Transcript Microscopy, Staining

Chapter 4
Microscopy,
Staining, and
Classification
© 2012 Pearson Education Inc.
Lecture prepared by Mindy Miller-Kittrell
North Carolina State University
Table 4.1 Metric Units of Length
Figure 4.2 Light refraction and image magnification by a convex glass lens-overview
Light
Glass
Air
Focal point
Specimen
Convex
lens
Inverted,
reversed, and
enlarged
image
Figure 4.3 The limits of resolution of the human eye and of various types of microscopes
Diameter
of DNA Ribosomes
Proteins Viruses
Atoms
Amino
acids
Typical bacteria
and archaea
Flea
Chloroplasts
Mitochondrion
Large
protozoan
(Euglena)
Chicken
egg
Human red
blood cell
Scanning tunneling microscope
(STM) 0.01 nm–10 nm
Transmission electron microscope (TEM)
0.078 nm–100 µm
Scanning electron microscope (SEM)
0.4 nm–1 mm
Atomic force
microscope (AFM)
1 nm–10 nm
Compound light microscope (LM)
200 nm–10 mm
Unaided human eye
200 µm–
Microscopy
• General Principles of Microscopy
– Contrast
– Differences in intensity between two objects, or
between an object and background
– Important in determining resolution
– Staining increases contrast
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Microscopy
• Light Microscopy
– Bright-field microscopes
– Simple
– Contain a single magnifying lens
– Similar to magnifying glass
– Leeuwenhoek used simple microscope to
observe microorganisms
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Microscopy
• Light Microscopy
– Bright-field microscopes
– Compound
– Series of lenses for magnification
– Light passes through specimen into
objective lens
– Oil immersion lens increases resolution
– Have one or two ocular lenses
– Total magnification (objective lens X ocular
lens)
– Most have condenser lens (direct light
through specimen)
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Figure 4.4 A bright-field, compound light microscope-overview
Ocular lens
Line of vision
Remagnifies the image formed by
the objective lens
Body
Transmits the image from the
objective lens to the ocular lens
using prisms
Arm
Objective lenses
Primary lenses that
magnify the specimen
Stage
Holds the microscope
slide in position
Condenser
Focuses light
through specimen
Diaphragm
Controls the amount of
light entering the condenser
Illuminator
Light source
Coarse focusing knob
Moves the stage up and
down to focus the image
Fine focusing knob
Base
Ocular lens
Path of light
Prism
Body
Objective
lenses
Specimen
Condenser
lenses
Illuminator
Microscopy
• The microscopes we will be using are parfocal,
which means that once you have it focused at
one magnification using the coarse adjustment,
you should only have to use the fine adjustment
for the other objectives.
Figure 4.5 The effect of immersion oil on resolution-overview
Microscope
objective
Refracted light
rays lost to lens
Microscope
objective
Lenses
More light
enters lens
Glass cover slip
Glass cover slip
Slide
Slide
Specimen
Light source
Without immersion oil
Immersion oil
Light source
With immersion oil
Microscopy
• Electron Microscopy
– Light microscopes cannot resolve structures
closer than 200 nm
– Greater resolving power and magnification
– Magnifies objects 10,000X to 100,000X
– Detailed view of bacteria, viruses, and large
atoms
– Two types
– Transmission electron microscopes
– Scanning electron microscopes
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Figure 4.13 SEM images-overview
Microscopy
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Rotate nosepiece to 4x objective
Raise stage completely using coarse adjustment.
Find area of specimen on slide with naked eye
Place specimen over circle of light (adjust to area of interest using
stage dials)
Lower stage until specimen comes into focus.
Switch to the 10x objective. Once focused here, switch to 40x
objective.
Once focused at 40x, move 40x objective out of the way.
Place oil directly on slide and rotate objective labeled 100x (oil).
Use ONLY fine focus (small knob) only to bring into view through
ocular.
Aseptic Technique
• Required for all microbiology preparations to
assure that contaminants are not introduced.
• On a personal note, aseptic technique assures that
infectious agents are not spread to you, fellow
students, or the laboratory surfaces.
Disinfection
Disinfection:
The use of a physical or chemical procedure to
virtually eliminate all recognized pathogenic
microorganisms but not all microbial forms
(bacterial endospores) on inanimate objects.
Decontamination
Sterilization: The use of physical or
chemical procedures that destroy all
microbial life forms, including highly
resistant bacterial endospores.
Autoclave: Pressurized steam at 15 psi
and 121oC for an average of 20 min (10 –
40 min depending on bulk and load)
Microbiology Lab Equipment
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Microscope (with accessories)
Inoculation loops
Source of flame (Bunsen burner)
Microscope slides and Cover slips
Gram staining kits
Petri dishes and proper growth media
Incubators
Autoclave (pressure cooker)
Clorox bleach, like you buy at the supermarket, diluted to 5-10% or disinfectant
provided in lab.
Growth media
Broth—liquid media
Agar plates
Agar slants
Media Types
• General purpose
• Selective
• Differential
Media Types
• General purpose:
• Supports growth of most non fastidious organisms
• Tryptic Soy Agar
• Selective:
• Favors the growth of one type of microorganisms and
inhibits the growth of others
• Saboraud Dextrose Agar (SDA)—grows fungi
• Differential Media:
• Distinguishes between different groups of bacteria on
the basis of biochemical characteristics
Aseptic Technique
• Required for all microbiology preparations to
assure that contaminants are not introduced.
• On a personal note, aseptic technique assures that
infectious agents are not spread to you, fellow
students, or the laboratory surfaces.
General rules of microbiology laboratory
• The inoculating loop is usually used for making transfers of bacterial
cultures (see next few slides for technique).
• Allow the loop to cool sufficiently so that any organisms to be tested
will not be killed by the hot wire, but do not allow the loop to contact
anything during the cooling period or contamination will result.
• Learn to remove and replace the caps or lids efficiently without setting
them on the countertop or leaving the cover off too long.
• After the transfer is completed the loop must be sterilized again.
Follow the procedure outlined on the following slides to prevent
splattering of infectious materials.
• It may be easier to work while sitting down.
• Attention to details and practice will allow you to work both rapidly
and accurately.
How to hold an Inoculating Loop
FLAMING A LOOP
FLAMING A LOOP
FLAMING A LOOP
Flaming tubes
Transferring Microorganisms to Slant Test Tubes
Streaking a slant
Procedure for Transferring Microorganisms To/From Test Tubes
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Hold loop with dominant hand
Flame the loop
Hold culture tube in other hand
Remove cap with pinkie of dominant hand
Flame mouth of culture tube
Place loop into tube
Flame mouth of culture tube and close
Put the tube back in the rack.
Open new culture tube or plate. If tube, flame the mouth.
Inoculate
Flame mouth of tube and close or close plate
Flame loop
Procedure for transferring microorganisms to/from plate
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Flame loop
Pick up bottom of plate in non-dominant hand.
Touch the loop to a bacterial colony.
Replace the bottom of the plate on the top.
Pick up the bottom of a new plate.
Touch loop to plate.
Replace the bottom of the plate on the top.
Flame the loop.
Staining
• Principles of Staining
– Staining increases contrast and resolution by
coloring specimens with stains/dyes
– Smear of microorganisms (thin film) made prior
to staining
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Figure 4.15 Preparing a specimen for staining
Spread culture in
thin film over slide
Air dry
Pass slide through
flame to fix it
Staining
• Simple Stains
• Differential Stains
– Gram stain
– Acid-fast stain
– Endospore stain
• Special Stains
– Negative (capsule) stain
– Flagellar stain
© 2012 Pearson Education Inc.
Figure 4.16 Simple stains-overview
Gram Stain
Color of
Gram-Positive Cells
Color of
Gram-Negative Cells
Primary Stain:
Crystal Violet
Purple
Purple
Mordant:
Iodine
Purple
Purple
Decolorizing Agent:
Alcohol-Acetone
Purple
Colorless
Counterstain:
Safranin
Purple
Red
Figure 4.17 The Gram staining procedure-overview
Slide is flooded with crystal
violet for 1 min, then rinsed
with water.
Slide is flooded with iodine
for 1 min, then rinsed with
water.
Result: All cells are stained
purple.
Result: Iodine acts as a
mordant; all cells remain
purple.
Slide is flooded with solution
of ethanol and acetone for
10–30 sec, then rinsed with
water.
Slide is flooded with safranin
for 1 min, then rinsed with
water and blotted dry.
Result: Smear is decolorized;
Gram-positive cells remain
purple, but Gram-negative
cells are now colorless.
Result: Gram-positive cells
remain purple, Gram-negative
cells are pink.
Figure 4.18 The Ziehl-Neelsen acid-fast stain
Figure 4.19 Schaeffer-Fulton endospore stain of Bacillus anthracis
Figure 4.20 Negative (capsule) stain of Klebsiella pneumoniae
Bacterium
Capsule
Background
stain
Figure 4.21 Flagellar stain of Proteus vulgaris
Flagella