Bacterial Enumeration

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Enumeration. GoalTo determine the number of bacteria per milliliter of sampleCells/mlMethodsDirectStaining and counting under microscopeHeterotrophic plate countCount colonies and assume each colony represents 1 cell in original cultureIndirect: estimate based on another propertyMPNStatistical estimate based on growth patterns in mediaSpectroscopyEstimate based on turbidity (or transmittance of light).

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Bacterial Enumeration

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1. Bacterial Enumeration

2. Enumeration Goal To determine the number of bacteria per milliliter of sample Cells/ml Methods Direct Staining and counting under microscope Heterotrophic plate count Count colonies and assume each colony represents 1 cell in original culture Indirect: estimate based on another property MPN Statistical estimate based on growth patterns in media Spectroscopy Estimate based on turbidity (or transmittance of light)

3. Enumeration of Bacteria Methods Viable: Live cells only Heterotrophic plate count MPN Total: Live and dead Spectroscopy Staining w/ microscopy

4. Direct Count Indirect Count Advantage More accurate Disadvantage More time Uses To get accurate counts of cells in clinical or environmental samples Advantage Quicker to do Disadvantage Less accurate Uses To get quick and dirty count in controlled circumstances

5. Viable Total When you are concerned about only living and metabolically active organisms EX) CLINICAL SAMPLES When you need to know a number of all organisms EX) Microbial Ecology

6. Spectrophotometer Measuring turbidity or optical density of a solution by measuring % transmittance of light OD= mathematical way of expressing turbidity As OD increases; turbidity increases and Transmittance decreases As transmittance increases… Incident light into culture at specific wavelength 540, 600 or 660 nm Some light scatters; more scatter with more cells The non-scattered light exits and is read by a photocell %T

8. OD Optical Density OD=2-(log %T) For our E. coli culture 1 OD = 1.25 x 106 cells/ml This conversion is different depending on species!

9. Guidelines for Spec 20 Use clean cuvettes Wipe with KimWipes before inserting Zero your spec with a blank before you measure. Solutions should be diluted first Readings are not accurate when the culture is too turbid

10. Heterotrophic Plate Count Spread a sample evenly across a plate, incubate 24 hours, and count colonies Each colony represents 1 single cell that was in original sample These require dilutions prior to plating The plate should have between 30 and 300 colonies <30 will give statistically inaccurate count >300 TNTC (colonies too clumped)

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