Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Bls 206 lecture 3


Published on

Published in: Technology
  • Be the first to comment

Bls 206 lecture 3

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