Exercise 2

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Exercise 2

• Staining
• Begin Purification of
• Environmental Unknown

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Staining

• When using Brightfield illumination, bacterial
  cells are often stained to provide contrast and
  make them easier to see

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Stains can be

• ANIONIC (negatively charged) these will bind to
  positively charged cell walls and are often used
  in negative staining techniques
• NEGATIVE STAIN Used to determine morphology
  and cellular arrangement in bacteria that are too
  delicate to withstand heat fixing. The negative
  charge on the surface of the cell repels the
  stain, thus the cell remains unstained while the
  background becomes dark
• Eosin, nigrosin, and methylene blue are examples
  of such stains.

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Stains can be

• CATIONIC (positively charged) these stains bind
  to negatively charged cell walls
• GRAM STAIN An important differential stain that
  reveals fundamental differences in the nature of
  the cell wall due to differences in the amount of
  peptidoglycan
• Gram-positive cells appear purple in color.
  Gram-negative cells appear pink in color

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Dry mount preparation

• Emulsify
• mix thoroughly a small amount of bacteria in a
  drop of water
• Allow the slide to air dry
• the thinner you spread the emulsion, the faster
  it will dry
• Heat-fix
• gentle heating of the slide by passing it through
  the flame of a Bunsen burner 3 or 4 times

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SIMPLE STAIN

• SIMPLE a single dye is used
• This stain reveals the basic cell morphology and
  arrangement
• Other stains may be used such as Crystal Violet
  or Safranin
• (Cells have been heat-fixed and a dry mount
  prepared before staining)

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Methylene Blue Stain

• Is a SIMPLE STAIN using Methylene Blue
• Reveals the basic cell morphology and arrangement
  of an organism
• Bacillus megaterium is a large, Gram- positive
  rod used for this stain

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Methylene Blue Stain

• Simple stain of
  a Bacillus spp.

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Methylene Blue Procedure

• Prepare a dry mount of a culture
• Flood slide with Methylene Blue, let stand for 1
  minute
• Gently rinse stain off slide with distilled water
• Blot dry with Bibulous paper
• Observe under 100X oil immersion

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DIFFERENTIAL Stain

• DIFFERENTIAL STAIN A staining procedure where
  two or more dyes are used to distinguish between
  two different organisms or between two different
  parts of an organism
• Gram stains, spore stains, and acid-fast stains
  are examples of this type of stain

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Gram Stain

• GRAM STAIN a differential stain that is widely
  used to aid in identification of bacteria
• Originally devised by Hans Christian Joachim
  Gram, a Danish doctor
• Gram stains differentiate between two major cell
  wall types  

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Gram -
Gram
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Gram Stain
Proteus vulgaris G- rod, Opportunistic
Streptococcus pneumoniae G cocci, Pathogenic
Staphylococcus epidermidis G cocci, Opportunistic
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Gram Stain

• Gram-negative rods, Gram-positive rods, and
  Gram-positive cocci will be available for
  staining
• Two cultures (e.g. a Gram-negative rod and a
  Gram-positive coccus) are prepared on the same
  slide and stained simultaneously
• Prepare a dry mount mixing a loopful (inoculating
  loop) of each of these cultures

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Gram Stain Procedure

• Prepare a dry mount
• Flood with Crystal Violet 1 minute
• Rinse with water
• Flood with Gram's Iodine let stand 1 minute
• Pour off iodine and decolorize with 70 ethanol
  (ten seconds MAX)

• gently rinse with water
• Flood with Gram's Safranin and let stand 1
  minute
• Rinse with water
• Blot dry G
• Observe first 40x, 100x, then 100X with oil.

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Encapsulated Bacteria

• Capsule staining is performed to identify a
  pronounced gelatinous, slimy layer called a
  capsule. Some capsules appear to be made of
  glycoprotein, others contain polypeptides. All
  appear to be water-soluble. Many potential
  pathogens have a capsule.

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Capsule Stain Procedure

• Emulsify Klebsiella pneumoniae into 1 drop of
  Congo Red (do not heat fix )
• Spread out the emulsion over the entire slide
  using another slide and allow to air dry (similar
  to blood smear)
• Flood the slide with Maneval's Stain and let
  stand for 1 minute
• Rinse with water and blot dry
• Observe under 100X oil immersion

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Environmental Isolate

• Check your environmental isolate
• Look at page 2-3 and record configuration, margin
  and elevation for each of your isolates
• If you have streaked your plates at least 3 times
  make a wet mount of your bacteria and inspect for
  purity
• Have me check your wet mount