Supplemental Lecture (98/04/03 update) by Stephen T. Abedon (abedon.1@osu.edu)

  1. Chapter title: Counting Microbes
    1. A list of vocabulary words is found toward the end of this document
  2. Methods of cell quantification
    1. Counting microorganisms:
      1. The determination of the number of cells or concentration of cells in a sample plays numerous and important roles in microbiological characterization and experimentation.
      2. Methods of cell quantification can be divided between
        1. direct counts, including:
          1. viable counts
          2. total counts
        2. estimations of cell number
  3. Direct count
    1. Actual counting of microorganisms:
      1. A method by which a reasonably statistically precise measurement of cell quantification is achieved.
      2. The basis of a direct count is the actual counting of every organism (or every living organism---often limited to a single type of microorganism ) present in a sub-sample of a population.
    2. Direct counts include:
      1. plate counts (a viable count)
      2. most probable number method (a viable count)
      3. direct microscopic count (a total count)
  4. Viable count
    1. A viable count is a direct counting method in which only viable cells are counted.
    2. Viable counts can be accomplished by such techniques as:
      1. pour plating
      2. spread plating
      3. most probable number method
  5. Total count
    1. A total count is a direct counting method in which all cells are counted, whether dead or alive .
    2. Generally taking total counts requires the employment of microscopes.
  6. Estimation of cell number
    1. Correlates to cell number:
      1. Estimates of cell number use various correlates of cell number rather than direct counting .
      2. Such methods are often preferable either for convenience or because direct counting is difficult or even impossible in many situations (for example, when quantifying filamentous organisms ).
    2. Estimates of cell number include determinations of:
      1. turbidity
      2. metabolic activity
      3. dry mass
  7. Plate counts [pour plate, spread plate]
    1. Colonies founded by cells:
      1. Colonies grown in petri dishes by various methods (not including streaking ) may be used to determine the count of viable microorganisms .
      2. Plate counts assume that every colony is founded by a single cell.
      3. That cell must have been alive in order to grow and form a colony .
    2. Caveats:
      1. Problems with plate counts are:
        1. they require lengthy incubation for colonies to become visible
        2. cell clumping can lead to an undercount of viable cells
        3. it is very easy to have too many or too few colonies on a plate to accurately measure viable count
        4. prevention of crowding often requires serial dilution*
        5. too few cells requires concentrating, e.g., by centrifugation or filtration**
      2. *Depending on your organism, and other circumstances, you will want to have no more than 300 to 500 colonies per plate (e.g., for big colonies 300 will be many, for small colonies 500 may still work).
      3. **Typically one uses a minimum number which is 10-fold smaller than the maximum number, but greater than 30.
      4. Note that none of these caveats are unique to plate counts.
  8. Filtration
    1. Concentrating cells:
      1. If too few colonies are present then the original culture must be concentrated prior to determining its plate count.
      2. Filtration, which sieves microorganisms out of the medium, is one method of concentrating microorganisms .
  9. Most probable number method [MPN]
    1. No plate viable count:
      1. The most probable number method is a way of determining approximate viable count by diluting cultures then growing the dilution cultures in broth tubes.
      2. At the dilution at which broth becomes turbid or not with similar likelihood, the culture has been dilution to the point that the broth tubes were inoculated with on the order of only a single microorganism (turbid ) or fewer (not turbid ).
      3. The concentration of the culture is then taken to be equal to the amount of dilution necessary to have reached this point.
    2. Advantageous where broth is advantageous:
      1. MPN is especially useful in situations where there is an advantage to using broth over solid medium .
      2. For example, many organisms are not good at forming colonies, such as highly motile organisms.
    3. "When samples contain too few organisms to give reliable measures of population size by the standard plate count method, as in food and water sanitation studies, or when organisms will not grow on agar, the most probable number method is used." (p. 143, Black, 1996)
  10. Direct microscopic count
    1. Direct microscopic count is a determination of the number of microorganisms found within a demarcated region of a slide known to hold a certain volume of culture .
    2. This total count method of cell quantification is very rapid but has the problem of requiring high cell concentrations (e.g., 107 / ml) as well as potentially counting dead and living cells with equal probability.
  11. Turbidity
    1. The cloudiness or turbidity of a culture is caused by the individual cells scattering light.
    2. Degree of turbidity is a direct correlate of cell mass.
    3. Estimator of cell number:
      1. The average cell mass of individual cells in a culture must first be determined if the turbidity of a culture is to be used to estimate cell count .
      2. This standardization usually works best for cultures growing in exponential phase .
  12. Metabolic activity
    1. The metabolic output or input of a culture may be used to estimate viable count. This method has the same caveats as turbidity methods.
    2. Examples include: "The rate at which metabolic products such as gases and/or acids are formed by culture reflects the mass of bacteria present. . . The rate at which a substrate such as glucose or oxygen is used up also refelcts cell mass." (p. 144, Black, 1996)
  13. Dry mass
    1. Estimation of cell count:
      1. Determinations of dry mass has all of the problems of turbidity and metabolic activity with the added problem of having to dry cultures before employing it.
      2. "To calculate the dry weight of cells, they must be separated from the medium by some physical means such as filtration or centrifugation. The cells are then dried, and the resulting mass is weighed." (p. 144, Black, 1996)
    2. For filamentous organisms this method works sufficiently well compared to the other methods listed that dry mass determination is employed.
  14. Viable
    1. Alive. See discussions on cell death .
  15. Clumping
    1. Clumping is when two or more viable cells are stuck together, for whatever reason.
    2. Counted as single cell:
      1. When clumped both or more cells are counted as only a single cell in a viable count.
      2. This is one disadvantage of employed viable counts for cell quantification .
  16. Vocabulary
    1. direct count
    2. dry mass
    3. estimating cell number
    4. metabolic activity
    5. most probable number method
    6. plate count
    7. total count
    8. turbidity
    9. viable count
  17. Practice questions
    1. Three of four tubes are turbid. Each represents a million-fold dilution of a culture. You conclude that the original culture has a concentration of approximately one million per ml. You are employing what method of microorganism quantification? [PEEK]
    2. You would tend to prefer the use of dry mass as a method of quantification if (circle only one correct answer) [PEEK]
      1. your microorganism will not grow in broth
      2. your culture is in stationary phase
      3. you are quantifying a filamentous microorganism
      4. your microorganism is not resistant to desiccation
      5. all of the above
      6. none of the above
    3. Describe a general procedure you would have to employ to determine the viable count (i.e., direct count), via the pour plate method, of a solution where the concentration of viable cells is less than 1 per liter. (hint: "using a very large petri dish and a whole lotta' agar" is not an acceptable answer) [PEEK]
    4. Describe a situation in which the Most Probable Number method would be a necessary alternative to a plate count for determination of viable count. [PEEK]
    5. Turbidity measurements are legitimate means for estimating actual cell concentrations but only so long as __________ has previously been determined? [PEEK]
    6. Dry mass determination as a means of organism enumeration is particularly suited to the quantification of __________. (choose best answer) [PEEK]
      1. organisms found at low density.
      2. filamentous organisms.
      3. intact but dead bacilli.
      4. viruses.
      5. viable organisms.
      6. turbid cultures.
    7. Describe (or sketch) a log-linear graph of death phase in terms of total cell count (assume that dead cells are not distinguishable from living cells by direct microscopic count). [PEEK]
    8. Describe the determination of viable cell count via the MPN method where your determination of the cell concentration of a pure culture yields an answer of 105 cells/ml. Be sure to indicate what dilution was used to obtain what result which yielded the culture concentration indicated (don't bother going into detail about how that dilution was technically or algebraically achieved). [PEEK]
    9. What kind of thingy does one employ to do a total count? [PEEK]
    10. Which of the following is not employed in estimating cell number? [PEEK]
      1. Turbidity
      2. plate counts
      3. culture cloudiness
      4. culture metabolic activity
      5. dry mass
      6. rate of liberation of CO2
    11. You dilute a culture 100-fold and place one ml of this diluted culture into each of four tubes, each tube already containing 9 ml of sterile broth. After 48 hrs, two of those tubes are sparkling clear and two are very turbid. A 10-fold dilution of the original culture divvied up as above yields four turbid tubes. A 1000-fold dilution yields four sparkling clear tubes. You assume that contamination did not play a significant role in this experiment. What is the cell concentration in the original culture? [PEEK]
  18. Practice question answers
    1. most probable number method
    2. iii, you are quantifying a filamentous microorganism. See p. 164, Tortora et al., 1995.
    3. Some kind of concentrating method such as filtration.
    4. Microorganism does not form colonies.
    5. per cell turbidity .
    6. ii, filamentous organisms.
    7. A straight, horizontal line or, if some replication continues even in death phase, then a line which increases with time. You would observe qualitatively the same thing on a linear-linear graph.
    8. A 105-fold dilution of the culture was inoculated into sterile, crystal clear broth tubes and resulted, following appropriate incubation, in approximately half of the tubes exhibiting turbidity and half not.
    9. A microscope (thingy).
    10. ii, plate counts
    11. approximately 100 cells per ml
  19. References
    1. Black, J.G. (1996). Microbiology. Principles and Applications. Third Edition. Prentice Hall. Upper Saddle River, New Jersey. pp. 140-144.
    2. Tortora, G.J., Funke, B.R., Case, C.L. (1995). Microbiology. An Introduction. Fifth Edition. The Benjamin/Cummings Publishing, Co., Inc., Redwood City, CA, pp. 147-154, 158-166.