Counting Bacteria - University of Windsor

Counting Bacteria - University of Windsor

1 Counting Bacteria Filename: CelCount.ppt Hugh B. Fackrell 02/07/20 2 Presentation Outline Direct Counts

Microscopic Electronic counter Dilution series Membrane filtration Indirect Counts Optical density Weight of culture Chemical 02/07/20 3 Direct Counts: Microscopic

Calibrated stained smear Counting chamber Epifluorescence Need high numbers of bacteria in the sample to be useful 02/07/20 4 Calibrated Stained Smear 02/07/20 5 Petroff- Hauser Counting Chamber

02/07/20 6 Epifluorescence Microscopy Nucleic acid specific dye Binds between base pairs of DNA or RNA 3-6 tetramethyldiamino acridine Intercalation

Fluoresces when bound DS-DNA -> Green SS-DNA -> Red Orange Degraded DNA -> Red Orange 02/07/20 7 Epifluorescence Approximate viable cells /total cells

Viable cells green Dead cells orange Count low numbers cells fluorscent against a black background 02/07/20 8 Dilution Series: dilution 1ml 1ml 1ml

1ml 1ml 9ml broth 1/10 1/1,000 1/100 1/100,000 1/10,000 02/07/20 9 Dilution Series: plating 1ml

1/10 1ml 1ml 1ml 1/100 1/1,000 1/10,000 1ml 1/100,00 0 02/07/20 10 Dilution Series: Colony counts 1/10

1/100 1/10,000 1/1,000 1/100,00 0 02/07/20 11 Membrane filtration Membrane filter

Filter known amount of fluid fixed pores 0.45 m 0.22 m bacteria trapped on filter ALL bacteria trapped on membrane Place filter in Petri dish with appropriate medium Incubate Count number of colonies 02/07/20 12 Membrane filtration

Number of Viable cells Sensitive eg filter entire bottle of pop <1 bacterium / bottle Growth media Each colony from a single bacterium

selective or differential Combine with Epifluorescence Black polycarbonate filters 02/07/20 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 02/07/20

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