Lab 4 Lab 3 Goals and Objectives

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Lab 4 Lab 3 Goals and Objectives

• Learn methods of differential staining
• Use two or more stains and categorize cells into
  groups
• Gram Staining
• Separates bacteria in two different groups Gram
  positive and Gram negative
• Important first test for bacterial identification
• Acid Fast Staining
• Detects the high mycolic acid content of certain
  bacterial cell walls, like those of Mycobacteria
• Mycolic acids are long fatty acids found in the
  cell walls of a group of bacteria that includes
  Mycobacterium tuberculosis.
• Important diagnostic tool

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GRAM STAIN 1. Crystal Violet Primary Stain
stains all cell walls purple 2. Iodine Mordant
combines with crystal violet to create large
insoluble dark purple complex 3. Alcohol
Decolorizer solubilizes Gram negative outer
membrane flushing iodine/crystal violet complex
out of thin wall, thick Gram positive cells
resist decolorization 4. Safranin Counterstain
stains cell walls pink (obscured by purple walls
of Gram positive, but Gram negative, who had
become colorless, now are pink)
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Figure 15.2
Figure 15.1
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Use wax pencils (China Marker) Sharpie will
wash off with alcohol! Be sure to resuspend
broth cultures completely! Exercise 15 Gram
Stain Each pair make slides and stain At
least 2 triple smear slides with mixes from
broths 1. Staphylococcus aureus Pseudomonas
aeruginosa 2. Bacillus subtilis
Klebsiella pneumoniae No need to Gram stain
Mycobacterium smegmatis
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Gram Staining
Fig 15.3 pg 110
Add step (3.5) Wash 2 seconds
(until water runs clear)
(until water runs clear)
immediately!!!
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Gram stain
Staphylococcus aureus Pseudomonas
aeruginosa
Bacillus subtilis Kelbsiella pneumonia
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Exercise 17 Acid Fast Staining

• 1. Carbolfuchsin primary stain
• 2. Aid-alcohol decolorizer
• 3. methylene blue counter-stain
• 
  
• 
  Staphylococcus aureus
• 
  Mycobacterium smegmatis

• Each pair make minimum one acid fast slide with
• Staphylococcus aureus (use 2-4 loops of broth
  on slide first)
• Mycobacterium smegmatis (slant, use needle, get
  tiny amount and mix into
• broth on slide very well break up all clumps)

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Fig 17.1 pg 118
Acid Fast Stain
30sec
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• Make all smears on slides first so they can
  dry before heat fixing!!!
• Split the work to save time but make sure
  everyone gets to see all slides!
• Each pair needs for lab 4
• 1 broth culture Staphylococcus aureus
• 1 broth culture Pseudomonas aeruginosa
• 1 broth culture Bacillus subtilis (replaces B.
  megaterium)
• 1 broth culture Klebsiella pneumonia (replaces M.
  catarrhalis)
• 1 wet slant 5 day culture Mycobacterium smegmatis
• Crystal violet
• Grams iodine
• 95 Alcohol (Ethanol)
• Safranin
• Methylene blue
• Acid-alcohol
• Carbolfuchsin