This document discusses various methods for counting bacteria, including direct microscopic counts, dilution series plating, membrane filtration, and electronic measurement. Direct microscopic counts can be done with stained smears under a microscope or using an electronic counting chamber. Dilution series involve making serial dilutions of a sample and plating them to obtain isolated colonies that can be counted. Membrane filtration uses filters of defined pore sizes to trap bacteria from a sample, which are then cultured and counted. Electronic methods measure turbidity from bacterial growth in broths or use cell counters with sensors to obtain cell counts.

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Counting of Bacteria
(Pharm. Microbiology)

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

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

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Petroff- Hauser CountingPetroff- Hauser Counting
ChamberChamber

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Epifluorescence MicroscopyEpifluorescence 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

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

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

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

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Dilution Series: Colony countsDilution Series: Colony counts
1/100,000
1/100 1/1,0001/10
1/10,000

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Membrane filtrationMembrane 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

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Membrane filtrationMembrane 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

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Electronic MeasurementElectronic 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