Lab Procedures for exam

A set of flashcards summarizing key lab procedures, including preparation and staining techniques for bacterial cultures.

Steps to make a bacterial smear from solid culture/Ex. 7 — Making a Smear.

1. Place 1 drop sterile water on slide. 2. Flame loop, cool, touch colony, mix in drop. 3. Spread thin film. 4. Let air dry fully. 5. Heat fix by quickly passing slide 2–3× through flame.

Steps to make a bacterial smear from broth culture/Ex. 7 — Making a Smear.

1. Flame loop, cool, dip into broth. 2. Place a small loopful on slide, spread thin. 3. Let air dry fully. 4. Heat fix by quickly passing slide 2–3× through flame.

Steps for a Gram stain

1. Start with heat-fixed smear. 2. Flood with crystal violet – 1 min, rinse. 3. Flood with Gram’s iodine – 1 min, rinse. 4. Decolorize with ethanol/acetone until run-off is clear (≈5–10 sec), rinse. 5. Flood with safranin – 1 min, rinse. 6. Blot dry, view with oil immersion.

Purpose & result of Gram stain

Gram + → purple (thick peptidoglycan keeps crystal violet). Gram − → pink/red (CV washes out, safranin shows).

Steps for KOH test

1. Place drop of 3% KOH on slide. 2. Mix a heavy sample of colony with loop for 30 sec. 3. Lift loop: Stringy slime = Gram negative. No string = Gram positive.

Steps for Vancomycin disk test

1. Streak plate with unknown bacteria. 2. Place Vancomycin disk on agar. 3. Incubate. Clear zone = sensitive = Gram positive. No zone = resistant = usually Gram negative.

Steps for Kirby-Bauer antibiotic test

• Make a lawn: Swab the whole agar plate in 3 directions with the bacteria.

• Add antibiotic disks (with the dispenser or sterile forceps).

• Incubate 18–24 hours.

• Look at the clear zones around disks (zones of inhibition):

  • Big clear zone = bacteria are sensitive (antibiotic works).

  • Little or no clear zone = resistant (antibiotic doesn’t work).

• Broad-spectrum = works on many types of bacteria.
  Narrow-spectrum = works on only a few.

• Bactericidal = kills bacteria.
  Bacteriostatic = just stops them from growing.

measure the clear zone (halo) in millimeters with a ruler.

Steps for a streak plate (isolation)

1. Flame loop, cool. 2. Streak 1st quadrant heavily. 3. Flame loop, drag a few lines into 2nd quadrant. 4. Flame, drag into 3rd, repeat to 4th. 5. Incubate → isolated colonies appear in later quadrants.

Ways to test motility

Motility agar (semi-solid): stab straight down → fuzzy spread = motile, straight line = non-motile. Wet mount / hanging drop: living cells swim if motile; non-motile just jiggle (Brownian).

Steps for a capsule stain

1. Place drop of India ink/nigrosin on slide. 2. Mix small bacteria sample. 3. Use clean slide to spread thin film. 4. Air dry (do NOT heat fix). 5. Flood with crystal violet (or safranin) 1 min, rinse gently. Background dark, cells colored, capsule clear halo.

Steps for a negative stain

1. Drop of nigrosin near edge. 2. Mix bacteria into drop. 3. Use other slide to spread smear. 4. Air dry (no heat). Cells clear/white, background dark.

Steps for a simple stain

1. Prepare heat-fixed smear tiny bit of colony + one drop of water.. 2. Flood with single dye (crystal violet / methylene blue / safranin) 1 min. 3. Rinse, blot dry. Cells one color → shows size, shape, arrangement.