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Flashcards in LAB: Differential stains Deck (15):
1

Gram Stain Procedure

  1. Prepare a smear of the bacteria
  2. Apply Crystal violet. Sit for 1 minute and rinse with distilled water
  3. Apply Gram's Iodine. Sit for 1 minute and rinse with distilled water
  4. Rinse gently with ethyl alcohol. Do not over rinse - rinse for few seconds. Rinse with distilled water for 10-15 seconds
  5. Apply Safranin. Sit for 1 minute and rinse with distilled water
  6. Blot dry with bibulous paper
  7. Observe on high power
  8. Sketch and label Gram + control and Gram - control

2

Gram Stain Procedure cont...

  • Purple indicates Gram-positive (Staphylococcus; cocci in clusters)
  • Pink indicates Gram-negative (E. coli; individual rods)

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3

Gram-Positive Composition of cell wall

  • thick peptidoglycan
  • no outer membrane
  • stained purple
    • cells are stained
    • uncolored around the cell is the capsule

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Gram-Negative Composition of Cell Wall

  • thin peptidoglycan
  • outer membrane (highly negative)
  • stained pink

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5

Capsule Stain

  • background purple stained
  • capsule is halo-like

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Importance of capsule

  • Hides bacteria from immune system; gives some bacteria its virulence
  • adhesion
  • made of polysaccharides
    • thick rigid layer

7

​Endospore Stain

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  • differentiate between spores and vegetative cells
  • pink stained cells = vegetative cells
  • light greened stained cells = endospores
  • Schaeffer-Fulton endospore slide 

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​Central location

  • Central endospores are located in the middle of the vegetative cell

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Terminal location

  • Terminal endospores are located at the end of the vegetative cell

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Examples of bacteria that forms endospores

  • Bacillus
  • Clostridium

11

Differential Stains

  • use 2 different dyes
  • differentiate between cell types or cell structures

12

Primary Stain

  • Crystal Violet - stains all cells purple

13

​Mordant

  • Gram's Iodine - fixes crystal violet into cell wall of G+ cells

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​Decolorizer

  • Ethyl Alcohol -  removes crystal violet from G- cells

15

​Counterstain

  • Safranin - ​stains G- cells pink