, kills organism, preserves shape and size
–Heat
fixation- briefly pass slide through flame
–Chemical
Fixation- dip slide in methanol or formalin |
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Simple
Stains: |
|
•Used
to determine size, shape, and
arrangement of
cells
•Composed
of a single basic dye
–Combine
with and stain negatively charged structures
–Crystal
violet, safranin, or methylene blue
- 1 dye -neg cells attract pos dye |
| |
Differential
Dyes: |
|
•Used
to distinguish between different types of
cells or structures
•Use
more than one dye |
| |
Types
of Differential Stains (3): |
|
–Gram
stain
–Acid-fast
- -Endospore |
| |
Gram
Stain is used for: |
|
•Used
to differentiate between two large
groups of microorganisms (Gram – and Gram +)
•Typically,
the first stain used to identify a bacterial pathogen |
| |
Clinical
Relevance of the Gram stain: Presence of
Gram negative organisms may indicate: |
|
–Serious
illnesses
•Endotoxins are part of the
outermost LPS layer in Gram – bacteria (Shigella, Salmonella)
–Resistant
to antibiotics
•Outermost
LPS layer protects the peptidoglycan layer from
antimicrobials
–Presence
of a coliform
•Indicator
of unsanitary conditions of food or
water (E.
coli) |
| |
Gram
positive bacteria: |
|
–Final color: purple
–Thick layer of peptidoglycan in cell wall
–Retains dye-mordant
complex (purple) |
| |
Gram
negative bacteria: |
|
–Final color: red/ pink
–Much thinner layer of peptidoglycan in cell wall
–Does not retain
dye-mordant complex, needs counterstain (red) |
| |
Acid-Fast
Stain: |
|
-have hydrophobic layer
•Waxy
layer of acid-fast bacterial cells will
retain primary dye and stain red/ pink
•Non-acid-fast bacteria will not
retain primary dye and will counterstain blue
|
| |
Endospore: |
|
dehydrated
structure formed inside a bacterial cell in response to harsh environmental
conditions |
| |
Characteristics of Endospore (5): |
|
–Usually found in Gram
+ cells
–Dormant,
non-reproductive
–Tough cell wall is
impermeable to most chemicals (stains)
– Difficult to destroy
(need to autoclave)
–Examples: Bacillus (anthrax) and Clostridium
(botulism) species |
| |
Endospore Stain, what color does it stain? |
|
•Malachite
green is retained in spore
•Vegetative (growing) cells will counterstain red/ pink with safranin
|
| |
Special
Stains
Negative
(capsule) stain: |
|
•Negative
stains are repulsed by negative charges on cells
•Stain the background and not the cell
•Cells
are counterstained with another dye
•Useful
to determine the presence of a capsule |
| |
Special
Stains
Flagellar stain: |
|
•Used
to determine the presence, number, location and arrangement of flagella
•Flagella
are normally invisible with light microscopy
•fatens up flagella |
| |
•Staining for Electron Microscopy |
|
-stain in the form of a metal
•Prevent
electrons from passing through (TEM)
•Excited
by primary electrons and release secondary electrons to create image (SEM)
–Stains
may bind molecules in specimens or the background
|
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