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bmic topic 3.3 - Coggle Diagram

- - - - Makes a total count (both viable and dead cells are counted)
        
        Using special counting chamber called the Petroff-Hauser counting chamber
        
        Count the number of cells present in specific volume of culture, then finally expressed as cells/ml
        
        Pros and cons
        
        Rapid with immediate results
        
        However difficult to count if cells are motile or clumped together
        
        Tedious and tiring
        
        Large errors are likely
        
        Insensitive – requires at least 10^6 cells/ml
    - - Sample is passed over a membrane and the cells are concentrated and trapped on the membrane
      - The membrane is then placed on a suitable agar medium, and the cells grow into colonies which can be counted
      - pros and cons
        
        Large volumes of samples can be processed
        
        Especially good for samples with low cell densities
        
        tedious and expensive
    - - Make a viable count (only living cells will be counted)
        
        Begin with serial dilutions of the sample
        
        Count only plates with 25 – 250 cfu
        
        To calculate cell density (cfu/ml):
        (Average cfu X Dilution factor)/Volume of inoculum
      - pros and cons
        
        Reasonable sensitivity and does not require complicated instruments
        
        often used for environmental and food samples
        
        slow and tedious
        
        Accuracy depends on counting large numbers of cfu
    - - A statistical approach – the more cells in a sample, the more dilutions are needed to reduce the numbers to zero
        
        1.Begin by making 3 consecutive serial dilutions of the sample
        
        2.For each of these dilutions, inoculate into 3 or 5 tubes of suitable growth medium (array of 3 X 3 or 3 X 5 tubes)
        
        3,Incubate and score the number of positive (with growth) tubes for each dilution===based on turbidity
        
        Read the results from a MPN table
      - pros and cons
        
        Only provide an estimated range of microbial numbers
        
        often used for environmental, food and water samples
        
        used for samples with low cell densities
        
        Often used for the enumeration(establishing number of) of coliforms and E. coli using combination of specific media and incubation conditions
        
        Coliforms grow and produce gas in lauryl tryptose broth at 35oC; others may grow but do not produce gas
  - - - Turbidity measurement
        
        Microbial growth in broth results in turbidity
        
        Higher the cell density, the more turbid is the broth
        
        A beam of light is passed through the broth to reach a sensor
        
        The cells scatter the light such that less light reaches the sensor  increases the optical density
        
        measurement tool: spectrophotometer
        
        pros and cons
        
        Easy and quick
        
        Useful to follow growth of culture
        
        Turbidity can be correlated to plate counts under identical growth conditions  extrapolate to cell density
        
        However, both dead and living cells contribute to turbidity
        
        Not too sensitive – requires at least around 10^6 cells/ml to be detectable
      - Dry and wet weight
        
        Separate cells from growth medium by filtration or centrifugation
        
        Wash with buffer or water
        
        Weigh for wet weight
        
        For dry weight, dry cells in oven (110oC for 8 h, or 80oC for 24 h) then weigh
        
        pros and cons
        
        Fairly insensitive; require > 106 cells
        
        require at least 50 mg
        
        However, it’s good for filamentous microorganisms that do not plate well as colonies may not be formed from single cells. Filamentous microorganisms float on the surface or settle in broth, so its difficult to measure the turbidity
      - Nitrogen content
        
        Measures amount of organic nitrogen in cell mass eg. using the Kjeldhal method
        More cells make more proteins, therefore higher organic nitrogen content
      - Metabolic activities
        
        Microbial growth is characterized by increased metabolic activity. Measures consumption of substrates, accumulation of metabolic waste products e.g. carbon dioxide and acid are directly proportional to bacterial numbers