Exercise 1'2

1
Exercise 1.2

• Safety Quiz
• Microscope Functions and Care
• Familiarization of the Microscope
• Pond Water
• Environmental Isolate

2
Safety Quiz

• 1. When coming in to check cultures, you DO NOT
  have to wear your lab coat.
• TRUE FALSE
• You must ALWAYS wear your lab coat when in
  the lab.
• 2. Eating, drinking and applying lip-gloss while
  in the laboratory IS PERMITTED.
• TRUE FALSE
• Eating, drinking, and applying lip-gloss is
  NEVER permitted in the laboratory.
• 3. If your microscope is NOT in good working
  condition, you should try and fix it yourself.
• TRUE FALSE
• Bring microscope problems to the attention
  of your TA.

3
Safety Quiz

• 4. You should assume that the inoculating
  loop/needle is contaminated until it is
  sterilized in the flame.
• TRUE FALSE
• 5. If an accident occurs where culture is
  spilled, you should clean it up immediately.
• TRUE FALSE
• Notify TA immediately of any culture spills.
• 6. When using culture tubes, it is acceptable to
  place them in the student bins with the stains
• TRUE FALSE
• ALWAYS place culture tubes in racks.

4
Safety Quiz

• 7. Under certain circumstances, students may
  remove cultures from the laboratory.
• TRUE FALSE
• NEVER remove cultures from the laboratory.
• 8. You must wash your hands with soap and water
  upon arriving and before leaving the laboratory.
• TRUE FALSE
• 9. You must wash you work bench with Osyl and a
  damp sponge upon arriving and before leaving the
  laboratory.
• TRUE FALSE

5
Safety Quiz

• 10. It is NOT necessary to wear safety glasses
  when working with bacterial cultures.
• TRUE FALSE
• You are to wear safety glasses whenever
  you are working with bacterial cultures.
• 11. Gloves MUST be worn when working with stains
  and reagents that may be toxic to the skin.
• TRUE FALSE
• 12. If you are pregnant, or otherwise
  immunocompromised (i.e. HIV/AIDS, chemotherapy
  treatment, etc.), you may be at greater risk than
  otherwise.
• TRUE FALSE

6
Safety Quiz

• 13. Each student is held responsible for the
  equipment that is either assigned to or used by
  them.
• TRUE FALSE
• 14. If a culture tube is dropped or broken, the
  student should clean it up immediately and then
  notify the TA.
• TRUE FALSE
• Immediately notify your TA if culture
  tubes are spilled, dropped, or broken.
• 15. Broken glass, such as slides or cover slips,
  should be disposed of into a waste basket or the
  nalgene buckets provided on the lab bench.
• TRUE FALSE
• Slides and cover slips are ALWAYS placed
  in slide trays at the ends of the benches.

7
Safety Quiz

• 16. Open wounds should be covered before coming
  into the lab.
• TRUE FALSE
• 17. Mouth pipetting is allowed only if the TA is
  assisting you.
• TRUE FALSE
• You should NEVER mouth pipette.
• 18. Your lab coat should be laundered and
  bleached periodically during the semester.
• TRUE FALSE

8
Safety Quiz

• 19. Open-toed shoes are permitted in the
  laboratory during summer sessions only.
• TRUE FALSE
• Open-toed shoes are NEVER permitted in
  the laboratory.
• 20. Failure to follow directions from the
  laboratory instructor may result in dismissal
  from the class (both lecture and lab).
• TRUE FALSE

9
MICROSCOPY

• Bright-field
• The most common form of light microscopy.
  Extensively used for the visualization of
  microorganisms usually necessary to stain
  specimens for viewing.

Enterococcus
Anthrax spore stain
10
MICROSCOPY

• Dark-field Used for viewing live, unstained
  material. The specimen appears bright on a dark
  background. For low- or medium-power work.

Diatom
Rotifer
Cyanobacteria Nostoc
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MICROSCOPY

• Phase contrast Microscopy used when a colorless
  specimen, which absorbs little light, (e.g. a
  non-pigmented living cell) is not clearly visible
  by bright-field microscopy

Bacteria
Amoeba
Rotifer
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MICROSCOPY

• Fluorescence microscopy
• Fluorochrome treated specimens (fluorescent
  stained) are irradiated with ultra-violet
  radiation and the light emitted forms the image
  of the specimen in a manner similar in principal
  to that in bright-field microscopy. The
  fluorescent scope is designed so that the
  specimen can be illuminated at one wavelength of
  light and observed by light emitted at a
  different wavelength.

Yersinnia pestis
Rhizopus rot-Black bread mold
13
MICROSCOPY

• 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. It produces high contrast images of
  unstained, transparent specimens in what appear
  to be three dimensions.

Heliozoan Actinomycies
Amoeba Nucleus and Vacuole
14
MICROSCOPY

• Electron microscopy microscopy in which an
  electron beam interacts with a specimen and
  contributes to an image of the object. Electron
  microscopy is used for examining viruses,
  macromolecules, and the ultra structure of cells.
  (electronically colored)

 Vibrio parahaemolyticus
Mixed Bacteria
15
The three factors in obtaining an image

• 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.

16
Magnification The extent 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).

17
Resolution The 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 (Pg 1-3 in lab manual)
• 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.

18
Contrast The use of elements, such as colors,
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, and/or the iris
  diaphragm.
• 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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To Enhance Contrast Alter Optics by Adjustment
of

• 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.

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THE MICROSCOPE
21
THE MICROSCOPE its parts, their function,
maintenance and use
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THE MICROSCOPE its parts, their function,
maintenance and use

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THE MICROSCOPE its parts, their function,
maintenance and use
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THE MICROSCOPE its parts, their function,
maintenance and use
25
THE MICROSCOPE its parts, their function,
maintenance and use
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THE MICROSCOPE its parts, their function,
maintenance and use
27
Familiarization

• Observation of typed text.
• Follow instructions as outlined in your lab
  manual.
• RememberStart at the 10X objective, then to 40x.
  We will not be using the 100X objective for this
  exercise.

28
Pond Water

• Observation of organisms from local pond water.
• Again, follow instructions as outlined in your
  lab manual.
• Use the 10X, then the 40X objective.
• Have me or the TAA come over BEFORE you go to
  the 100X. Proper use of the 100X objective will
  be critical to your grade. Each student will be
  shown this individually.

29
Environmental Isolate

• Choose a partner with whom you wish to work
• Do not open the plates until inspected by your
  laboratory instructor (MOLD)
• Inspect plates for the presence of growth
• Record the numbers and different types of
  colonies
• As a team, select two suitable colonies

30
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)
• Students 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
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Environmental Isolate

• Label plates (not lids but sides)
• Initials
• Section
• Date
• Store lid side down
• Check daily until growth appears note hours or
  days