1 Counting Bacteria u Filename Cel Count ppt
1 Counting Bacteria u Filename: Cel. Count. ppt u Hugh B. Fackrell 11/23/2020
2 Presentation Outline u Direct Counts u Microscopic u Electronic counter u Dilution series u Membrane filtration u Indirect Counts u Optical density u Weight of culture u Chemical 11/23/2020
3 Direct Counts: Microscopic u Calibrated stained smear u Counting chamber u Epifluorescence Need high numbers of bacteria in the sample to be useful 11/23/2020
4 Calibrated Stained Smear 11/23/2020
5 Petroff- Hauser Counting Chamber 11/23/2020
6 Epifluorescence Microscopy u Nucleic u 3 -6 u Binds acid specific dye tetramethyldiamino acridine between base pairs of DNA or RNA u Intercalation u Fluoresces when bound u DS-DNA -> Green u SS-DNA -> Red Orange u Degraded DNA -> Red Orange 11/23/2020
7 Epifluorescence u Approximate viable cells /total cells u Viable cells green u Dead cells orange u Count u cells low numbers fluorscent against a black background 11/23/2020
8 Dilution Series: dilution 1 ml 1 ml 1 ml 9 ml broth 1/10 1/1, 000 1/100, 000 1/10, 000 11/23/2020
9 Dilution Series: plating 1 ml 1/100 1 ml 1/1, 000 1 ml 1/100, 000 11/23/2020
10 Dilution Series: Colony counts 1/100 1/10, 000 1/100, 000 11/23/2020
11 Membrane filtration u Membrane filter u fixed pores 0. 45 m 0. 22 m u bacteria trapped on filter u Filter known amount of fluid u ALL bacteria trapped on membrane u Place filter in Petri dish with appropriate medium u Incubate u Count number of colonies 11/23/2020
12 Membrane filtration u Number of Viable cells u Each colony from a single bacterium u Sensitive u eg filter entire bottle of pop u <1 bacterium / bottle u Growth media u selective u Combine u Black or differential with Epifluorescence polycarbonate filters 11/23/2020
13 Electronic Measurement u Turbidity Broths with many bacteria become turbid u turbidity increases light scattering u increased absorbance in spectrophotometer u u Cell counters u fluid pumped through a micro pipette u pumped past an electronic sensor u records number of cells 11/23/2020
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