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Counting Micro Organisms


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Counting Micro Organisms

  1. 1. Counting Micro-organisms Counting elephants is easy – counting bacteria is not quite as easy!
  2. 2. Counting Micro-organisms <ul><li>A single tiny drop of nutrient broth incubated overnight may contain 5 000 000 cells – this is a lot to count. </li></ul><ul><li>1cm 3 may contain 10 8 cells. </li></ul><ul><li>In order to estimate numbers it is necessary to dilute the sample. </li></ul>
  3. 3. SERIAL DILUTION 1ml into 9ml = 1:10 dilution conc. 10-1 10-2 10-3 10-4 10-5 10-6 Counting Micro-organisms
  4. 5. Counting Micro-organisms <ul><li>It is possible to dilute a cell suspension in known steps in order to plate a specific range of cells. This set of plates contain a 1 to 10 dilution series. </li></ul>
  5. 6. TOTAL COUNTS <ul><li>Direct counts </li></ul><ul><li>The haemocytometer is a specialized microscope slide used to count cells. </li></ul><ul><li>The centre portion of the slide has etched grids with precisely spaced lines. </li></ul>
  6. 7. TOTAL COUNTS <ul><li>To get an accurate count there should be between 40 and 70 cells in a 1 mm square. </li></ul><ul><li>If not you dilute or concentrate the cell suspension as necessary . </li></ul>
  8. 9. TOTAL COUNTS <ul><li>Indirect counts </li></ul><ul><li>These depend on the effects of the organisms to estimate their numbers. </li></ul><ul><li>As organisms grow they make the nutrient broth turbid. </li></ul><ul><li>This turbidity can be measured with a colorimeter </li></ul>
  9. 10. TOTAL COUNTS <ul><li>The more organisms the greater the optical density of the solution. </li></ul>
  10. 11. TOTAL COUNTS <ul><li>Indirect count </li></ul><ul><li>The Coulter counter is a probe which measures the change in conductivity of a solution as a bacteria passes through a narrow gap. </li></ul>
  11. 12. TOTAL COUNTS <ul><li>Problem with INDIRECT COUNTS and DIRECT COUNTS is that they can not tell living cells apart from dead cells </li></ul><ul><li>Advantage of INDIRECT COUNT is that the process can be automated </li></ul>
  12. 13. VIABLE COUNTS <ul><li>Count only those cells capable of growing </li></ul><ul><li>Viable counts can be accomplished by such techniques as pour plating. </li></ul><ul><li>Assumption each viable cell gives rise to a colony. </li></ul>
  13. 14. VIABLE COUNTS <ul><li>Advantages: </li></ul><ul><li>The method can be made to be very sensitive. </li></ul><ul><li>One can count subsets of the population </li></ul><ul><li>Disadvantages </li></ul><ul><li>Colony-forming units may underestimate cell numbers because of clumping or chains of cells. </li></ul><ul><li>Counts require at least a few hours, usually overnight, for incubation. </li></ul>