Announcements

1
Announcements

• Consolidate the trash cart.
• Your environmental isolate is your job
• Continue to work on your stains
• Remember to clean the stains off the desk top
• All bottles need to be cleaned

2
Exercise 3.2

• Sterile transfers
• Dental plaque
• Gram stain quiz
• Environmental unknown streak/storage conditions
• Finish differential staining

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Today in Microbiology
4
Reviewing Sterile Transfers

• Purpose
• To become proficient at proper lab technique
• To transfer liquids by pipetting sterile liquids
  from one tube to another aseptically
• To transfer exact volume of liquids by pipette
  from one tube to another

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Sterile Transfers

• Important Points
• This exercise is exactly is identical in
  procedure to last weeks aseptic transfers. The
  difference being a nutrient medium is used in
  place of the H20.
• This exercise requires (and teaches) dexterity
  and technical skill.
• You will be graded on your technique. Use your
  concentration and pay attention to detail, both
  the aseptic transfers as well as accuracy in
  volume.

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Sterile Technique Review

• The blue Pipette Pump is for 1 ml pipettes the
  green one is for 5 and 10 ml pipettes
• After the lid is removed, flame the mouth of the
  bottle or tube
• Do not place bottle caps or culture tube closures
  on your lab bench hold them in your hand
• Use pipette pump to remove or dispense liquid
• Flame the mouth of the bottle or tube before
  returning the lid

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Sterile Technique Procedure

• Write your initials and lab section on
• A bottle of 50ml Trypticase soy broth (TSB)
• 6 sterile culture tubes
• Note use tape or wax pencil on the side of the
  bottle/tube. Do not label the white area on the
  bottle/tube.
• Perform the transfers described on page 3-3
• Remaining media and culture tubes will be checked
  for growth (contamination!!) and correct volume

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How do you know if you pass?

• Evidence of Contamination-Turbidity
• TSA Broth is Transparent , but when bacterial
  cells are present, the color becomes Opaque

Turbid Culture
Clear Culture
I will have your results next week
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Checking the Environmental Isolate

• Check your plates for growth in the 3rd quadrant.
• Check for visual appearance of contamination by
  other bacteria.
• Streak another plate to for at least one more
  attempt at purification.
• If you feel that your plates are pure, Please
  follow the next slide for other procedures.

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Think your Unknown is Pure?

• Prepare a wet mount of your isolate and examine
  under 100X w/ oil
• Is there more than one organism?
• Prepare a simple stain of your isolate
• Is there more than one morphology?
• If not, what is the morphology of your organism?

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SoYour unknown isnt pure?

• What to do
• Keep streaking the TSA plates
• Continue to do wet mounts to check purity
• Make sure you are doing these things as often as
  possible

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IMPURE
PURITY
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SoYour Unknown is Pure

• 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

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Storage Conditions

• Get two storage condition tubes, Label with tape
  on the side of tube.
• 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)

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Storage Conditions for Unknown

• 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, then put the 4C tube in
  the refrigerator (leave the RT tube at room
  temperature)
• DO NOT disturb the tubes for two full weeks

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Storage Conditions

• 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
• Which organism regenerates better?
• Which storage conditions are best for your
  organism?
• NOTE This exercise is performed to determine
  the storage conditions best suited to your
  environmental organism not "where the culture
  grows best
• Sub-culture every week

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Storage Conditions
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Dental Plaque

• Continuous accumulation of a soft deposit on the
  teeth

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Dental Plaque Procedure

• Observe your oral microfauna
• Rub tip of toothpick under gum line
• Mix into drop of DI water on slide
• Cover w/ cover slip
• Observe under phase contrast (100X w/oil)

• Gram stain your oral microfauna
• Take a new sample with another toothpick
• Prepare a dry mount
• Gram stain your sample
• Observe on brightfield (100X w/oil)

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Review for Gram Stain Unknown

• 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 (correct terminology and
  spelling count)
• DO NOT call me until you are ready to hand in
  your quiz
• You can not move around your slide. All organisms
  must be viewable.

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ONCE THE QUIZ BEGINS

• YOU MAY NOT
• You MAY NOT use your lab manual
• You MAY NOT ask your neighbors for help

• I WILL NOT tell you how to do the stain, how to
  find the organisms, how to focus on the
  organisms, how to fill out your quiz correctly,
  or what is wrong with your spelling unless you
  decide not to complete the quiz today then we
  can discuss pointers

• YOU MAY
• You MAY use the laminated sheets in your drawers

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What to do if finished?

• 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
• Gram stain your dental plaque
• Work on Environmental Unknown

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NEXT WEEK

• MWAssignment 4
• Continue Environmental Unknown isolation
• Perform appropriate stains on Unknown
• You will be doing a gram stain on your Unknown,
  however, you have to have a colony that is only
  24-36 hours old.
• Dont forget to start Storage Conditions
  experiment
• Exercise 4- Enumerating Bacteria
• MAJOR DANGER???