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) cells Includes bacteria, fungi, algae, viruses, and protozoans 2 Microorganisms in the Lab Growth Media 1
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 2
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 Counting Microorganisms 3
Methods Turbidity measurements: Optical density Direct counts Viable counts MPN 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 11 Turbidity measurements Spectrophotometer (A600): Measuring optical density Light Detector.reading 600nm Different reading 12 4
Turbidity measurements O.D. 600nm 2.0 % Transmission 0 1.0 Inverse relationship 50 0 Cellular density 100 13 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 Direct Counts Advantages: Quick Growth is not required No information about organism required Limits: Does not discriminate between live and dead May be difficult to distinguish bacteria from detritus 15 5
Using a hemacytometer Using a hemacytometer (Cont d) Hemacytometer This slide has 2 independent counting chambers 18 6
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 20 Determining the Direct Count (Cont d) 1mm 1mm Depth: 0.1mm Calculate the volume of a square: = 0.1cm X 0.1cm X 0.01cm= 1 X 10-4 cm 3 or ml Divide the average number of cells by the the volume of a square Therefore 7/ 1 X 10-4 ml = 7 X 10 4 cells/ml 21 7
Problem A 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? Viable Counts A viable cell: a cell which is able to divide and form a population (or colony) 1. A viable cell count is done by diluting the original sample 2. Plating aliquots of the dilutions onto an appropriate culture medium 3. Incubating the plates under appropriate conditions to allow growth Colonies are formed 4. Colonies are counted and original number of viable cells is calculated according to the dilution used 23 Serial dilutions of sample Viable Counts 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 24 8
Dilution of Bacterial Sample 25 Dilution of Bacterial Sample 26 Dilution of Bacterial Sample 27 9
Dilution of Bacterial Sample 28 Plating of Diluted Samples 5672 57 4 29 Viable Counts The total number of viable cells is reported as Colony-Forming Units (CFUs) rather than cell numbers Each single colony originates from a colony forming unit (CFU) A plate having 30-300 colonies is chosen Calculation: Number of colonies on plate X reciprocal of dilution (dilution factor) = Number of CFU/mL Ex. 57 CFU/0.1mL X 10 6 = 5.7 X 10 7 CFU/mL 30 10
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 10 5 = 6.3 x 10 7 /ml in original sample What if there were 100 ml in the flask? 31 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 Only living cells develop colonies Clumps or chains of cells develop into a single colony? CFU one bacteria? = = Ex. One CFU of Streptococcus one of E.coli Most probable Number: MPN Based on Probability Statistics Presumptive test based on given characteristics Broth Technique 11
Most Probable Number (MPN) Begin with Broth to detect desired characteristic Inoculate different dilutions of sample to be tested in each of three tubes Dilution -1-2 -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 MPN/g (ml) 0.10 0.01 0.001 3 2 1 150 Multiply result by middle dilution factor» 150 X 10 2 = 1.5 X 10 4 /ml» Since you have 1g in 10mL must multiply again by 10» 1.5 X 10 5 /g Microscopy Staining 12
Positive staining Simple 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 13
Axes of division Cocci (Coccus) 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. 14
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 15
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 Staining- Principal Uses a combination of two stains Primary stain - Crystal violet Purple Secondary stain Safranin Red Gram positive The cell wall traps the 1 o stain Gram negative Cell wall does not allow 1 o stain to be trapped 48 16
Cell Wall 49 Method Primary Stain 1. Staining with crystal violet 2. Add Gram s iodine (Mordant) + + + + + + + + + + + + + + + Wall: peptidoglycan Plasma membrane - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - LPS Gram positive Gram negative 50 Method Differential Step 3. Alcohol wash Cell wall is dehydrated and less permeable Stai + iodine complex is trapped LPS layer is dissolved Cell wall is dehydrated, but permeable Complex is not trapped Wall: peptidoglycan Plasma membrane - - + - - - + - - + - - - + - - + - - - + + - - + - - - + - - + - - - + - - + - - - + + Gram positive Gram Negative LPS 51 17
Method Counter Stain 4. Staining with Safranin + + + + + + + + + + + + + + + Wall: peptidoglycan Plasma membrane - - + - - - + - - + - - - + - - + - - - + + - - - - - - - - - - - - - - - LPS Gram positive Gram Negative 52 Summary Fixation Primary stain Crystal violet Wash Decolorization Counter stain Safranin 53 Acid Fast Staining Diagnostic staining of Mycobacterium Pathogens associated with Tuberculosis and Leprosy Cell wall has mycoic acid Waxy, very impermeable 18
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 19
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) 20