Exercise 3 Part I

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Exercise 3 Part I

• Aseptic Technique
• Environmental Unknown
• Differential Staining Continued
• Work on Simple, Capsule, and Gram if they
  have
• not been checked off yet
• Acid-Fast stain
• Endospore stain
• Gram Stain Unknown
• QUIZ NEXT WEEK

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Aseptic Technique

• Aseptically transferring liquids by pipetting
  sterile media from one container to another
• This exercise requires (and teaches) dexterity
  and technical skill
• Practice with tap water today
• Appropriate manipulation of the equipment takes
  PRACTICE! The only way to learn these skills is
  to physically handle the objects
• Next week you will use an enriched medium to
  transfer and contamination will count

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Page 3-3 in Lab Manual

• Practice performing the transfers described in
  Sterile (Aseptic) Technique (Part II) with water
• These steps must be performed precisely next week
  with sterile broth for your Aseptic Technique
  Quiz
• Point values
• 6 tubes _at_ 0.25pt. for correct volume and 0.25pt.
  for sterility 3pt.
• Wheaton bottle _at_ 1pt. for correct volume and 1pt.
  for sterility 2pt.
• Total Quiz value 5pt. (Technique Points)

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Sterile Technique Procedure

• Insert the pipette pump into the pipette as shown
  in figure 3.1
• Gently twist to make sure pipette is fit
  correctly
• Prime the pump as demonstrated in class

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REMEMBER

• Make sure the pipette and Pipette Pump have a
  good seal, but DO NOT jam them together
• DO NOT place bottle or culture tube caps or
  pipettes on the lab bench You must hold these
  in your hands at all times in order for them to
  remain sterile
• Hold the pipette at an angle so that the liquid
  does not wet the cotton plug
• Always flame the lips of bottles and culture
  tubes BEFORE and AFTER each transfer
• Work as quickly (but accurately) as you can, and
  keep materials within the heat field of your
  Bunsen burner
• Place pipette in wrapper and then into Nalgene
  bucket as demonstrated in class

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Environmental Unknown

• Inspect your first attempt at purifying your
  Environmental Unknown.
• Your Unknown should be purified AT LEAST twice
• Select an isolated colony from the 3rd sector
  (ideally) of your isolation streak plate and
  re-streak it (isolation streak) onto a new TSA
  plate to confirm purification
• Be sure to label the plate appropriately and SAVE
  the first isolation streak plate as well as the
  original source plate

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Environmental Unknown

• Leave plates to incubate at room temperature and
  check everyday until isolated colonies grow in
  the 3rd sector
• When this occurs, you may make another (third)
  isolation streak plate from one of those colonies
• Save all plates until next weeks lab (parafilm
  older plates as needed)
• Next week you and your partner will choose a
  single organism (each) and we will try to confirm
  purification

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Until 715 / 215

• Practice Aseptic Technique
• Make Environmental Unknown isolation streak
  plates
• Work on stains from last week (Simple, Capsule,
  Gram) Make sure you are checked off for each of
  these (as well as bacteria under Phase Contrast)
• Practice for Gram Stain Unknown Quiz next week
  (even if you have already been checked off for
  this stain)

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Differential Staining(cont.d)

• Acid-Fast stain
• used to identify organisms of the genera
  Mycobacterium and Nocardia, including
  Mycobacterium tuberculosis
• contain waxes (mycolic acids) in their cell walls
  
• impervious to dyes such as those used in a Gram
  stain
• dyes can be driven into these organisms with
  heat
• Include a non-acid-fast control on the slide
  (e.g. Bacillus spp.)

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What is a Control ?

• CONTROL ORGANISM An organism with a known
  reaction to a specific test that is used in
  comparative analysis
• WHEN TO USE A CONTROL ORGANISM Always!

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Acid-Fast Bacterial Cell Wall Structure
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Acid-Fast Mycobacterium
Mycobacterium avium (pink cells)
Mycobacterium tuberculosis (pink cells)
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ACID-FAST STAIN (Photographic Atlas p. 29)

• 1.Clean Slide
• 2. Prepare a dry mount of your organism
  Mycobacterium smegmatis (heat fix)
• 3. Place a piece of filter paper over the slide,
  hold it in place with a clothespin and saturate
  the paper with Carbol Fuchsin
• 4. Gently heat the slide for a FULL 5min.,
  adding more Carbol Fuchsin as the filter paper
  dries out (golden metallic sheen)
• 5. Remove paper and gently rinse slide with
  distilled water (DO NOT leave filter papers in
  sinks they go in the trash cans)
• 6. Decolorize slide with Acid Alcohol for 5-10
  seconds
• 7. Rinse with distilled water
• 8. Counterstain with Methylene Blue for 1min.
• 9. Rinse with distilled water and blot dry

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Endospore Stain

• Endospore stain (Schaffer and Fulton)
• Some microorganisms (e.g. Bacillus megaterium)
  are able to form endospores. In contrast to
  vegetative cells (actively growing cells),
  endospores are resistant to heat, dessication,
  and chemicals (including stains)
• Procedure for Endospore stain is VERY SIMILAR to
  the Acid-Fast procedure

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Endospore Formation

• Bacteria that are resistant to environmental
  extremes frequently produce spores to allow
  survival during adverse conditions.
• The process of condensation of bacterial DNA
  within a series of membranes that accumluate
  calcium, dipicolinic acid and protein layers is
  known as SPORULATION

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Endospore Placement

• Placement can be
• terminal
• subterminal
• central
• What is the difference between an endospore and
  an exospore?

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Endospore Stain
Bacillus anthracis spores
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(No Transcript)
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ENDOSPORE STAIN (Photographic Atlas p. 32)

• Clean Slide
• Prepare a dry mount of your organism Bacillus
  megaterium (heat fix)
• Place a piece of filter paper over the slide,
  hold it in place with a clothespin and saturate
  the paper with Malachite Green
• Gently heat the slide for a FULL 5 min., adding
  more Malachite Green as the filter paper dries
  out (maroon metallic sheen)
• Remove the paper and rinse the slide gently, but
  thoroughly with water
• Counterstain with Safranin stain for up to 2min.,
  then briefly rinse again with water
• Blot dry and examine under 100X oil immersion
  spores will appear green in pink cells

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NEXT WEEK

• Mini-Writing Assignment 3 due
• Aseptic Technique Quiz
• PRACTICE!!!
• Gram Stain Unknown Quiz
• PRACTICE!!!
• Confirmation of Environmental Unknown ISOLATION
• Microscopy catch-up
• (Use of Phase Contrast and all stains)

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Example of Gram Stain Quiz

• Unknown
• Directions Perform a Gram Stain and fill out the
  blanks as you go. Be patient and stay calm, as
  you will be given ample time to practice and
  finish. If your technique doesnt work out the
  first time, clean another slide and start again.
  There are a minimum of 2 and a maximum of 3
  genera in each sample.
• Remember Spelling counts and THIS IS A QUIZ
  therefore no talking or helping your neighbor.
  Raise your hand when you are finished and I will
  come to you. Good Luck!
• 1.The NUMBER of different genera in your unknown
  is, (circle the correct number).
• 1 2 3
• 2. The MORPHOLGY, GRAM STAIN, AND GRAM REACTION
  of the different genera are,
• Morphology
  Gram Stain Gram Reaction
• Cocci or Rod
  Color (pink or purple) G or G-
• A._________________ _________________
  ________________
• B._________________ _________________
  ________________
• C._________________ _________________
  ________________
• 3. The names of the different genera are, (Genus
  and species).
• A.________________________