Microbiology Lab

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Microbiology Lab

• October 9
• October 10

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Exercise 3.2Overview

• Dental Plaque observation
• Environmental Unknown
• Isolation confirmation
• Begin storage conditions
• Microscopy catch-up

• Assignments
• return MWA 3
• due pre-lab 3.2 and MWA 4
• Aseptic Technique Quiz
• Gram Stain Unknown Quiz

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Schedule for Today

• Aseptic technique quiz
• Gram stain quiz
• Dental plaque observation
• Env. Unknown
• Microscopy catch-up

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Quiz

• Aseptic Technique
• Gram Stain Unknown

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Aseptic Technique Quiz

• Remember-sterile/aseptic technique is critical in
  microbiology and health professions
• The goal of sterile technique is to prevent the
  introduction of microbes into an
  environment-whether liquid or solid media or
  someones body
• This is a 5 point quiz
• No cheating

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Aseptic Technique Quiz

• Perform transfers of broth medium aseptically as
  described in the lab manual (3-3)
• Remember you cannot set tubes and bottles down
  once you have picked them up
• Pay attention to pipette gradations prime the
  pump
• 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)
• When finished, place tubes (in order) in the
  class tube racks provided

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Gram Stain Unknown Quiz

• You must at least attempt a stain/quiz today
• If you are unsuccessful, try again during MY open
  hours or next week in class
• 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
• Complete a Gram stain on at least one dry mount
• If you can handle multiple slides at a time you
  may complete the stains all at the same time

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Gram Stain Unknown Quiz

• If you are confident with your stain, fill out
  the quiz completely
• Check for spelling, terminology, and that you
  have written the organism in the correct
  scientific fashion
• DO NOT call me until you are ready to hand in
  your quiz
• I MUST see your stain/slide
• If you take it off the microscope without me
  seeing it AND approving it you will have to start
  over

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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 or the sheet I
  handed out
• You MAY NOT ask your neighbors for help
• At this point I cannot
• tell you how to do the stain
• focus on the organisms
• explain how fill out your quiz correctly or
• tell you what the correct spellings for your
  organisms are

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Normal FloraDental Plaque
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Normal Flora

• Norma flora of the human body
• Composed of hundreds of species
• Grow on or in the body
• Categories
• Benign
• Beneficial
• Functions
• Direct vs. indirect
• Protection
• Nutrient production/absorption

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Normal Flora

• Three major areas of the human body we are often
  concerned about in terms of normal flora are
  the
• Skin
• generally Gram positive bacteria
• Divided into resident and transient populations
• Gastrointestinal tract
• Varies considerably, often dependent upon diet
• Oral cavity
• A few main species contribute to the production
  of dental plaque
• Formation of dental plaque can create habitats
  for other organisms
• When these organisms are disrupted, there can be
  serious consequences

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Normal Flora of the Oral Cavity

• The microbial community of the oral cavity is
  very complex and heterogeneous
• Though there are often many nutrients present,
  some components are not beneficial to microbial
  growth
• Ex saliva contains nutrients but also contains
  lysozyme- an enzyme especially damaging to Gram
  positive organisms
• If lysozyme is so damaging to Gram positive
  organisms, what molecule/structure do you think
  it works on?

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Normal Flora of the Oral Cavity

• In order to colonize teeth, bacterial must be
  able to attach
• They do this by attaching to a glycoprotein layer
  formed as a result of saliva
• This initial colonization is done by the
  following organisms
• Streptococcus sanguis, S. sobrinus, S. mutans,
  and S. mitis
• When there is extensive growth of these organisms
  dental plaque is formed

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Normal Flora of the Oral Cavity

• If this plaque is not removed, it continues to
  get thicker as a result of further colonization
  by other organisms
• Fusobacterium spp. is a filamentous bacteria that
  grows within the matrix created by the
  Streptococcus spp.
• Also present may be spirochetes (Borrelia spp.),
  Gram positive rods and Gram negative cocci

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Oral Cavity

• The bacteria that form plaque produce acid as a
  byproduct of metabolism
• This acid can cause the calcium in the enamel of
  the tooth to deteriorate
• Table sugar (a.k.a. sucrose) helps speed this
  process because bacteria can ferment this
  molecule to acids

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Oral Cavity Microbes

• In addition to tooth decay, recent research has
  indicated microbes found in the mouth can be
  associated with heart problems
• This is why you may be instructed to take
  antibiotics before dental work if you have a
  heart murmur

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Dental Plaque Observation

• In light of the importance of the microbial
  community of the mouth, we will be observing our
  own flora
• To do this we will obtain organisms from our
  mouths and prepare a wet mount as well as
  complete a Gram stain of our samples
• It is very important to use sterile technique and
  be careful when obtaining your samples
• Use your own tube of toothpicks -no sharing
• Once you have taken the toothpick out of the tube
  it should immediately be used for obtaining a
  sample
• DO NOT set it down anywhere
• If you drop it, USE ANOTHER ONE

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Dental Plaque Observation Procedure

• 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 and Gram stain it

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Environmental Unknown
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Environmental Unknown

• Check plates for isolated colonies in the 3rd
  sector
• Choose one plate between lab partners (DO NOT
  dispose of other plates yet)
• Make and view a wet-mount of an isolated colony
• What type of microscopy should you be using for
  this?
• Examine your wet-mount for uniformity of size and
  shape-see me or another TA to confirm status

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Environmental UnknownAfter Confirmation of
Purity

• To confirm purity, perform a Gram Stain (with
  controls) if time permits and IF you have a fresh
  culture
• Generally between 24-48 hours depending on your
  organism
• If you do not have a fresh culture, re-streak a
  plate and come in at the appropriate time
• If your chosen Environmental Unknown is confirmed
  to be a pure culture, keep all plates in its
  lineage
• Refrigerate plates from past streaks
• Dispose of all unrelated plates ONLY once you
  have confirmed purity
• Remember to watch for fungus and parafilm if
  necessary

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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

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Environmental UnknownStorage Conditions

• The experiment will take two full weeks to
  complete, but you will not be behind if you need
  to start later
• DO NOT waste media tubes or your time if you are
  not certain you have a pure culture yet

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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)

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Environmental UnknownStorage Conditions (Contd.)

• 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 screw caps all the way down
• Do not disturb the tubes for two full weeks
• That is, dont take culture from them for any
  reason

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Environmental UnknownStorage Conditions (Contd.)

• 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

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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

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NEXT WEEK

• Exercise 4 Enumeration of Bacteria (Hamburger)
• Mini-Writing Assignment 4 will be returned,
  pre-lab 4 due
• Continue Environmental Unknown isolation if
  necessary
• Perform appropriate stains on your organism
• You must have fresh cultures for this
• To do an endospore stain you must make a stress
  plate-see me for directions
• Dont forget to start Storage Conditions
  experiment once you have confirmed purity