stain, observe and identify the microbes
TRANSCRIPT
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In Chapter 4 and Lab 1, 2 and 3 you
will learn the basic skill sets
necessary to
Stain, Observe and Identify the Microbes
ACGM/CSLO goals:
1. Use and comply with laboratory safety rules, procedures, and
universal precautions.
2. Demonstrate proficient use of a compound light microscope.
3. Describe and prepare widely used stains and wet mounts, and
discuss their significance in identification of microorganisms.
4. Perform basic microbiology procedures using aseptic techniques for
transfer, isolation and observation of commonly encountered, clinically
significant bacteria.
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MICRO CLASS/LAB PREPARATIONS
Required Item:
TEXTBOOK,
MASTERING BIO,
ACCESS CODE,
LAB Manual/Journal
Scantrons 882-E, Scantron No.2 pencil
USEFUL Items: RED PEN
Colored Pencils
Purple
Pink
Red
Blue
Green
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BIOHAZARD Rules
All laboratory supplies (which touches specimen) and specimen must be placed in the BIO HAZARD
bin.
Please inform students that NO TRASH items should be placed in the BIOHAZARD bins
NO EXCEPTIONS. Do not put soda cans , regular trash in the biohazard bin (NO FOOD ALLOWED).
Very important
VIOLATORS will lose points
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MICRO LAB PREPARATIONS
Lab Performance
My Review
Peer Review Form
Lab Safety Form Signup
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PowerPoint® Lecture
Presentations prepared by
Mindy Miller-Kittrell,
North Carolina
State University
C H A P T E R
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Microscopy,
Staining, and
Classification
4
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Figure 3.4 Approximate size of various types of cells.
Red Blood Cells
~10 um
15001.5mm
1500 um
Width of penny
=
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Figure 4.3 The limits of resolution (and some representative objects within those ranges) of the human eye and of various types of microscopes.
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Magnification, the ratio of an object’s image
size to its real size
Resolution, the measure of the clarity of the image,
or the minimum distance of two distinguishable
points
Contrast, visible differences in brightness between
parts of the sample
Official definitions
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Microscopy
General Principles of Microscopy
Magnification (enlarge)
Resolution (tell apart 2 objects close together)
Contrast (Differences in intensity between two
objects)
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Smaller the wavelength
smaller the object you can see
(small objects need small hands)
Think about giant fingers and texting
General rule for any microscopy/detector
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Figure 4.1 The electromagnetic spectrum.
VIBGYOR
Smaller the object = Use smaller wavelength
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Microscopy
Microscopes are used to visualize cells
In a light microscope (LM), visible light is passed
through a specimen and then through glass lenses
Glass lenses focuses light and enlarges and
resolves objects
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Light
Air Glass
Focal point
Specimen Convexlens
Inverted,
reversed, and
Enlarged
image
5 50
Magnification = 50/5 = 10X
Lenses refract (bend) the light, so that the image is magnified
Magnify using lenses
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Microscopy
Light Microscopy
Bright-field microscopes
Simple – single lense
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 – multiple lenses
More than one - Series of lenses for magnification
Light passes through specimen into objective lens
Have one or two ocular lenses
Total magnification = magnification of objective lens X
magnification of ocular lens
E.g. 10 times X 10 times = 100 times
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Figure 4.4 A bright-field, compound light microscope.
Coarse focusing knob
Moves the stage up and
down to focus the image
Illuminator
Light source
Diaphragm
Controls the amount of
light entering the condenser
Condenser
Focuses light
through specimen
Stage
Holds the microscope
slide in position
Objective lenses
Primary lenses that
magnify the specimen
Body
Transmits the image from the
objective lens to the ocular lens
using prisms
Ocular lens
Remagnifies the image formed by
the objective lens
Line of vision
Ocular lens
Path of light
Prism
Body
Objective
lenses
Specimen
Condenser
lenses
Illuminator
Fine focusing knob
Base
Arm
objective lens
occular lens
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Figure 4.5 The effect of immersion oil on resolution.
Glass cover slip
Slide
Specimen Light source
Without immersion oil
Lenses
Immersion oil
Glass cover slip
Slide
Light source
With immersion oil
Microscope
objective
Refracted light
rays lost to lens
Microscope
objective
More light
enters lens
Special type of objective lens = oil immersion lens
Increases magnification and resolution
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Microscopy
General Principles of Microscopy
Contrast
Differences in intensity between two objects, or an
object and its background
Increase contrast by staining
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Figure 6.3ab
Brightfield(stained specimen)
50 µ
m
Staining increases contrast
Brightfield(unstained specimen)
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Figure 4.16 Simple stains.
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Staining
Principles of Staining
Dyes used as stains are usually salts
Chromophore is the colored portion of the dye
Acidic dyes (negatively charged) stain alkaline
structures
Basic dyes (positively charged) stain acidic
structures
Basic dyes are more commonly used since inside of
most cells is negatively charged
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Staining
Simple stains
Differential stains
Gram stain
Acid-fast stain
Endospore stain
Special stains
Negative stain
Flagellar stain
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Staining
Most microorganisms are difficult to view by bright-
field microscopy
Coloring specimen with stain increases contrast
and resolution
Specimens must be prepared for staining
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Figure 4.15 Preparing a specimen for staining.
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paramecium
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penicillium
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spirogyra
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Figure 3.12 Bacterial shapes and arrangements.
2) strepto
1) coccus 2) bacillus
3 Shapes
3) spirillum
2 main arrangements
1) staphylo
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Microbe Naming Rules
Genus speciesGenus species
Genus species
Genus species
X
X Escherichia coli
Escherichia coli
E. coli
E. coliG. species
G. species
Genus speciesX
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Microbe Naming Rules
Based on shape and arrangement
Arrangement+shape species
Staphylococcus species
Streptobacillus species
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cocci bacilli
arrangement
shape
Streptococci
Staphylococci
Streptobacilli
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Negative staining
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This large (0.7-1.0 mm), nonpathogenic gram
negative spirillum exhibits polar amphitrichous
flagellation and a characteristic red pigment.
Rhodospirillum rubrum
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Rhodospirillum rubrum
Negative staining
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Rhodospirillum rubrum
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Next time we meet there will
be a short pre-lecture quiz based
on lab 1,2 and 3