Happy Valentines Day

1
Happy Valentines Day!

• This bacteria is all alone on Valentines day and
  needs a dinner datecan you guess who it is?

2
Today Exercise 3 (Part 2)

• Assignments
• Collect MWA 4, Pre-lab 3.2
• Return MWA 3, Pre-lab 3.1
• Sterile Technique Quiz
• Dental Plaque
• Environmental Isolate
• Microscopic examination
• Staining
• Storage conditions
• Gram Stain Unknown Quiz

3
Notes

• Clean your scopes before AND after using them.
• If even one scope is dirty, the whole class will
  miss points.
• Put empty bottles of stain in front of the stain
  bins so the techs can fill them.
• There have been a lot of stain messes left after
  lab. I will mark off points if there is stain on
  or around your bench.
• We all need to respect the hard working custodian
  and do our part.

4
McMillan Ch.4

• Theres a lot to do today, so Im not going to
  lecture on it right now.
• You should still read it!
• Its fair game on the next quiz
• Helpful for your first paper, which is coming up!

5
Sterile Technique

• This exercise is exactly identical in procedure
  to last weeks aseptic transfers. The difference
  being a nutrient medium is used in place of the
  H20.
• You will be graded on your technique. Concentrate
  and pay attention to detail.
• You will be graded on both the aseptic transfers
  as well as accuracy in volume.

6
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

7
Sterile Technique Quiz

• Write your initials and lab section on
• A bottle of 50ml trypticase soy broth (TSB),
  write on tape. DO NOT WRITE ON THE BOTTLE!
• 6 sterile culture tubes. You may write directly
  on the glass tube, or on tape.
• Perform the transfers described on page 3-3
• Remaining media and culture tubes will be checked
  for growth (contamination!!) and correct volume

8
Sterile Technique Quiz

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

Turbid Culture
Clear Culture
9
Dental Plaque

• Soft deposit that continuously accumulates on
  teeth
• Complex microbial community
• gt 1010 bacteria per milligram
• As many as 400 distinct bacterial species
• Small number of epithelial cells, leukocytes, and
  macrophages

10
Dental Plaque

• Observe your oral microfauna
• Rub tip of toothpick under gum line
• Mix into drop of water on slide
• Cover w/ cover slip
• Observe under phase contrast (100X w/oil)
• REMEMBER Phase Contrast requires the use of the
  Phase dial on the Condenser.

11
Dental Plaque

• This is a great opportunity to practice Gram
  stain technique before quiz
• Gram stain your oral microfauna
• Take a new sample
• Prepare a dry mount
• Gram stain
• Observe on brightfield (100X w/oil)

12
Dental Plaque

• Notice the different Gram responses
• Pink (-)
• Purple ()
• Yeasts will stain black and will be much larger.
• Also note the various morphologies you are seeing.

13
Environmental Isolate Purity

• 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?

14
Environmental Isolate Purity

• If your isolate is not pure (two different
  morphologies of organisms are present), do
  another streak or pick other isolate.
• If pure, determine storage conditions

15
Environmental Isolate Storage
Using sterile technique use the colony to
inoculate two TSA slants (Straight streak, not
zigzag)
Today IF PURE
Lids unscrewed 1/2 turn from fully tightened.
Since what you sampled came from outdoors it is a
safe assumption that they need oxygen to live.
TSA TSA
16
Environmental Isolate Storage
CHECK DAILY!!
Incubate at room temperature
Store at 4C in frig.
As soon as grown up Tighten lids and.
Store at room temp.
TSA TSA
17
Environmental Isolate Storage

• Recap
• Verify purity of environmental unknown
• Inoculate 2 TSA slants w/ single colony
• Store at room temperature
• Check cultures daily
• Once cultures have grown, tighten caps
• Store one slant at room temp.
• Store one slant in refrigerator

18
Environmental Isolate Storage

• We will re-streak onto fresh TSA slants in two
  weeks.
• Make sure your tubes are properly labeled with
  tape (leave tab).
• Tubes lost now mean you no longer have your
  isolate.

19
Gram Stain Quiz

• Each sample has a minimum of two and not more
  than three organisms.
• Each will have a Gram and Gram organism.
• Perform a gram stain on your unknown (you may use
  the staining guide in your drawer)
• Determine the organisms in your unknown
• CAREFULLY fill out your quiz! This is an all or
  nothing quiz!
• Work at your own pace, but try to get done today

20
Gram Stain Quiz Tips

• 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

21
Gram Stain Quiz

• 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

• Unless you decide not to complete the quiz today,
  I WILL NOT tell you
• how to do the stain
• how to focus on the organisms
• how to fill out your quiz correctly
• what is wrong with your spelling

22
Next Week

• Assignments
• Pre-lab 4
• Exercise 4
• Enumerating Bacteria spread and pour plates
• Environmental Isolate (cont.)
• Microscopic examination
• Staining
• Storage conditions
• Repeat Gram Stain Quiz (if necessary)