mcbl lab-1

• Disinfect the bench


  1. Spray the bench with ZED disinfectant

  2. Wipe the bench with a paper towel to ensure that the disinfectant covers the bench and absorbs the disinfectant

  3. Put the paper towel into the red biohazard can
• Pipettes


  Allow us to measure and transfer small solutions
• Do not force anything
• Be sure that the pipette isn't locked when attempting to change the volume
• Put the pipettes into drawer A when finished
• Aseptic technique


  1. Flame sterilize the inoculating loop

  2. Wait ~10 seconds for the loop to cool down

  3. Hold the loop in one hand and take the tube of bacteria into the other

  4. Use the hand holding the loop to take off the cap; keep the cap in your hand, pointed down

  5. Briefly pass the top of the tube of bacteria over the flame to sterilize it

  6. Dip the inoculating loop into the solution; hold the culture tub on an angle

  7. Do not touch the shaft of the inoculating loop to the inside of the tube because it isn't sterile

  8. Flame the top of the tube again and put the cap back on
• Streak plating


  1. Rub/streak the loop across the surface of the agar media

  2. Flame sterilize the loop between each set of streaks

  3. Incubate the plates inverted
• Spread plating


  1. Pipette solution with bacteria onto agar media

  2. Put Petri dish on turn table

  3. Lift the lid of the Petri dish

  4. Spin the turn table

  5. Hold the cell spreader on the surface of agar media to spread the solution
• The objective of this lab is to isolate and characterize an antibiotic producing organism
• Gram stain


  Differentiates bacteria based on differences in cell wall
• Gram-positive bacteria

  • Large amount of peptidoglycan in cell wall
• Gram-negative bacteria

  • Relatively little peptidoglycan in cell wall; the cell wall has an outer membrane made of LPS and protein
• Gram stain process


  1. Crystal violet and iodine are applied to bacterial cells, producing a purple colored complex inside the cells

  2. Alcohol is applied to the cells - removing the purple complex from gram-negative cells, but not from gram-positive cells

  3. Safranin is then applied to the cells - which is a molecule that binds cell membranes - turning the gram-negative bacteria a pinkish red color
• Endospores


  Survival structures formed by microbes such as Bacillus and Clostridium
• Endospore structure


  • Coat: the outer protein rich coat provides much of the chemical and enzymatic resistance capabilities
  • Cortex: has specialized peptidoglycan that contributes to heat resistance capabilities
  • Core contains DNA, ribosomes, and small acid soluble proteins (SASPs) that protects DNA from UV light and DNA damaging chemicals
• Endospore staining


  1. Heating and steaming the bacteria (to break down the coat) for 5 minutes - which alters the endospore structure enough so that the malachite green stain can penetrate the outer coat

  2. The endospores are then cooled restoring the endospore structure such that the stain gets trapped inside - giving them a green color

  3. Safranin binds to cell membranes and it will therefore stain the vegetative cells a pinkish red color
• Viable Plate Counts


  Concentration of microbes are determined by spreading a specific volume of a sample on agar media and then counting the number of colonies that grow
• Colony forming unit (CFU)


  One cell produces a single colony
• Turbidity


  Cloudiness of a solution caused by particles suspended in the solution
• Direct Microscopic Counts


  Directly counting the microbes using a hemocytometer
• Axenic culture


  Pure culture
• Non-axenic culture


  Mixed culture
• Selective media


  Media that enhances the growth of specific organisms while inhibiting the growth of other organisms
• Differential media


  Media that creates a visual difference between specific bacteria - usually taking advantage of a biochemical difference between different types of bacteria
• MacConkey Agar is a selective medium that contains bile salts which inhibit gram-positive bacteria
• Nutrient Agar is neither selective nor differential medium
• Comparison of cell concentration methods

  • Live cells
  • Dead cells
  • Rapid method
  • Requires another method
  • Direct
  • Viable
  • Turbidity
• Antibiotics


  Substances or compounds that kill or inhibit the growth of bacteria
• Antibiotic resistance


  Ability of an organism to grow or survive in the presence of an antimicrobial compound
• Mutation


  Heritable changes in the nucleotide sequences of the genetic material (DNA or RNA)
• Horizontal transfer


  The exchange of genes between and among cells
• Transformation


  Transfer of bacterial genes involving free DNA (chemically transmitted - ex. heat)
• Transduction


  Transfer of bacterial genes from one cell to another by a virus
• Conjugation


  Transfer of bacterial genes from one prokaryotic cell to another by a mechanism involving cell-to-cell contact and a plasmid
• Antibiotic paradox


  Antibiotic use can be beneficial by reducing or eliminating infectious organisms, but can also be harmful by promoting antibiotic resistance
• Resistant organisms increase in population when antibiotics eliminate non-resistant organisms
• Dose


  The quantity of antibiotic taken
• Duration


  The length of time the antibiotic is taken
• To slow down the development of resistance, use a high enough dose for a long enough duration, use multiple drugs with different modes of action, limit antibiotic use, and identify or create new antibiotics
• Most microbes don't grow on laboratory media, which is known as "The Great Plate Count Anomaly"