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Counting Microorganisms Methods • • • • 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 3 Turbidity measurements • Spectrophotometer (A600): – Measuring optical density Light Detector….reading 600nm Different reading 4 Turbidity measurements O.D. 600nm 2.0 1.0 0 % Transmission 0 Inverse relationship Cellular density 50 100 5 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 6 Serial Dilutions Bacterial culture • 63 CFU/0.1ml of 10-5 CFU CFU CFU • 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? 7 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 Direct Counts • The sample to be counted is applied onto a hemacytometer slide that holds a fixed volume in a counting chamber • The number of cells is counted in several independent squares on the slide’s grid • The number of cells in the given volume is then calculated Using a hemacytometer Using a hemacytometer (Cont’d) Using a hemacytometer (Cont’d) Determining the Direct Count • Count the number of cells in three independent squares – 8, 8 and 5 • Determine the mean – (8 + 8 + 5)/3 =7 – Therefore 7 cells/square 13 Determining the Direct Count (Cont’d) 1mm Depth: 0.1mm 1mm • Calculate the volume of a square: = 0.1cm X 0.1cm X 0.01cm= 1 X 10-4cm3 or ml • Divide the average number of cells by the the volume of a square – Therefore 7/ 1 X 10-4 ml = 7 X 104 cells/ml 14 Problem • A 500μl sample is applied to a hemacytometer slide with the following dimensions: 0.1mm X 0.1mm X 0.02mm. Counts of 6, 4 and 2 cells were obtained from three independent squares. What was the number of cells per milliliter in the original sample if the counting chamber possesses 100 squares? Most probable Number: MPN – Based on Probability Statistics – Presumptive test based on given characteristics – Broth Technique Most Probable Number (MPN) • Begin with Broth to detect desired characteristic • Inoculate different dilutions of sample to be tested in each of three tubes -1 Dilution -2 -3 -4 -5 -6 3 Tubes/Dilution 1 ml of Each Dilution into Each Tube After suitable incubation period, record POSITIVE TUBES (Have GROWTH and desired characteristics) MPN - Continued • Objective is to “DILUTE OUT” the organism to zero • Following the incubation, the number of tubes showing the desired characteristics are recorded • Example of results for a suspension of 1g/10 ml of soil • Dilutions: -1 -2 -3 -4 • Positive tubes: 3 2 1 0 – Choose correct sequence: 321 and look up in table Pos. tubes 0.10 0.01 0.001 3 2 1 MPN/g (mL) 150 – Multiply result by middle dilution factor » 150 X 102 = 1.5 X 104/mL » Since you have 1g in 10mL must multiply again by 10 » 1.5 X 105/g Microscopy Staining Simple Staining • Positive staining – Stains specimen – Staining independent of the species • Negative staining – Staining of background – Staining independent of the species Method • 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. Microscopy Differential Staining Differential Staining Gram Stain • Divides bacteria into two groups • Gram Negative & Gram Positive • Stained Purple – Rods • Genera Bacillus and Clostridium – Coccus • Genera Streptococcus, Staphylococcus and Micrococcus Gram Negative • Stained Red – Rods: • Genera Escherichia, Salmonella, Proteus, etc. – Coccus: • Genera Neisseria, Moraxella and Acinetobacter Rules of thumb • If the genus is Bacillus or Clostridium = Gram (+) rod • If the genus name ends in coccus or cocci (besides 3 exceptions, which are Gram (-)) = coccus shape and Gram (+) • If not part of the rules above, = Gram (-) rods Gram + Cell Wall Vs Gram - Peptidoglycan wall Plasma Membrane Absent Lipopolysaccharide layer 31 Method – Primary staining 1. Staining with crystal violet 2. Addition of Gram’s iodine (Mordant) + + + Wall:peptidoglycan Plasma membrane + + + + + + + + + + + + LPS --------------Gram positive --------------Gram negative Method – Differential step 3. Alcohol wash Wall is dehydrated – Stain + iodine complex is trapped Wall: peptidoglycan Plasma membrane Wall is not dehydrated – Complex is not trapped LPS - - +- - -+- - +- - -+- - + - - -+ + Gram positive - - +- - -+- - +- - -+- - + - - -+ + Gram negative Method – Counter Stain 4. Staining with Safranin + + + + + + + Wall:peptidoglycan Plasma membrane + + + + + + + + LPS - - +- - -+- - +- - -+- - + - - -+ + Gram positive --------------Gram negative 34 Summary Gram Positive Gram Negative Fixation Primary staining Crystal violet Wash Destaining Counter staining Safranin 35 Acid Fast Staining • Diagnostic staining of Mycobacterium – Pathogens associated with Tuberculosis and Leprosy – Cell wall has mycoic acid • Waxy, very impermeable Method • Basis: – High level of compounds similar to waxes in their cell walls, Mycoic acid, makes these bacteria resistant to traditional staining techniques Method (Cont’d) • Cell wall is permeabilized with heat • Staining with basic fuchsine – Phenol based, soluble in mycoic layer – Cooling returns cell wall to its impermeable state • Stain is trapped • Wash with acid alcohol – Differential step • Mycobacteria retain stain • Other bacteria lose the stain Spore Stain • Spores: – Differentiated bacterial cell – Resistant to heat, desiccation, ultraviolet, and different chemical treatments • Thus very resistant to staining too! – Typical of Gram positive rods • Genera Bacillus and Clostridium – Unfavorable conditions induce sporogenesis • Differentiation of vegetative cell to endospore – E.g. Anthrax Malachite Green Staining • Permeabilization of spores with heat • Primary staining with malachite green • Wash • Counter staining with safranin Sporangium (cell + endospore) Vegetative cells (actively growing) Spores (resistant structures used for survival under unfavourable conditions.) Endospore (spore within cell)