Counting Bacteria Filename: CelCount.ppt Hugh B. Fackrell

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

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

Calibrated Stained Smear

Petroff- Hauser Counting Chamber

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

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

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

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

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

Membrane filtration Membrane filter Filter known amount of fluid 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

Membrane filtration Number of Viable cells Sensitive Growth media 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

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