1 Counting of Bacteria 2 Presentation Outline Direct

1 Counting of Bacteria

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

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

4 Petroff-Hauser Chamb Counting er

5 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

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

7 1 ml Dilution Series: 1 ml dilution 1 ml 1 ml 9 ml broth 1/10 1/1, 000 1/100, 000 1/10, 000

8 Dilution Series: plating 1 ml 1/100 1 ml 1/1, 000 1/10, 000 1 ml 1/100, 000 08/28/18

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

1 0 Membrane 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

1 1 Membrane 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

1 2 Electronic Turbidity Measureme nt Broths with many bacteria become 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