BIO244 Lab Sample Outline
(Your outline does not need to be typed!)
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Lab 2 - Goals and Objectives : Learn aseptic technique and pure culture
isolation
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Exercise 9 and 10
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Wash bench with disinfectant
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Exercise 9: Aseptic Technique
1. Label all tubes using tape with name, date, name of organism, and “Ex.9”
2. Perform a broth culture to broth culture aseptic transfer using E. coli: follow
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directions in figure ….. and ……. pages ………. Incubate at 37°C.
3. Perform a slant culture to slant culture aseptic transfer using E. coli: follow
directions in figure ……. pages ………. Incubate at 30°C.
4. Perform a plate culture to slant culture aseptic transfer using S. marcescens:
follow directions in figure ……. page ……….. Incubate at 30°C.
5. incubate at correct indicated temperature.
6. Remove tape from E. coli and S. marcescens cultures and discard.
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Exercice 10: Pure Culture Technique
1. Label plates directly with marker with name, date, “mixed culture”, and “Ex.10”
and
2. Perform a streak for isolation using Quadrant Method B.
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Start with directions in figure …… on pages …….., then perform isolation as
described on page 85: Quadrant Streak (Method B)
3. Repeat on a second plate.
4. incubate at 25°C
5. Remove tape from mixed culture and discard.
6Return all materials and wash bench with disinfectant
Conclusions:
1,
2.
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Materials: Broth culture E. coli
Slant culture E. coli
Plate culture S. marcescens
1 sterile BHI broth
2 sterile BHIA slants
Flame, loop, tape, marker
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Materials: Mixed culture (contadins E.
coli, M. luteus, S. marcescens)
2 sterile BHIA plates
Flame, loop, marker
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Lab 1 Goals and Objectives: Learn how to use microscope and
measurements
Microscopy lecture: Text Chapter 3
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Exercise 1: Brightfield Microscopy
Exercise 5: Microscopic Measurements
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Exercise 7: Ubiquity of Bacteria (Replacement activity in packet p. 55)
– Homework: “Testing Household Surface” handout,
– Take home plates, swabs, and sterile water
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READ DIRECTIONS CAREFULLY
International measurement system
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Microorganisms are measured in micrometer range - µm
meter- m - Standard unit of length=3.38 ft or 1.09 yd
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Prefix
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1 m = 100 cm = 1000 mm = 1,000,000 µm = 1,000,000,000 nm = 10,000,000,000 Ǻ
(centimeter) (millimeter) (micrometer)
(nanometer)
(Angstroms)
cells
viruses
small molecules
large organelles
small organelles
atoms
Symbol
Factor
Gram
Liter
Meter
milli
micro
nano
m
μ
n
mm
µm
nm
Numerically
Name
1
10-3
10-6
10-9
0.001
one thousandth
0.000 001
one millionth
0.000 000 001 one billionth
Microscopy: The Instruments
A microscope (Greek: μικρόν (micron) = small + σκοπεῖν (skopein) =
to look or see
1. Magnification – the ability to make larger
– 10X, 20X, 40X, 100X, 200X, 1000X
2. Resolution - the ability of the lenses to distinguish two points.
– A microscope with a resolving power of 0.4 nm can distinguish between two
points ≥ 0.4 nm.
– Shorter wavelengths of light provide greater resolution.
Microscopy
Microscopes can largely be separated into three classes:
1. Light microscopes
- Visible wavelengths of light
- UV light
- Magnification - up to 2000X
- Resolution – 0.2 µm (200 nm)
2. Electron microscopes
– Uses beams of electrons instead of light.
– The shorter wavelength of electrons
gives greater resolution.
– Magnification limit – 10,000 -100,000 X
– Resolution limit – 2.5 nm (0.0025 µm)
3. Scanning probe microscopes
- A physical probe is used either in close contact to the sample or nearly
touching it
- Resolution 1/100 of an atom.
Figure 3.2 Microscopes and Magnification.
Unaided eye
≥ 200 m
Light microscope
200 nm – 10 mm
Tick
Actual size
Scanning
electron
microscope
10 nm – 1 mm
Red blood cells
Transmission
electron
microscope
10 pm – 100  m
E. coli bacteria
T-even bacteriophages
(viruses)
Atomic force
microscope
0.1 nm – 10nm
DNA double helix
Compound light microscope
• The image from the objective lens is magnified again by the ocular lens.
• Total magnification = objective lens  ocular lens
Limits:
Magnification – 1000-2000X
Resolution – 0.2 µm
Figure 3.1b
Compound light microscope
• Refractive index is the
light-bending ability of a
medium.
• The light may bend in air so
much that it misses the small
high-magnification lens.
• Immersion oil is used to
keep light from bending.
• 100X objective lens
• 1000x total magnification
Figure 3.3
Brightfield Illumination Darkfield Illumination Phase-Contrast Microscopy
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Dark objects are visible
against a bright
background.
– Light reflected off
the specimen does
not enter the
objective lens.
Light objects are visible against a
dark background.
Light reflected off the
specimen enters the
objective lens.
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Accentuates diffraction of the light
that passes through a specimen.
Good resolution
Figure 3.4a, b
Fluorescence Microscopy
• Uses UV light.
– Fluorescent substances absorb UV
light and emit visible light.
– Cells may be stained with fluorescent
dyes (fluorochromes).
• Confocal Microscopy
– Uses fluorochromes and a laser
light.
• The laser illuminates each plane in
a specimen (a slice)
• produce 2D or 3D image.
Figure 3.6b
Transmission Electron Microscopy (TEM)
• Ultra thin sections of specimens.
• Light passes through specimen, then an electromagnetic lens, to a
screen or film.
• Specimens may be stained with heavy metal salts.
• 10,000-100,000X; resolution 2.5 nm
Figure 3.8a
Scanning Electron Microscopy (SEM)
• An electron gun produces a beam
of electrons that scans the surface
of a whole specimen.
• Secondary electrons emitted from
the specimen produce the image.
• 1000-10,00X; resolution 20 nm
Figure 3.8b
Scanning-Probe Microscopy
• Scanning tunneling microscopy uses a
metal probe to scan a specimen.
– Resolution 1/100 of an atom.
• Atomic force microscopy uses a metal
and diamond probe inserted into the
specimen.
– Produces 3D images.
Figure 3.9a
Preparation of Specimens for Light Microscopy
• Live or unstained cells have little contrast with the
surrounding medium.
– However, researchers do
make discoveries about cell
behavior looking at live
specimens.
• Specimen preparation
1.
2.
3.
Smear - A thin film of a
microbes suspension on a
slide
Dry the smear – to prevent
cell lysis
Fix the smear - to attach the
microbes to the slide
Smears staining
• Stains consist of a positive and negative ion.
• In a basic dye, the chromophore is a cation.
• In an acidic dye, the chromophore is an anion.
• Staining the background instead of the cell is called negative
staining.
General categories of stain
1. Simple stain- Use of a single basic
dye
– Methylene blue, carbolfucsin, crystal
violet
– can see the size and shape of the
bacteria
2. Differential Stains - use two or
more stains and categorize cells into
groups
– Gram stain
– Acid-Fast Stain
General categories of stain
3. Special Stains
– Negative staining - the background is
stained, leaving the actual specimen
untouched, and thus visible
– Capsule staining - is useful for capsules.
– Heat is required to drive a stain into
endospores.
– Flagella staining requires a mordant to
make the flagella wide enough to see.
Figure 3.12a-c
BIO244 Lab 1 Microscopy
Students will need:
• microscope and cord (sign out number, use all semester)
• 1 “e” slide
• 1 stage micrometer
• immersion oil: share one bottle per student pair
• lens tissue & methanol
• laboratory supplemental packet
• lab manual or photocopied pages
• BIO244 Microscope Worksheet
• Chapter 3 Notes outline
• 2 sterile agar plates, stack and tape to keep lids shut
• 4 sterile swabs
• 1 vial sterile water
• Exercise 1: Brightfield Microscopy
– Learn how to use the microscope (guide in suppl.
packet p.51)
1. Diopter and interocular adjustments
2. Light adjustments (iris diaphragm)
3. Oil immersion
• Exercise 5: Microscopic Measurements
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Calibrate the ocular micrometer (suppl. packet p.52)
Microscope Worksheet
Measure the provided specimens
Complete the microscope worksheet and turn it in
Microscope Measurement Concept Practice Homework:
complete
– Measurement practice sheet in supplemental packet, self
check:
answers in packet (p. 53-54)
Exercise 5: Microscopic Measurements
Calibrate the ocular micrometer (suppl. packet p.52)
1. Make sure left ocular is turned fully counter-clockwise
2. Start with 10X objective
3. Get stage micrometer (2mm ruler on slide) in focus:
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you will see the ocular micrometer with labels in multiples of 10 (0,
10, 20, 30)
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the stage micrometer with labels in multiples of 0.1 (0.0, 0.1, 0.2)
stage micrometer
ocular
micrometer
3. Line up the zero lines of the two micrometers (you can turn the top of the ocular to
position the ocular micrometer and the mechanical stage controls to move the stage
micrometer)
At 100X total magnification they should line up as shown below
“0.1” on stage micrometer is 0.1mm on that actual 2mm ruler. This distance
corresponds to 10 “units” (spaces between lines) on the ocular micrometer (0-10): the
0.1 line of the stage ruler lines up with the 10 line on the ocular one. How long in
millimeters is one unit on the ocular micrometer with the 10X objective in place?
0.1mm = 10 “units”
1 unit = 0.1mm ÷ 10
1 unit = 0.01mm
to convert mm to µm, multiply by 1000
When the microscope is set to 100X total
magnification, one unit on the ocular
micrometer is worth ________µm
4. Repeat for the 40X objective:
0.1mm = 40 “units”
1 unit = 0.1mm ÷ 40
1 unit = 0.0025mm
to convert mm to µm, multiply by 1000
When the microscope is set to 400X total
magnification, one unit on the ocular
micrometer is worth ________µm
When the microscope is set to 100X total
magnification, one unit on the ocular
micrometer is worth ____10____µm
When the microscope is set to 400X total
magnification, one unit on the ocular
micrometer is worth ____2.5____µm
When the microscope is set to 1000X total
magnification, one unit on the ocular
micrometer is worth ____1____µm
Dinoflagellate
10Xobjective
(100X)
Czura 2005
4Xobjective
(40X)
40Xobjective
(400X)
Lilium Pollen
10Xobjective
(100X)
Czura 2005
4Xobjective
(40X)
40Xobjective
(400X)
Frog Blood
10Xobjective
(100X)
Czura 2005
4Xobjective
(40X)
40Xobjective
(400X)
Streptococcus faecalis
40X objective
(400X)
Czura 2005
100X objective
(1000X)
Dinoflagellate
Frog Blood
Lilium Pollen
Streptococcus faecalis
• Exercise 7: Ubiquity of Bacteria (Replacement
activity in packet p. 55)
– Homework: “Testing Household Surface” handout,
– Take home plates, swabs, and sterile water
• READ DIRECTIONS CAREFULLY