Exercise 3'2 Overview

1
Exercise 3.2Overview

• Hand back MWA 2, turn in pre-lab and MWA 3
• Assign MWA 4 (pass out paragraph and discuss)
• Aseptic Technique
• Environmental Unknown
• ISOLATION confirmation
• Dental Plaque observation
• Environmental Unknown
• Storage Conditions
• Gram Stain Unknown Quiz
• Microscopy catch-up

2
Aseptic Technique Quiz

• Perform transfers of Broth Medium aseptically as
  described in the lab manual (3-3)
• Only one set of materials will be given to each
  student
• Stay calm and work accurately
• Label all tubes and Wheaton bottle (DO NOT write
  on Wheaton bottles use masking tape)
• Place tubes (in order) in the class tube racks
  provided

3
Environmental Unknown

• Check plates (3rd isolation streak) for isolated
  colonies in the 3rd sector
• Choose one preferred plate between lab partners
  (DO NOT dispose of other plates yet)
• View wet-mount of an isolated colony under Phase
  Contrast Look for uniformity of size and shape
• Perform a Gram Stain (with controls!) if time
  permits
• If your chosen Environmental Unknown is confirmed
  to be a pure culture, keep all plates in its
  lineage and dispose of all unrelated plates)

4
Dental Plaque Observation

• Sterilize forceps with alcohol and flame
• Obtain sterile toothpick and gently sample
  gingival crevice as demonstrated-do NOT cause
  yourself to bleed
• Place a drop of water on a slide and mix with
  toothpick
• Top with a cover slip and view using Phase
  Contrast
• Sample your gingival crevice again to make a dry
  mount slide so you can practice Gram staining

5
Environmental UnknownStorage Conditions

• If you and your lab partner have a PURE culture
  you may begin the storage conditions experiment
  today
• This will show you the best place to STORE your
  organism it will NOT tell you whether or not
  your organism can GROW in the refrigerator
• The experiment will take two full weeks to
  complete, but you will not be behind if you need
  to start late
• DO NOT waste media tubes if you are not certain
  you have a pure culture yet

6
Environmental UnknownStorage Conditions (Cont.d)

• Choose a SINGLE colony from your PURE
  Environmental Unknown plate
• Take ½ of the colony with your inoculating loop
  and make a straight-line streak on a TSA slant
  (screw-cap ) labeled 4C
• Take the other ½ of the colony and do the same
  thing with a TSA slant labeled RT (room
  temperature)

7
Environmental UnknownStorage Conditions (Cont.d)

• Leave both screw-caps about ½ turn unscrewed to
  allow air to enter the tubes
• Wait until both tubes have shown decent growth
  and tighten the caps to about ¼ turn unscrewed,
  then put the 4C tube in the refrigerator (leave
  the RT tube at room temperature)
• DO NOT disturb the tubes for two full weeks
• In two weeks, re-streak the culture from each
  tube onto new TSA slants (leaving caps ½ turn
  unscrewed) and check each day to see which tube
  has the culture that recovers the best
• This will tell you how best to STORE your culture
• It WILL NOT tell you if your culture GROWS better
  in the refrigerator or at room temperature

8
Gram Stain Unknown Quiz

• Work at your own pace, but try to get done today
• Choose one Gram Stain Unknown and write the
  number on your quiz immediately
• Prepare a few dry-mounts at the same time it
  will save you time if your first stain doesnt
  work out
• If you are confident with your stain, fill out
  the quiz completely (make sure you use the
  correct terminology and that organism names are
  spelled correctly)
• DO NOT call me until you are ready to hand in
  your quiz

9
Gram Stain Unknown Quiz(Cont.d)

• Once the quiz begins
• You MAY use the laminated sheets in your drawers
• You MAY NOT use your lab manual
• You MAY NOT ask your neighbors for help
• At this point I cant
• tell you how to do the stain
• how to focus on the organisms
• how to fill out your quiz correctly or
• what the correct spellings for your organisms are

10
Microscopy Catch-up

• Make sure you have looked at bacteria under oil
  using Phase Contrast
• Dental plaque and Environmental Unknown organisms
  can fulfill these requirements
• Make sure you have completed all five staining
  techniques from Ex. 2 and Ex. 3.1
• Simple Stain
• Capsule Stain
• Gram Stain
• Acid-Fast Stain
• Endospore Stain

11
NEXT WEEK

• Exercise 4 Enumeration of Bacteria (Hamburger)
• Mini-Writing Assignment 4 due
• Complete this exercise with the sheet I give you,
  not the sheet in your lab manual
• Continue Environmental Unknown isolation if
  necessary
• Perform appropriate stains on Unknown
• Dont forget to start Storage Conditions
  experiment