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Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
Bls 206 lecture 3
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Bls 206 lecture 3

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  • 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. Mixed Culture from Raw Poultry
  • 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. The Goal -Isolated Colonies to Start Pure Cultures
  • 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. 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. Microbes will surprise you each chance they get !
  • 8. Isolation Requires Aseptic Technique
  • 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. 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 <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. <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. <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. Colony Morphology
  • 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. 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. 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. Streak Plates
  • 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. 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. 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. <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. <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. <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:

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