BIO244 Lab Sample Outline (Your outline does not need to be typed!)

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BIO244 Lab Sample Outline(Your outline does not
need to be typed!)

• Lab 2 - Goals and Objectives Learn aseptic
  technique and pure culture isolation
• Exercise 9 and 10
• Wash bench with disinfectant
• 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
• directions in figure .. and . pages .
  Incubate at 37C.
• 3. Perform a slant culture to slant culture
  aseptic transfer using E. coli follow directions
  in figure . pages . Incubate at 30C.
• 4. Perform a plate culture to slant culture
  aseptic transfer using S. marcescens follow
  directions in figure . page .. Incubate at
  30C.
• 5. incubate at correct indicated temperature.
• 6. Remove tape from E. coli and S. marcescens
  cultures and discard.
• 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.
• 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 25C
• 5. Remove tape from mixed culture and discard.

• 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
• 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

• Exercise 1 Brightfield Microscopy
• Exercise 5 Microscopic Measurements
• 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

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International measurement system

• Microorganisms are measured in micrometer range -
  µm
• meter- m - Standard unit of length3.38 ft or
  1.09 yd
• 
  
  
• Prefix Symbol
  Factor Numerically Name
•   Gram
• Liter
  
  
• Meter
  1
•  
•  milli m mm
  10-3 0.001
  one thousandth
•  micro µ µm
  10-6 0.000 001
  one millionth
•  nano n nm
  10-9 0.000 000 001 one
  billionth
• 1 m 100 cm 1000 mm 1,000,000 µm
  1,000,000,000 nm 10,000,000,000 ?
• (centimeter) (millimeter)
  (micrometer) (nanometer)
  (Angstroms)
• 
  cells viruses
  small molecules
• 
  large organelles small organelles
  atoms

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Microscopy The Instruments

• A microscope (Greek µ????? (micron) small
  s??pe?? (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.

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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.

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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
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Light Microscopy

• The use of any kind of microscope that uses light
  to observe specimens
• Types of light microscopy
• Compound light microscopy
• Dark field microscopy
• Phase-contrast microscopy
• Differential interference contrast microscopy
• Fluorescence microscopy
• Confocal microscopy

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Figure 1.2
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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
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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
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Brightfield Illumination Darkfield Illumination
Phase-Contrast Microscopy

• 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.

• Accentuates diffraction of the light that passes
  through a specimen.
• Good resolution

Figure 3.4a, b
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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
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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
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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
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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
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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
• 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

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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.

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General categories of stain

• 1. Simple stain- Use of a single basic dye
• methyleneblue, 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

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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
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• 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

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• 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

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• Exercise 5 Microscopic Measurements
• 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)

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Exercise 5 Microscopic Measurements Calibrate
the ocular micrometer (suppl. packet p.52)
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• Make sure left ocular is turned fully
  counter-clockwise
• Start with 10X objective
• Get stage micrometer (2mm ruler on slide) in
  focus
• you will see the ocular micrometer with labels in
  multiples of 10 (0, 10, 20, 30)
• the stage micrometer with labels in multiples of
  0.1 (0.0, 0.1, 0.2)

stage micrometer
ocular micrometer
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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?
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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
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4. Repeat for the 40X objective
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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
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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
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4Xobjective (40X)
Dinoflagellate
40Xobjective (400X)
10Xobjective (100X)
Czura 2005
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Lilium Pollen
4Xobjective (40X)
40Xobjective (400X)
10Xobjective (100X)
Czura 2005
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Frog Blood
4Xobjective (40X)
40Xobjective (400X)
10Xobjective (100X)
Czura 2005
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Streptococcus faecalis
40X objective (400X)
100X objective (1000X)
Czura 2005
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Lilium Pollen
Dinoflagellate
Streptococcus faecalis
Frog Blood
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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
• READ DIRECTIONS CAREFULLY