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Counting Bacteria

Counting Bacteria. Filename: CelCount.ppt Hugh B. Fackrell. Presentation Outline. Direct Counts Microscopic Electronic counter Dilution series Membrane filtration Indirect Counts Optical density Weight of culture Chemical. Direct Counts: Microscopic. Calibrated stained smear

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Counting Bacteria

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  1. Counting Bacteria • Filename: CelCount.ppt • Hugh B. Fackrell

  2. Presentation Outline • Direct Counts • Microscopic • Electronic counter • Dilution series • Membrane filtration • Indirect Counts • Optical density • Weight of culture • Chemical

  3. Direct Counts: Microscopic • Calibrated stained smear • Counting chamber • Epifluorescence Need high numbers of bacteria in the sample to be useful

  4. Calibrated Stained Smear

  5. Petroff- Hauser Counting Chamber

  6. Epifluorescence Microscopy • Nucleic acid specific dye • 3-6 tetramethyldiamino acridine • Binds between base pairs of DNA or RNA • Intercalation • Fluoresces when bound • DS-DNA -> Green • SS-DNA -> Red Orange • Degraded DNA -> Red Orange

  7. Epifluorescence • Approximate viable cells /total cells • Viable cells green • Dead cells orange • Count low numbers • cells fluorscent against a black background

  8. 1ml 1ml 1ml 1ml 1ml 1/1,000 1/10 1/100,000 1/100 1/10,000 Dilution Series: dilution 9ml broth

  9. 1ml 1ml 1ml 1ml 1ml 1/1,000 1/10,000 1/10 1/100 1/100,000 Dilution Series: plating

  10. 1/1,000 1/100 1/10 1/10,000 1/100,000 Dilution Series: Colony counts

  11. Membrane filtration • Membrane filter • fixed pores 0.45 m 0.22 m • bacteria trapped on filter • Filter known amount of fluid • ALL bacteria trapped on membrane • Place filter in Petri dish with appropriate medium • Incubate • Count number of colonies

  12. Membrane filtration • Number of Viable cells • Each colony from a single bacterium • Sensitive • eg filter entire bottle of pop • <1 bacterium / bottle • Growth media • selective or differential • Combine with Epifluorescence • Black polycarbonate filters

  13. Electronic Measurement • Turbidity • Broths with many bacteria become turbid • turbidity increases light scattering • increased absorbance in spectrophotometer • Cell counters • fluid pumped through a micro pipette • pumped past an electronic sensor • records number of cells

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