1. Counting Bacteria
Filename: CelCount.ppt
Hugh B. Fackrell

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

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

4. Calibrated Stained Smear

5. Petroff- Hauser Counting Chamber

6. 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

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

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

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

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

11. 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

12. 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

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