Transcript CFU
Microbiology The Study of Microorganisms Definition of a Microorganism • Derived from the Greek: Mikros, «small» and Organismos, “organism” – Microscopic organism which is single celled (unicellular) or a mass of identical (undifferentiated) – Includes bacteria, fungi, algae, viruses, and protozoans 2 Microorganisms in the Lab Growth Media Goals • • • • Growth under controlled conditions Maintenance Isolation of pure cultures Metabolic testing Types • Liquid (Broths) – Allows growth in suspension – Uniform distribution of nutrients, environmental parameters and others – Allows growth of large volumes • Solid media – Same as liquid media + solidification agent • Agar: Polysaccharide derived from an algae Growth in Broths Non inoculated clear Turbid + sediment Turbid Clear + sediment Growth on Agar • Growth on solid surface • Isolated growth • Allows isolation of single colonies • Allows isolation of pure cultures Single colony Solid Media (Cont’d) • Slants – Growth on surface and in depth – Different availabilities of oxygen – Long term storage • Stab – Semi-solid medium – Long term storage – Low availability of oxygen Microscopy Staining Simple Staining • Positive staining – Stains specimen – Staining independent of the species • Negative staining – Staining of background – Staining independent of the species Methods • Simple stain: – One stain – Allows to determine size, shape, and aggregation of bacteria Cell Shapes • Coccus: – Spheres – Division along 1,2 or 3 axes – Division along different axes gives rise to different aggregations – Types of aggregations are typical of different bacterial genera Cocci (Coccus) Axes of division Arrangements Diplococcus Streptococcus (4-20) Tetrad Staphylococcus Hint: if name of genus ends in coccus, then the shape of the bacteria are cocci Cell Shapes (Cont’d) • Rods: – Division along one axis only – Types of aggregations are typical of different bacterial genera The Rods Axes of division Arrangements Diplobacillus Streptobacillus Hint: if name of bacteria genus is Bacillus, then the shape of the bacteria are rods If it doesn’t end in cocci, it’s probably a rod. Counting Microorganisms • • • • Turbidity measurements: Optical density Viable counts MPN Direct counts Turbidity measurements • Measures the amount of light that can go through a sample • The less the amount of light which goes through the sample the denser the population • Mesures optical density or percent transmission 17 Turbidity measurements • Spectrophotometer (A600): – Measuring optical density Light Detector….reading 600nm Different reading 18 O.D. 600nm 2.0 1.0 0 % Transmission 0 Inverse relationship Cellular density 50 100 19 Viable Counts • Serial dilutions of sample • Spread dilutions on an appropriate medium • Each single colony originates from a colony forming unit (CFU) • The number of colonies represents an approximation of the number of live bacteria in the sample 20 Bacterial culture CFU CFU CFU • 63 CFU/0.1ml of 10-5 • 630 CFU/1.0ml of 10-5 • 630 CFU/ml X 105 = 6.3 x 107/ml in original sample What if there were 100 ml in the flask? 21 Viable Counts • Advantages: – Gives a count of live microorganisms – Can differentiate between different microorganisms • Limits: – No universal media • Can’t ask how many bacteria in a lake • Can ask how many E. coli in a lake – Requires growth ? – CFU one bacteria ? = = • Ex. One CFU of Streptococcus one of E.coli