Microbiology Lab October 2 -- October 3 Week 4 Overview Turn

1
Microbiology Lab

• October 2 -- October 3

2
Week 4 Overview

• Turn in MWA 3, pre-lab 3.1
• Return quiz 2, pre-lab 2 and MWA 2
• Discuss MWA 4

• Review Staining
• Staining Part 2
• Acid-Fast
• Endospore
• Aseptic Technique
• Part 1
• Quiz Next Week!!
• Gram Stain Quiz Next Week!
• Env. Isolate
• Continue with purification

3
Common Problems with MWA2

• Appropriate title Effects of Leucine on E.
  coli growth
• Put the answers in plain site on your graph, both
  on the graph and written out
• Strain B is at saturation therefore it would be
  gt 50 mM of leucine

4
Hand in assignments

• MWA 3
• Pre-lab 3

5
Mini-writing 4

• Objective To concisely and correctly summarize a
  scientific article
• This is a critical skill when writing reports
• You need to be able to re-read your own work and
  catch mistakes great time to work on grammar,
  spelling and syntax
• Make sure to hit on the main points of the lab

6
Review Staining

• What are the two major categories of staining we
  discussed last week and provide an example of
  each
• The acid-fast and endospore stains are examples
  of what category of staining (hint think about
  what these stains tell us)?

7
Review Staining Techniques

• Describe in detail how you would prepare a dry
  mount

8
Todays Stains

• Acid-Fast
• Endospore

9
History/development of the Acid-fast stain

• Developed in 1880s
• Scientists noticed certain dyes react in very
  specific ways with organs
• The specificity of dyes for microbial
  cells/cellular components was subsequently
  studied
• Research along these lines was continued by
  examining chemicals and their abilities to kill
  certain microorganisms
• Work of this nature was some of the first to
  allow us to understand different agents might be
  useful in killing microbes without hurting humans
  (weve talked about this in a previous lecture)

Chemical Archievers http//www.chemheritage.org/c
lassroom/chemach/pharmaceuticals/erlich.html
10
Acid-fast Stain

• What specific cellular component is the basis for
  the acid-fast staining procedure?
• that is, what component of the cell requires we
  use this procedure rather than, for example, a
  Gram stain?

11
Acid-fast stain contd.

• Used to identify organisms of the genera
  Mycobacterium and Nocardia, including M.
  tuberculosis and M. leprae
• Mycobacterium contain waxes called mycolic acids
  in their cell walls
• These molecules are impervious to dyes such as
  those used in the Gram stain
• The mycolic acids are one of the main
  pathogenicity/virulence factors of these
  organisms
• Certain dyes can be driven into these organisms
  with heat Include a non-acid-fast control on the
  slide (e.g., Bacillus spp.)

12
Acid-fast stain Importance

• Historical perspective Tuberculosis
• Spinal columns of Egyptian mummies show decay due
  to tuberculosis (2400 BCE)
• Hippocrates identified the disease as early as
  460 BCE
• 17th century records have accurate descriptions
  of the disease
• 1865-French military doctor demonstrated the
  disease could be passed from humans to cows to
  humans
• In 1880s the acid-fast stain allowed us to view
  this microbe

13
Acid-fast stain Importance

• Historical perspective
• Leprosy
• 1873- Dr. G. H. A. Hansen discovered the
  bacterial agent involved in leprosy
• Throughout the 1900s (1941-1950) a variety of
  treatments were developed and used
• By 1970 a multi-drug treatment had been developed
  and is still in use
• What current infection is often controlled with a
  multi-drug treatment?

14
Acid-fast stain Importance

• These two disease are responsible for millions of
  deaths over the centuries
• When the acid-fast stain actually allowed us to
  view these organisms we were better able to treat
  and control them
• Although Kochs postulates do not necessarily
  rely on being able to see the organism, it
  certainly helps as a means of identifying what
  organism is responsible or at least related to
  the disease

15
Acid-Fast vs. Gram Positive Cell Wall Structure

• Note the differences in molecular structure

Gram positive cell wall image taken from
http//www.prism.gatech.edu/gh19/b1510/grampos.gi
f
16
Acid-Fast Stains of Mycobacterium spp.

• Which are the acid-fast organisms and which are
  the non-acid fast organisms? Look at your
  procedure.

17
Acid-fast stain procedure

• Clean slide and prepare a dry mount of the
  organism Mycobacterium smegmatis (plate) and
  Bacillus megaterim (broth) (dont forget to heat
  fix)
• Place several wet paper towels around the Bunsen
  burner
• You will lose several technical points if you do
  not do this
• Place a piece of filter paper over the slide,
  hold it in place with a clothespin and saturate
  the paper with carbol fuschin
• DO NOT mix up filter paper with parafilm!!!!
• Gently heat the slide for 7 minutes. Add more
  carbol fuschin as the filter paper starts to dry
  out

18
Acid-fast stain procedure contd.

• Remove paper and gently rinse slide with
  distilled water
• Paper should be disposed of in contaminated waste
• Decolorize slide with acid alcohol for 5-10
  seconds
• Rinse with distilled water
• Counterstain with methylene blue for 1 minute
• Rinse with distilled water and blot dry

19
Endospore Stain

• Endospore stain (Schaffer and Fulton staining
  method)
• The staining procedure is very similar to the
  acid-fast procedure
• 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

20
Endospores

• Many bacteria, especially members of the Bacillus
  spp. are able to form endospores
• 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

21
Endospore Formation
22
Endospore Placement
Terminal
Subterminal
Central
23
Example of an endospore stain
Which is the spore and which is the vegetative
cell in this picture?
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Endospore stain procedure

• Clean Slide and prepare a dry mount of the
  organism Bacillus megaterium (plate) (remember to
  heat fix)
• Place several wet paper towels around the Bunsen
  burner
• Again- loss of major technical points if you
  dont!!!
• Place a piece of filter paper over the slide,
  hold it in place with a clothespin and saturate
  the paper with malachite green (wear gloves with
  this stain!)
• Gently heat the slide for 5 minutes. Add more
  malachite green as the filter paper starts to dry
  out

25
Endospore stain procedure contd.

• 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.

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Aseptic Technique

• Part 1

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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
• There will be a 5 point quiz next week

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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

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
Gram Stain Unknown Quiz

• Next week

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Example of Gram Stain Quiz for Next Week

• Unknown
  
  Name
• 
  
  Date Sec
  
• Gram Stain TEST
• 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

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What is this?
http//slph.state.nc.us/public2/images/Yersinia_pe
stis_microscopic.jpg
33
How about this one?
http//medlib.med.utah.edu/kw/derm/mml/24850028.jp
g
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and this one?
http//www.wzw.tum.de/micbio/cms/UserFiles/Image/n
euhaus3.jpg
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Environmental Unknown
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Environmental Isolate

• Continue with purification
• Re-streak as often as possible
• You should also start keeping track of 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?
• When you think you have pure cultures, do a wet
  mount of each and have a TA verify purity

37
Next Week

• MWA 4 Pre-lab 3.2 due
• Gram stain quiz
• Aseptic technique quiz
• Dental plaque observation
• Env. Isolate purity confirmation, staining,
  storage conditions