Week 3 Overview

1
Week 3 Overview

• Return MWA 1
• Hand in pre-lab assignment
• Discuss MWA 2
• Review graphing
• Due next week
• Environmental Isolate Project
• Observe plates and write down observations
• Re-streak for purity if necessary
• Staining
• Discuss staining methods and why they are used
• Perform simple stain, Gram Stain, capsule stain

2
Mini Writing Assignment 2

• Hand graphed, not on the computer
• Line of best fit
• See pages 38-50 in McMillan for an overview of
  graphing

3
Environmental Isolate

• Observe the plates
• They were allowed to grow for about 48 hours and
  then refrigerated to slow growth
• Write down your observations about growth and
  draw a picture of your plate/colonies for future
  reference
• Refer to page 2-3 in your manual to describe the
  colonies
• Re-streak for purity
• If colonies appear to be composed of more than
  one organism
• Or if you do not have any growth in the 2nd or
  3rd sections

4
Review

• Three main factors of microscopy
• Magnification
• Resolution
• Contrast
• Adjust the factors by altering the organism or
  the microscope
• Last week we viewed organisms by adjusting the
  contrast (phase contrast microscopy)
• This week will alter contrast by staining the
  organisms (dont use your phase ring-set it to
  zero to use brightfield)

5
Staining

• The purpose of staining is to increase contrast
  so you can see organisms or specific parts of
  organisms parts organisms
• Staining can be used to determine the morphology
  of an organism (simple staining) and/or
  differentiate the organisms from one another
  (differential staining)

6
Staining

• Two types of stains cationic and anionic
• Cationic stains bind to negatively charged
  structures
• Anionic stains bind to positively charged
  structures
• Generally, bacterial membranes are negatively
  charged and cationic stains are used

7
Dry Mount Procedure (broth)

• Obtain and clean a slide and coverslip
• Obtain culture of organism
• Vortex culture
• Take one or two loop-fulls of culture and place
  on the slide (remember your sterile technique)

8
Dry Mount Procedure cntd.

• Spread the loopfull of culture on the slide The
  thinner your spread it, the faster it will dry
• Let the slide air dry
• Fix the bacteria to the slide by passing the
  slide above the flame two or three times
• Do not stick the slide in the flame and leave it
  there
• Once it is heat fixed, you are ready to stain

9
Dry Mount Procedure (plate)

• Add one drop of water to the slide
• Use your loop to pick some culture off the plate
• Mix (emulsify) the culture with the water and
  spread out over the slide
• Allow to air dry
• Note Today when we take from the plate we will
  not use water for emulsification, we will
  actually use the stain Congo Red

10
Simple Stain

• This type of stain uses only one dye and is used
  to determine the morphology of the organism
• Today we will be using methylene blue to stain
  Bacillus megaterium (the same organism many of
  your saw last week using phase contrast
  microscopy)
• The methylene blue reacts with the cell wall of
  the organism so that you can view the cells under
  brightfield microscopy

11
Simple Stain

• Simple stain
• of Bacillus spp.

12
Simple Slide cntd.

• Prepare dry mount of B. megaterium
• Put several drops of methylene blue on the slide
• Let the stain sit for 1 minute
• Rise with distilled water
• Remember not to use ethanol!
• Blot dry with bibulous paper
• Remember not to rub the slide

13
Gram Stain

• History and significance of Gram stain
• Developed in 1883 by Hans Christian Gram
• The Gram stain essentially divides bacteria into
  two classes based on cell wall composition
• One of the most important stains in microbiology
• Mechanism
• Gram positive organisms have thick layers of
  peptidoglycan (peptide sugar)
• Gram negative organisms have some peptidoglycan
  but not nearly as much
• Additionally, in these organisms the
  peptidoglycan is beneath an outer membrane

14
Gram Positive vs. Gram Negative
http//www.slic2.wsu.edu82/hurlbert/micro101/imag
es/cellwall.gif
15
Gram Stain

• Crystal violet reacts with the peptidoglycan
• Grams iodine sets the crystal violet (acts as a
  mordant)
• Decolorizing washes away the crystal violet from
  the Gram negative cells
• Safranin reacts with the Gram negative cell walls
  and allows us to differentiate (counter-stain)

16
Gram Stain cntd.
http//water.me.vccs.edu/courses/ENV108/changes/gr
am.jpg
17
Gram Stain Procedure

• Prepare a dry mount of
• Bacillus megaterium (Gram positive rod),
• Escherichia coil (Gram negative rod), and
• Staphylococcus epidermidis (Gram positive cocci)
• You can mix them all together on the slide
• Remember to sterilize your loop in between
  culture tubes and flame the lip of the tube
  before and after taking broth
• Remember to heat fix the cells to the slide once
  dry

18
Gram Stain Procedure

• Add several drops of crystal violet to the
  slide-let stand 1 minute
• Rise with water
• Add several drops of Grams iodine-let stand 1
  minute
• Pour off iodine, decolorize with 70 ethanol (in
  the squeeze bottle, not the dropper bottle)

19
Gram Stain Procedure cntd.

• Add several drops of Grams safranin and let
  stand for 2 minutes
• Rinse with water, blot the slide dry

20
Capsule Stain

• Capsules
• Composition-most are made of polysaccharide, some
  composed of protein, and some of glycoproteins
• Function-often produced by pathogenic bacteria
  (one of the factors of pathogenicity) and help
  with attachment to host cell structures
• The molecules in the capsule repel the stain and
  only the background and the cytoplasm are stained

21
Capsule stain cntd.

• Example of capsule stain
• This is an example of
• a negative stain
• The dye has stained
• the background and
• the cell, but does not
• react with the capsule

http//www.rci.rutgers.edu/microlab/CLASSINFO/IMA
GESCI/capsulestain.htm
22
Capsule Stain Procedure

• Add one drop of Congo Red to the end of a slide
• Emulsify some Klebsiella pneumoniae into that
  drop
• Use a separate slide to drag the emulsion across
  the first slide
• Allow slide to air dry-DO NOT HEAT FIX
• Add a few drops of Manevals stain-let sit 1
  minute and rinse with distilled water

23
Summary
24
Microscopy

• After you finish making your slides, view them
  under each objective
• Before you go to oil, check with me
• Show me your oil immersion so I can check you off
• When you are done with your staining empty your
  stain trays in the front sinks and rinse with
  water and ethanol-set in sink to dry
• You may need to empty the trays a few times
  throughout the session

25
Wrap-up

• Clean microscope, let me check it off
• Write down the correct spellings of your Gram
  stain organisms, draw a picture of them and show
  me this before you leave today!
• MWA 2 is due next week
• Do pre-lab for exercise 3.1

26
Open Hours

• Monday-930-1030 (Robert)
• Tuesday-400-600 (Breanne/Maddy
  Camille/Robert)
• Wednesday-400-600 (Camille/Maddy Jean)
• Thursday-400-500 (Breanne/Jean)
• Friday-1200-200 (Alternating)