Viable Plate Counts & Microbial Cell Numbers PDF

Viable (plate) counts: measurement of living, reproducing population two main ways to perform plate counts – assuming when the cells were plated at the right concentration, each colony is formed by a single bacterial cell: spread-plate method pour-plate method...

Viable (plate) counts: measurement of living, reproducing population two main ways to perform plate counts – assuming when the cells were plated at the right concentration, each colony is formed by a single bacterial cell: spread-plate method pour-plate method Medium cooled to 50 °C  A serial dilution of the bacterial sample is used. The usual practice, which is most valid statistically, is to count colonies only on plates that contain between 30 and 300 colonies. Figure 5.15  Sources of error in plate counting depends on inoculum size, viability, culture medium, incubation conditions Mixed cultures grow at different rates. plating inconsistencies Reporting in colony-forming units instead of number of viable cells accounts for clumps. Applications quick and easy used in food, dairy, medical, and aquatic microbiology, and water analyses high sensitivity can target particular species in mixed samples  “The great plate count anomaly”: Direct microscopic counts of natural samples reveal far more organisms than those recoverable on plates. Why is this? Microscopic methods count dead cells, whereas viable methods do not - causing overestimation by the microscopic methods Different organisms may have vastly different requirements for growth – causing bias in the culture method  5.8 Turbidimetric Measures of Microbial Cell Numbers Cell suspensions are turbid (cloudy) because cells scatter light. Most often turbidity is measured with a spectrophotometer (Figure 5.16), and measurement is referred to as optical density (OD) at specified wavelength (e.g., OD540 for measurements at 540 nm [green light]). For unicellular organisms, OD is proportional to cell number within limits. To relate a direct cell count to a turbidity value, a standard curve must first be established. Figure 5.16  Bacterial growth phases E. coli can grow very fast in LB medium, doubling every 30 min under optimal growth conditions. The growth would typically experience three phases (in 1 day): lag phase, exponential phase, and stationary phase. 24 hrs  E. coli culture and turbidity E. coli is cultured at 37 °C with orbital shaking (~25 mm orbit diameter) at 200-250 rpm (for good aeration). Absorbance (A) at 600 nm is usually used for measuring bacterial density, especially E. coli. The absorbance (representing ‘turbidity’) is correlated with the concentration of the substance (in this case: bacterial cells). The mechanism of a spectrophotometer Cuvette can be made up of glass, plastics, or quartz. When the absorption is in the ultraviolet region, use quartz but not others.  Optical Density (OD) or Absorbance (A) I is the intensity of the transmitted light Io is the intensity of the incoming light This is what the spectrophotometer T (transmittance)=I/Io really measures Optical Density (OD) or Absorbance (A) = - log10T = -log100.1 -log101 wikipedia.org  How to calculate concentration? The Beer-Lambert Law A= A is in linear correlation with ɛ (a constant depends on the sustenance), ℓ (length of the light path) and sample concentration (mol/L). http://www.chemguide.co.uk/analysis/uvvisible/beerlambert.html Keep the solution at OD well below 1.0 The Beer-Lambert law equation only maintains linear correlation between absorbance and the concentration in a certain range (usually absorbance < 1). ~1 Need to dilute the bacterial samples when they are too dense before measuring OD600.  Advantages quick and easy to perform typically do not require destruction or significant disturbance of sample Same sample can be checked repeatedly Disadvantages sometimes problematic (e.g., microbes that form clumps or biofilms in liquid medium) – why?