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1 Counting Bacteria Filename: CelCount.ppt Hugh B. Fackrell 5/23/2017 2 Presentation Outline Direct Counts Microscopic Electronic counter Dilution series Membrane filtration Indirect Counts Optical density Weight of culture Chemical 5/23/2017 3 Direct Counts: Microscopic Calibrated stained smear Counting chamber Epifluorescence Need high numbers of bacteria in the sample to be useful 5/23/2017 4 Calibrated Stained Smear 5/23/2017 5 Petroff- Hauser Counting Chamber 5/23/2017 6 Epifluorescence Microscopy Nucleic 3-6 tetramethyldiamino acridine Binds RNA acid specific dye between base pairs of DNA or Intercalation Fluoresces when bound DS-DNA -> Green SS-DNA -> Red Orange Degraded DNA -> Red Orange 5/23/2017 7 Epifluorescence Approximate viable cells /total cells Viable cells green Dead cells orange Count cells low numbers fluorscent against a black background 5/23/2017 8 Dilution Series: dilution 1ml 1ml 1ml 1ml 1ml 9ml broth 1/10 1/1,000 1/100 1/100,000 1/10,000 5/23/2017 9 Dilution Series: plating 1ml 1/10 1ml 1ml 1ml 1/100 1/1,000 1/10,000 1ml 1/100,000 5/23/2017 10 Dilution Series: Colony counts 1/100 1/10 1/10,000 1/1,000 1/100,000 5/23/2017 11 Membrane filtration Membrane filter fixed pores 0.45 m 0.22 m bacteria trapped on filter Filter known amount of fluid ALL Place bacteria trapped on membrane filter in Petri dish with appropriate medium Incubate Count number of colonies 5/23/2017 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 Combine Black or differential with Epifluorescence polycarbonate filters 5/23/2017 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 5/23/2017
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