Microbiology LAB / Exam 2

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Steps: "How to make a SMEAR"
  1. Heat flame a loop, add distilled H​2O to loop
  2. Tap the d.H​​2​O from the loop onto 2 slides
  3. Heat loop, cool, remove small amount of bacteria from tube
  4. Add the bacteria to the d.H​2​O on both slides
  5. Spread the bacteria evenly across the slide.​ Purpose: So you can see the bacteria evenly under the microscope (not just blob).
  6. AIR DRY
  7. "Heat fix" slides by passing through a flame 3-4X. Purpose: To kill and adhere bacteria.
Steps: Simple Stain
  1. Place slides on staining tray
  2. To CV slide: Cover with Crystal Violet for 1 min.​ To S slide: Cover with Safrinan for 1 min.
  3. Rinse slide with distilled H​2O
  4. Blot dry with Bibulous paper
  5. View under microscope
  • 10x ---> 40x --oil-->100x (Record results)
  • When done: slides go in the sharps container and Bacterial cultures go in the Rack on the autoclave cart.
NOTE: The color of the slide takes up what you put on there.
Steps: Gram Stain
  1. ​Cover slides with C.V. for 1 min.
  2. Rinse slides with deionized H​2​O
  3. Cover slides with Iodine (mordant) for 1 min.
  4. Rinse slides with di.H​2​O
  5. Hold slide at 4​​5o angle​. Trickle alcohol (decolorizer) for 5-10 seconds until runoff is CLEAR.
  6. Rinse slides with di.H​2​O
  7. Cover slides with Safranin for 1 min.
  8. Rinse with di.H​2​O
  9. Blot dry with Bibulous paper
  10. View under microscope
  • 10x ---> 40x --oil--> 100x (Record results)
Cell Wall differences of:
  • Gram POSITIVE
  • Gram NEGATIVE
Gram POSITIVE:
  • Thick Peptidoglycan
  • Teichoic & Lipoteichoic acids
  • Inner membrane
Gram NEGATIVE:
  • Thin Peptidoglycan
  • Outer membrane has Lipopolysaccharides and porins
  • Inner membrane
Steps of the Gram Stain (CHART)
Gram POSTIVE:
  1. Crystal Violet ------------- Purple
  2. Iodine (mordant) --------- Purple​ (Forms the CV:I complex)
  3. Alcohol (decolorizer) ----- Purple​ (Dehydrates Peptidoglycan)
  4. Safranin (counterstain) --- Purple
Gram NEGATIVE:
  1. Crystal Violet -------------- Purple
  2. Iodine (mordant) ---------- Purple​ (Forms the CV:I complex)
  3. Alcohol (decolorizer) ------ COLORLESS (Reason: it washes away outer membrane and CV:I complex)
  4. Safranin (counterstain) ---- RED​ ​(Reason: it was colorless before hand, Safranin made it red
KOH String Test
Gram +: Result was WATERYGram - : Result was STICKY (VISCUS)
  • 3% KOH dissolves cell wall and DNA is released
Endospores
  • MICROSCOPE SLIDE:
  1. ​Negative (red rods)
  2. Endospores (blue circles)
  • ​Dormant form of bacteria
  • Form when cells are stressed
  • Resistant to staining and appear colorless in simple stain and gram stain
  • 2 Genera: Bacillus and Clostridium
Exosporium
Endospores are surrounded by a thick protein coat made up of keratin.
Acid Fast
  • MICROSCOPE SLIDE:
  1. red rods
  • Surrounded by a waxy coat of Mycolic Acid
  • 2 Genera: Mycobacterium and Nocardia
  • 2 Pathogens: Mycobacterium tuberculosis & Mycobacterium lepreae
Capsule
  • ​MICROSCOPE SLIDE:
  1. (​Negative stain) slide is "bluish", capsule is "clear"
  • ​Smooth glycocalyx: Help bacteria to adhere and evade phagocytosis​
  • MucoPolysaccharides / Mucopolypeptides
Flagella
  • MICROSCOPE SLIDE:
  • For Motility
4 Arrangements of Flagellums:
  1. Monotrichous: single polar flagellum
  2. Amphitrichous: flagella are at both ends of a bacterium
  3. Lophotrichous: more than one flagellum at one end of the cell
  4. Peritrichous: flagella surround the whole cell
Acidophiles
Below pH 5.5
Neutrophiles
Between pH 5.5-8.5
Alkaliphiles
Above pH 8.5
Pure water is _______
PH 7.0 (neutral pH)