Welcome to BIO 205 Laboratory

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Welcome to BIO 205 Laboratory

• Section H
• Class Number 6832
• Tuesday 600 850

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Welcome to BIO 205 Laboratory

• Section C
• Class Number 5552
• Wednesday 800 1050

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Welcome to BIO 205 Laboratory

• Section L
• Class Number 12069
• Wednesday 600 850

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Week 2 Overview

• 305W!!!
• Website
• Review Materials Needed for Lab
• Safety Review

• Exercise 1
• Microscopy
• Environmental Isolate Project

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Website

• Here is the site
• Updated weekly
• Weekly Announcements
• PowerPoint Presentations
• Key Terms
• Useful Links

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Review

• What do you do when you come in?
• Wash hands
• Put on labcoat
• Wipe down bench with 10 bleach and then follow
  with 70 Ethanol

• What do you need for lab?
• Lab coat
• Sharpie Marker
• Lab manual (read before lab) and related books
  need for exercise

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Safety Review

• Plate disposal
• Glove disposal
• Pipette disposal
• Notifying the TA
• Emergency numbers

• Fire extinguisher
• First Aid Kit
• Covering wounds
• Safety glasses
• Glass disposal
• Contaminated vs. uncontaminated

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Mini Writing Assignments

• Hand in Mini Writing Assignment 1
• Mini Writing Assignment 2
• Objective To create a graph and understand how
  datum points relate to one another
• This is not simply a connect the dots graph.
  You must draw a line of best fit
• This graph should be hand drawn on graph paper
  and labeled appropriately (see McMillan)
• Remember, you have two values to determine

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Exercise 1

• Environmental Isolate
• Microscopy

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Environmental Isolate

• Choose a partner
• DO NOT open the plates until inspected by your
  laboratory instructor
• Record number and types of colonies
• As a team, each person select two suitable colony
• Choose isolated colonies
• No fuzzy or yeasty colonies (absolutely NO
  fungus!)
• Choose colonies with decent amount of growth

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Before Streaking for Isolation

• Of the two chosen colonies Notice color,
  morphology, and size
• Pay attention to generation time ( good if they
  grow fairly quickly, e.g. 24-48 hours)
• Check EVERYDAY until isolated colonies have grown
  in the 3rd sector
• Remember that you are responsible for the care
  and maintenance of these organisms for the
  REMAINDER of the semester

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Environmental Isolate
ISOLATION STREAK PATTERN
Good, isolated growth
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Environmental Isolate

• Label plates (not lids, upside-down on the side
  of the agar half)
• Name
• Section
• Date
• Env. Iso. (or Unk.) 1st streak
• Store upside down

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Microscopy
Several Different Methods

• Bright-field
• Most common form of light microscopy
• Usually necessary to stain specimens for viewing

Enterococcus
Staphylococcus epidermidis
Anthrax spore stain
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Microscopy
Several Different Methods

• Dark-Field
• Used for viewing live, unstained material
• Specimen appears bright on a dark background
• Used for low- or medium-power work

Rotifer
Cyanobacteria Nostoc
Diatom
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Microscopy
Several Different Methods

• Phase Contrast
• Used when a colorless specimen (e.g. a
  non-pigmented living cell) is not clearly visible
  by bright-field microscopy
• Used for viewing live, unstained specimens

Rotifer
Bacteria
Amoeba
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Microscopy
Several Different Methods

• Fluorescence Microscopy
• Fluorochrome treated specimens (fluorescent
  stained) are irradiated with ultra-violet
  radiation
• The light emitted forms the image of the specimen
  in a similar way that bright-field microscopy does

Rhizopus rot-Black bread mold
Microcosm activity probe
Yersinia pestis
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Microscopy
Several Different Methods

• Nomarski Differential Interference Contrast
  Microscopy
• A combination of light waves that are out of
  phase with each other and produces interference
  that alters the amplitude of the light waves.
• Produces high contrast images of unstained,
  transparent specimens
• Produces a 3-D image

Amoeba Nucleus and Vacuole
Heliozoan Actinomycies
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Microscopy
Several Different Methods

• Electron Microscopy
• Electron beam is used rather than visible light
• Used for examining viruses, macromolecules, and
  the ultra structure of cells. (electronically
  colored)
• Specimens are killed, fixed, and impregnated with
  metal so the electrons will interact with them

 Vibrio parahaemolyticus
Mixed Bacteria
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What Types of Microscopy do We Use?

• Bright-field for stained organisms
• ex Gram stains, simple stains, etc.
• Phase contrast for live, unstained organisms
• ex hay infusion today

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What Determines the Image You See?

• Magnification The extent to which the image of
  an object is larger than the object itself.
• Resolution The ability of a microscope to reveal
  fine detail in a specimen.
• Contrast The use of elements, such as colors,
  light, forms, or lines, in proximity to produce
  an intensified effect.

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MagnificationThe factor to which the image of
an object is larger than the object itself.

• The total magnification is the product of the
  magnification of the powers of the two lenses.
• The magnification of the objective lens (10x,
  40x, 100x) multiplied by the magnification of the
  eyepiece lens (10x).
• The total magnification depends upon the focal
  lengths of these lenses (100x, 400x, 1000x
  respectively).

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ResolutionThe ability of a microscope to reveal
fine detail in a specimen.

• Resolving Power depends upon the wavelength of
  light and the property of an objective lens
  called the numerical aperture (NA). The higher
  the numerical aperture of an objective the better
  the resolving power.
• Refraction When light passes through a
  transparent material of one density into one of
  another density, the light is bent. The
  refractive index of glass is 1.5 and of air in
  1.0 by definition. Less light is lost if
  something is placed between the lens and the
  glass slide such as water or oil (which has a
  refractive index of 1.0) by this process less
  light is lost and by this process magnification
  is increased.

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ContrastThe use of elements, such as color,
light, forms, or lines in proximity to produce an
intensified effect.

• The most important aspect of successful
  microscopy is contrast. No matter how good
  magnification or resolution contrast is the key
  to successful microscopy.
• To enhance contrast you alter the optics of the
  scope by adjustment of the
• Condenser
• Rheostat
• Iris diaphragm (with Bright-field)
• Phase Contrast Condenser (with Phase contrast)

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To Enhance Contrast

• Condenser . . . knob on the left side of the
  scope underneath the stage
• Rheostat . . . the light adjustment located on
  the right hand side of the body of the scope
• Iris diaphragm . . . lever in front of the
  condenser (alters light intensity when using
  bright-field)
• Phase Contrast Condenser . . . dial with numbers
  on it underneath the stage (if available)

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The Microscope
In detail Parts and Maintenance
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Microscope Care and Maintenance

• Carry with two hands
• One on arm, the other underneath the base
• Place dust cover in drawer
• Do not drag microscope
• Get out a microscope and follow along

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Microscope Bit By Bit
Ocular or eyepiece
Diopter adjustment
Observation tube clamping screw Do
Not Touch
Binocular head with observation tube
Arm
Revolving nosepiece
Objective lens 10x, 40x, 100x
Mechanical Stage with slide clip
Condenser
Condenser height adjustment knob
Fine focus knob
Iris diaphragm lever
Course focus adjustment
Phase-contrast condenser ?? (Should be
here)
Rheostat
Power switch
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Parts of the Microscope
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Parts of the Microscope
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Parts of the Microscope
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Parts of the Microscope
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Parts of the Microscope
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Parts of the Microscope
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Parts of the Microscope
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Good Microscopy RequiresPatience and Practice

• SO PRACTICE SOME MICROSCOPY
• Turn on the power switch
• Place prepared slide or hay infusion specimen on
  the slide and then slide in the slide clip
• Turn the nosepiece in the correct direction
• Practice using the 10x and 40x objective first
  with prepared slides or hay infusion
• Adjust for best contrast, condensor, phase ring,
  rheostat.
• Only with TAs approval, go to 100x and
  demonstrate how to focus under oil immersion with
  bacteria (TA dispenses)
• Clean the scope thoroughly following the protocol
• STUDENTS DO NOT LEAVE UNTIL THEIR SCOPES HAVE
  BEEN CHECKED BY ME AND OFF THE LIST!!!

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Preparing a Wet Mount

• Clean slide
• Place sample on slide
• Place cover slip on at angle.
• View under scope

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What to View

• Letters
• String on prepared slide
• Pond water
• With and without oil!

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Next Week

• Pre-lab 2 due
• Assign Mini-writing assignment 2
• Start Staining, Exercise 2