Right Away

1
Right Away

• Wash Hands
• Lab Coat
• Wash Lab Bench

2
Quiz
3
Prior to Lab

• Due
• MW 3
• Pre-Lab 3.1
• Return
• MW 2
• Pre-Lab 2
• Quiz 2

4
MW 4

• Objective Be able to summarize
• This skill is a critical skill when writing
  reports
• You need to be able to re-read your own work
  and catch mistakes
• This assignment will help you practice with
  grammar, spelling and syntax
• The article is a bit more difficult subject
  matter. You may have to look topics up.

5
Review

• Microscopy used for dead, stained organisms?
• Differential Stain?
• Examples? (hint and - )
• More Safety!

6
Today

• Do two different differential stains
• Acid-Fast
• Endospore
• Aseptic (sterile) Technique Part 1
• Need to practice!
• Will be tested next week!
• Environmental Isolate
• Is it purified?

7
Exercise 3(Part 1)

• Differential Staining (cont.)
• Sterile Technique

8
Demo

• Bunsen Burner
• Paper Towels
• Clothes Pin
• Filter Paper
• Slide

9
More Staining!

• Acid-Fast
• Used to identify organisms in the genera
  Mycobacterium and Nocardia
• M. tuberculosis
• M. leprae
• Mycobacterium contain waxes in their cell walls
  called mycolic acids.
• These molecules are impervious to dyes such as
  the ones used in the gram stain
• The mycolic acids are one of the main
  pathogenicity/virulence factors of these
  organisms.

10
Acid-Fast

• Certain dyes can be driven into these organisms
  with heat.
• Include a non acid-fast control on the slide
• (e.g. Bacillus spp.)
• 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!

11
Acid-Fast Cell Wall Structure
12
Acid-Fast of Mycobacterium spp.
Mycobacterium avium (pink cells)
Mycobacterium tuberculosis (pink cells)
13
Acid-Fast Procedure

• Clean slide and prepare dry mount of the
  organism Mycobacterium smegmatis and Bacillus
  megaterium
• Heat Fix!!
• Wet paper towels around the base of the Bunsen
  burner
• You will lose technique points if you do not!
• Filter Paper

14
Acid-Fast Procedure

• Saturate the paper with carbol fuchsin
• DO NOT mix up filter paper with parafilm!
• GENTLY heat the slide for 5-7 min. Adding more
  carbol fuchsin as the paper starts to dry out.
• Do NOT allow it to become iridescent!!!
• Remove paper and GENTLY rinse with distilled
  water
• Paper should be disposed of in contaminated
  waste bin

15
Acid-Fast Procedure

• Decolorize with acid alcohol for 5-10 seconds
• (decolorizes B. megaterium)
• Rinse with distilled water
• Counter-stain with methylene blue for 1 minute
• Rinse with distilled water and blot dry
• Visualize under 100x oil immersion
• Acid-Fast cells are pinkish-purple, non
  acid-fast cells are blue

16
Endospore

• Characteristics/definition of an endospore
• An endospore is essentially a hard covering that
  contains the genetic material of the organism and
  is often produced under harsh environmental
  conditions
• Endospores are generally resistant to
  destruction by desiccation (drying out), heat,
  and staining
• We can use a combination of heat and malachite
  green to stain the spore
• Only a few genera of microorganisms (e.g.,
  Bacillus megaterium) are able to form
  endospores. These include Bacillus and
  Clostridium.

17
Endospore

• It is important to note that a bacterial spore
  and a fungal or plant spore are different
  structures with different functions
• A fungal or plant spore is often involved in
  sexual reproduction
• A bacterial spore is a means of protection and
  is not involved in sexual reproduction
• Many pathogenic organisms are spore-forming.
• E.g. B. anthracis, C. tetani, C. botulinum

18
Endospore Formation
19
Spore Placement
20
Endospore Placement
Terminal
Subterminal
Central
21
Ex. Of Endospore Stain
Bacillus anthrasis spores, Bacillus megaterium
spores
22
Endospore

• The resistance of the endospore is due to a
  tough outer covering made of the protein
  keratin.
• Keratin resists staining
• Malachite green is forced into the stain by
  steaming the bacterial emulsion.
• Malachite green has a low affinity for cellular
  material, so vegetative cells and spore mother
  cells can be decolorized with water and
  counterstained with Safranin.

23
Endospore Stain Procedure

• Clean Slide and prepare a dry mount of the
  organism Bacillus megaterium from a plate
  (emulsify) Heat Fix
• Wet paper towels around base of the Bunsen burner
• technical points
• Place a piece of filter paper over the slide,
  hold it in place with a clothespin and saturate
  the paper with malachite green (carcinogenic)
• Gently heat the slide for 5 minutes. Add more
  malachite green as the filter paper starts to dry
  out

24
Endospore Procedure

• Remove the paper and rinse the slide with WATER
  for 30 seconds.
• Counterstain with safranin stain for 20
  seconds, then briefly rinse again with water
• Blot dry and examine under 100X oil immersion
  spores will be green in pink cells.

25
Aseptic Technique Part 1

• One of the major goals of microbiology is not to
  contaminate our work
• By using special procedures we can generally
  keep our media free from microbes we do not
  want growing
• Today we are going to practice our techniques
• Next week there will be a 5 point quiz

26
Aseptic Technique Part 1

• Aseptically transferring liquids by pipetting
  sterile media from one tube to another.
• This exercise requires (and teaches) dexterity
  and technical skill. Are not using sterile
  media for the transfers.
• It is necessary to stress the importance of
  manipulating the equipment required to perform
  sterile transfers even though the medium used is
  water

27
Aseptic Technique Part 1

• Obtain all required materials and set up
  appropriately
• Once you start, you cant stop
• Loosen the lids of any screw-cap bottles/tubes
• Peel the paper surrounding the pipette slightly
  open
• Put the pipette pump on the end of the pipette

28
Aseptic Technique Part 1

• Insert the pipette into the Pipette Pump and
  gently, but firmly, push in and twist (Fig.
  3.1)
• Be sure not to contaminate the pipette by
  touching the tip with your hands
• The blue Pipette Pump is for 1ml pipettes the
  green one is for 5ml and 10ml pipettes

29
Aseptic Technique Part 1

• Pick up your bottle and tube
• Open both, not setting the caps down or turning
  them over and flaming the lips of both
• Insert pipette into liquid
• Draw out desired volume, flame sterilize lip of
  bottle, replace cap
• Dispense liquid from pipette into tube
• Flame sterilize the lip of tube, replace cap
• Put pipette into paper and discard

30
Aseptic Technique Part 1

• Practice transferring water from a culture
  bottle to tubes aseptically in various amounts
  with the three different sizes of pipettes (1, 5
  and 10 ml).
• Next week you will use an enriched media to
  transfer and contamination will count.

31
Gram Stain Unknown Quiz

• Unknown
  Name
  
• 
  
  Date Sec
  
• Gram Stain Quiz
• 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.
• B.
• C.

32
Environmental Isolate

• Please view a wet mount of your most promising
  organism
• Prepare a wet mount of your isolate and examine
  under 100X w/ oil
• Is there more than one organism? TA/TAA check
• Prepare a simple stain of your isolate
• Is there more than one morphology?
• If not, what is the morphology of your organism?
• Continue with purification
• - Re-streak as often as possible
• - You should know how fast each one grows
• - Ex do you have a lot of growth in 24 hours
  or does it take longer to get a decent amount of
  growth?

33
Unknown Isnt Pure.

• No Worries just keep streaking the TSA plates
• Continue to do wet mounts to check purity
• Make sure you do these as often as
• possible

34
IMPURE
PURITY
35
Next Week

• Due
• MW 4
• Pre-Lab 3.2
• Aseptic Technique Quiz
• Gram Stain Unknown Quiz
• Env. Isolate purity confirmation, staining,
  storage conditions