Bls 206 lecture 3
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  • 1. Isolation Technique
    • In nature microbial cultures are mixed
    • Identification relies upon isolating individual colonies
    • Testing requires pure cultures
    • As a result isolation technique provides an essential microbiological tool
  • 2. Mixed Culture from Raw Poultry
  • 3. Streak Plate Isolation Principle
    • An original inoculum containing a mixture of bacteria is spread into 4 quadrants on solid media.
    • The goal is to reduce the number of bacteria in each subsequent quadrant.
    • Colonies are masses of offspring from an individual cell therefore streaking attempts to separate individual cells.
    • Discrete colonies form as the individual cells are separated and then multiply to form isolated colonies in the later quadrants.
  • 4. The Goal -Isolated Colonies to Start Pure Cultures
  • 5. Can an isolated colony be considered pure?
      • This is generally assumed, however….
        • some colonies are very slow growers and may be too small to see.
        • some colonies may be growing under another colony
        • selective media may be preventing reproduction of some bacteria so they may be present but not visible
        • condensed water, capsules, slime, all represent areas where individual contaminant cells hide out.
  • 6. Any special considerations?
      • Different species of microbes represent challenges….
        • Encapsulated bacteria are sticky and don’t separate well.
        • Some species are motile and do not stay where you streak them spreading across the plate.
        • Fungal spores easily contaminate cultures within a plate.
        • Organisms can gain entrance to a Petri dish through water or the edges, or from the air currents while you are streaking.
  • 7. Microbes will surprise you each chance they get !
  • 8. Isolation Requires Aseptic Technique
  • 9. Isolation Requires Aseptic Technique
    • Aseptic technique is the process of:
    • Preventing contamination of a culture with environmental microbes
    • Preventing contamination of yourself or the environment with the organism in the culture
    • Remember everything is contaminated with a variety of environmental microbes.
    • Remember microbes are invisible, you must “see with your minds eye” during these procedures.
  • 10. Streaking the Quadrants
  • 11. Quadrant 1- Streak with broad narrow strokes in the upper half of the first quarter of the plate.
  • 12. Incinerate and cool the loop between the quadrants
  • 13. Quadrant 2 – Rotate the plate, enter the previous streak mark one or two times and then streak the upper portion of the second quarter of the plate with broad strokes.
  • 14. Incinerate and cool the loop between the quadrants
  • 15. Quadrant 3 – Rotate the plate, enter quadrant 2 one or two times and then streak with shorter more separated strokes from the top of the quadrant to the center.
  • 16. Incinerate and cool the loop between the quadrants
  • 17. Quadrant 4 – Enter quadrant 3 and then streak with broad S-shaped motions through the center of the plate.
  • 18. Streaking the Quadrants
  • 19. Isolated Colonies
  • 20. Culturing and Isolation Techniques
    • Bacteria require a constant nutrient supply to survive and grow
    • Acquire nutrients from their surroundings (free-living) or from a host (parasites)
    • Artificial media is used to grow bacteria in a lab (in vitro)
      • Agar is extracted from marine algae
        • A carbohydrate that cross-links to form a semi-solid mesh.
        • Melts at 100  C, solidifies at 42  C, but will remain a liquid at 60  C.
  • 21.
    • Most pathogenic bacteria have an optimum growth temperature of 37 °C (human body temp.)
    • Organisms grown in broth cultures are apparent through the turbidity that the large numbers of cells produce in the broth.
    • On agar, a solid medium, the bacterial cells form masses called colonies after about 18 – 24 hours of growth.
    • Colonies represent one viable cell or Colony Forming Unit (CFU) that came to rest on the agar surface.
      • This cell or CFU divides many times to form visible colonies on the agar
  • 22.
    • Isolated colonies =
      • those not touching other colonies
      • represent clones of the original cell or CFU since all the cells in the colony were derived from one cell or CFU and are genetically identical.
    • Isolated colonies are considered to be pure cultures of a particular bacterial species and strain.
  • 23. Colony Morphology
  • 24. Inoculation of a Broth Culture
    • Label the sterile nutrient broth with the source of the culture, your initials and the date.
    • Sterilize a loop in the Bacticinerator.
    • Using appropriate aseptic technique, remove a loop-ful of broth from the mixed culture tube.
    • Insert the loop into the sterile broth and gently swirl. Retract the loop and sterilize it in the Bacticinerator.
    • Incubate the broth at 37  C for 24 – 48 hours.
    • Observe the broth culture for turbidity. Record the results in the Table on page 30 in the lab book.
    Compare your inoculated broth tube to the un-inoculated control tube to determine the amount of turbidity. The more turbid the broth The higher the bacterial count per mL of broth.
  • 25. Inoculating an Agar Slant
    • Label the sterile nutrient agar slant with the source of the culture, your initials, and the date.
    • Sterilize the loop using the bacticinerator.
    • Using appropriate aseptic technique, remove a loopful of broth from the mixed culture tube.
    • Insert the loop into the sterile agar slant tube and starting at the base of the slant (closest to the bottom of the tube), very lightly draw the loop in a zig-zag motion up the slant. Do not dig into the agar. Sterilize the loop in the bacticinerator.
    • Incubate the slant at 37  C for 24 – 48 hours.
    • In the following lab observe the slant for growth. Record the results in the table on page 30 in the lab book.
  • 26. Streak Plates
    • Allow for the growth of isolated colonies on the surface of the agar.
    • Used to isolate clones of a particular bacterial species/strain.
    • An isolated colony, one that is not touching any other colonies, is assumed to be a pure culture.
    • May observe colony morphology that can be used to help identify the bacterial species.
    • Colonies of the same organism may grow differently on different media, e.g. the shape, color, growth pattern of the colony may differ on other types of media.
    • Colony Morphology Characteristics
      • Colony color
      • Type of hemolysis (if grown on Sheep Blood Agar)
      • Form
      • Elevation
      • Margin
  • 27. Streak Plates
  • 28. Pour Plating for Colony Counts
    • One of the most common methods of determining cell number is the viable plate count.
    • A sample to be counted is diluted in a solution that will not harm the microbe.
    • In most cases a volume of liquid from the sample is first diluted 10-fold.
    • In most cases, a 0.1-1.0 ml portion of this first dilution is then diluted a further 10-fold, giving a total dilution of 100-fold.
  • 29. Pour Plating for Colony Counts
    • This process is repeated until a concentration that is estimated to be about 1000 cells per ml is reached.
    • The sample serially diluted and individual cells are deposited in the molten agar and these give rise to colonies.
    • By counting each colony, the total number of colony forming units (CFUs) on the plate is determined.
    • By multiplying this count by the total dilution of the solution (dilution factor), it is possible to find the total number of CFUs in the original sample.
  • 30. Calculating CFU from dilution plating results
    • How does a count on a plates get converted to CFUs per gram or ml of sample? Let's illustrate the procedure with an example. Imagine that we perform the following experiment:
    • Five ml of milk are added to 45 ml of sterile broth. From this suspension, two serial, 1/100 dilutions are made, and 0.1 ml is plated onto Plate Count Agar from the last dilution. After incubation, 137 colonies are counted on the plate.
    • This problem may be illustrated as follows:
    The initial dilution is calculated As follows: So the initial dilution is 1/10 or 0.1 or 10 -1 .
  • 31.
    • Remember, there are many ways to make 1/10 and 1/100 dilutions.
    • A 0.1 ml to 0.9 ml dilution is the same as a 1 ml to 9 ml dilution and a 13 ml to 117 ml dilution.
    • Next, 1 ml of the first dilution is added to 99 ml to make the second dilution, that is a 1/100 dilution.
    • This is repeated with third dilution giving another 1/100 dilution.
  • 32.
    • Then 0.1 ml of the third dilution is plated out on a sterile plate.
    • The total dilution may be calculated mathematically as follows:
    • To obtain the concentration of bacteria in the original sample the “dilution factor” must be determined and then multiplied by the plate colony count.
  • 33.
    • The dilution factor is the inverse of the total dilution –
    Therefore, the “Dilution factor for our example is: 10 6 and the Total Colony Forming Units (CFUs) for this sample is: