BMS 90 L practical

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Cards (47)

  • Biohazardous material that must go into a Sharps Container (Red container)
    • Used swabs
    • Petri dishes
    • Slides
    • Broken Glass Tubes
  • Biohazardous material that can go into the Biohazard Can (White round container)
    • Gloves
    • Bench Paper ("Chucks")
    • Plastic Petri Dishes
  • Standard Operating Procedure (SOP)
    1. Dress appropriately for a laboratory environment
    2. No open toed shoes or sandals
    3. No clothing with baggy or flowing sleeves
    4. No shorts or short skirts
    5. Tied back long hair
    6. Always wear the lab coat while in the lab
    7. Always wear gloves when handling microbes
    8. Always Disinfect your bench
    9. Always handwashing right before you leave the class
  • The mouth, skin, and nose are major pathways of entry for microbes to enter the body
  • Things not allowed in the lab
    • No smoking
    • No eating
    • No drinking
    • No gum-chewing
    • No applying cosmetics
  • Streak Plate method

    1. Spread a loopful of culture over the surface of the agar plate in four quadrants
    2. This dilutes or reduces the number of bacterial cells through each quadrant, allowing for the isolation of discrete colonies
  • Colony

    A visible mass of microbes growing on the surface of an agar
  • Colony morphology characteristics

    • Form
    • Elevation
    • Margin
    • Surface
    • Optical Characteristics
    • Pigmentation
  • Microorganisms

    • Micrococcus luteus
    • Serratia marcescens
  • CFU/ml

    Colony forming unit per millilitre
  • Dilution factors

    • 1:10
    • 1:100
    • 1:1000
  • Fungal spore morphology

    • Asexual spore (only sporangiophores)
    • Sexual spore (only zygospore)
  • The majority of fungi produce sexual spores at some point in their life cycle, which increases genetic variation
  • Gram staining

    1. Add crystal violet (primary stain)
    2. Add the mordant (iodine)
    3. Add decolorizer (ethyl alcohol)
    4. Add counter stain (safranin)
  • Acid-fast staining

    1. Add the primary stain (carbol fuchsin)
    2. Heat fix cells
    3. Add the mordant (steam/heat or carbolic acid)
    4. Add decolorizer (acid alcohol)
    5. Add the counter stain (methylene blue)
  • Endospore staining

    1. Add the primary stain (malachite green)
    2. Add the mordant (steam/ pass the heat fixed slide through a flame)
    3. Add the decolorizer (water)
    4. Add counter stain (safranin)
  • Negative staining

    Used to see capsules of bacteria and fungi
  • If the aim is to see cell size, shape or arrangement, a simple stain can be performed
  • Differential staining uses two dyes to distinguish between cell types or parts
  • Gram positive and gram-negative bacteria
    • Gram negative bacteria are more resistant to disinfectants than gram positive bacteria because GN has an additional outer membrane (LPS) while GP just has the peptidoglycan layer
  • Kirby Bauer (antibiotics test)
    1. Disinfectant diffuses from the paper and kills the surrounding inoculated bacteria
    2. This creates a zone of no growth area around the disc called the Zone of inhibition
    3. The greater the zone of inhibition, the more effective the concentration of the disinfectant
  • Biohazardous material that must go into a Sharps Container (Red container)
    • Used swabs
    • Petri dishes
    • Slides
    • Broken Glass Tubes
  • Biohazardous material that can go into the Biohazard Can (White round container)
    • Gloves
    • Bench Paper ("Chucks")
    • Plastic Petri Dishes
  • Standard Operating Procedure (SOP)
    1. Dress appropriately for a laboratory environment
    2. No open toed shoes or sandals
    3. No clothing with baggy or flowing sleeves that could catch fire or hinder movements
    4. No shorts or short skirts
    5. Tied back long hair -fire hazard or a source of contamination
    6. Always wear the lab coat while in the lab
    7. Always wear gloves when handling microbes
    8. Always Disinfect your bench when you arrive and right before you leave
    9. Always handwashing right before you leave the class
  • The mouth, skin,and nose are major pathways of entry for microbes to enter the body
  • Things that should not be done in the lab
    • No smoking
    • No eating
    • No drinking
    • No gum-chewing
    • No applying cosmetics
  • Streak plate method

    1. A loopful of culture is spread over the surface of the agar plate in four quadrants
    2. This is done in such a way that the number of bacterial cells is diluted or reduced through each quadrant, allowing for the isolation of discrete (individual) colonies
  • Colony

    A visible mass of microbes growing on the surface of an agar
  • Colony morphology characteristics

    • Form
    • Elevation
    • Margin
    • Surface
    • Optical Characteristics
    • Pigmentation
  • Microorganisms

    • Micrococcus luteus
    • Serratia marcescens
  • Viable plate method

    • CFU/ml = (TVP on plate/volume plated) x dilution factor
    • CFU= colony forming unit
    • TVP = total viable plate count
    • Dilution factor: either 1:10, 1:100, or 1:1000 (multiply by one of these numbers)
  • Fungal spore morphology

    • Asexual spore (only sporangiophores)
    • Sexual spore (only zygospore)
  • The majority of fungi produce sexual spores at some point in their life cycle, which increases genetic variation
  • Plasmodium Falciparum slide shows merozoites in RBC
  • Gram staining

    1. Add crystal violet (primary stain) to cells → colors both GP and GN purple
    2. Add the mordant (iodine) to affix a dye in the cell → both GP and GN still purple, but the iodine + the crystal violet creates a complex that cannot leave thick peptidoglycan layer (which is seen in GP)
    3. Add decolorizer (ethyl alcohol) → the complex created between iodine and crystal violet is washed away in GN cells because of thin peptidoglycan layer (GN becomes clear) while the GP stays purple
    4. Add counter stain (safranin) → safranin is pink and it colors both GP and GN but is only seen in GN because the GP is still purple (therefore, the GN looks pink)
  • Acid-fast staining

    1. Add the primary stain (carbol fuchsin- deep red dye mixed in phenol solution)
    2. Add the mordant (steam/heat or carbolic acid) to maintain the color
    3. Add decolorizer (acid alcohol) → Removes the carbol fuchsin from cells that are not protected by mycolic acid, Acid fast remains red/pink while non acid fast becomes clear
    4. Add the counter stain (methylene blue) → Will not penetrate the waxy mycolic acid and will only stain the clear cells, Acid fast remains red/pink while non acid fast becomes blue
  • Endospore staining

    1. Add the primary stain (malachite green)
    2. Add the mordant → steam/ pass the heat fixed slide through a flame of bunsen burner 20 times to loosen up the spore coat so the malachite coat can easily permeate the endospore
    3. Add the decolorizer (water) → Endospores are resistant to de-staining = remain green, Vegetative cells lose the green since the dye is water soluble = becomes clear
    4. Add counter stain (safranin) → Safranin cannot penetrate the spore coat of the endospore = remains green, Easily stains the vegetative cells = become red/pink
  • Negative staining

    1. Put one drop of India ink (nigrosin) on the edge of a slide
    2. Inoculate the ink with the bacteria or fungi
    3. Smear the ink drop with a second slide
    4. Allow to air dry (do not heat or blot)
  • Negative staining is used to see the capsule of some bacteria such as Klebsiella pneumoniae and fungi like Cryptococcus neoformans
  • If the aim is to just see cell size, shape or arrangement, a simple stain can be performed