Lesson 3.5 Bacterial Growth.pdf

Lesson 3.5 Bacterial Growth Lotis M. Balala College Of Veterinary Medicine, Visayas State University  Learning Outcomes 1. Discuss the stages of bacterial growth. 2. Describe expression and measurement of bacterial growth. Bacterial Growth  orderly increase of all the chemical con...

Lesson 3.5 Bacterial Growth Lotis M. Balala College Of Veterinary Medicine, Visayas State University  Learning Outcomes 1. Discuss the stages of bacterial growth. 2. Describe expression and measurement of bacterial growth. Bacterial Growth  orderly increase of all the chemical constituents of an organism ✓ cellular constituents ✓ cellular structure  growth without cell division – increase in size and weight of the cell  for most microorganisms: growth followed by cell division and increase in cell number  increase in total mass – not necessarily a reflection of growth Divide by Binary Fission Origin of replication  not possible to distinguish resulting cells from the mother cell Phases of Growth Phases of growth: 1. Lag phase no immediate increase in cell number or mass cells are dormant but physiologically active synthesis of new protoplasmic constituents synthesis of new enzymes adaptation phase Varies considerably in length with the condition of the microorganisms and the nature of the medium. 2. Exponential or log phase microorganisms are growing and dividing at the maximal rate possible dependent on the following: genetic potential of the organism nature of the medium conditions under which they are growing rate of growth is constant microorganisms divide and double in number at regular intervals Cells nearly uniform in chemical composition and physiological characteristics. Balanced growth - all cellular constituents are manufactured at constant rates relative to each other Unbalanced growth - rates of synthesis of cell components vary relative to one another which is readily observed in either: - shift up – slow to fast growth - shift down – slowing down of growth 3. Stationary phase - no net increase or decrease in cell number (cryptic growth) - slow metabolic activity - cells enter this phase due to the following reasons: 1. nutrient limitation 2. accumulation of toxic products - cells are smaller than those in the log phase and do not have constant composition! 4. Death phase - death rate > growth rate - death is logarithmic but in most cases, slower than that of exponential growth Measuring Growth: 1. Cell count: a. direct method: direct counting of individual cells Advantages: - rapid and simple - requires minimum equipment - bacterial morphology can be observed Disadvantages: - can not be used for dilute suspensions - both viable and non-viable cells are counted make use of counting chambers  Petroff-Hausser Counting Chamber cells/ml = ave. # of cells x 25 squares x 50 x 1000  Hemacytometer cells/ml = ave. # of cells x CF x DF Electronic Particle Counter (Coulter counter) electronically measures the number of particles within a fixed size range based on the property that nonconductive particles will cause a disruption in an electric field as they pass through it b. Indirect Method (Viable Count Procedures) Assumptions: one organism gives rise to one colony bacterial suspension is homogenous no aggregates of cells present Advantages: sensitive easy to perform can be adapted to the measurement of a certain type of organism counting of viable cells only Disadvantages: aggregates of cells will form only one colony does not represent all the microorganisms 1. Plate Count Methods widely used for enumeration of viable microorganisms employ various media and incubation conditions dilutions are done before plating Main considerations: a. Composition of the medium b. Incubation conditions c. Period of incubation Advantage: Conditions can be adjusted so that members of a defined group can be enumerated.  Methods employed in inoculating Petri plates from the serial dilutions Computing for CFU/ml: CFU/ml = ave. # of colonies x DF volume plated Membrane Filter Method for samples of low density of viable cells ( < 10 organisms/ml) 1. known volume of sample 2. pass through a sterile membrane filter 3. place on a suitable medium 4. incubate 5. count number of colonies https://biologyreader.com/membrane -filtration-method.html Fig. 1: Colonies on CHROMagar with filter paper after 24hr incubation at 35C. 2. Most Probable Number Technique: multiple dilution to extinction approach 2. Cell Mass a. direct method Dry weight - microorganisms (dried to a constant weight) at 100 - 1050C Considerations: cells must be washed completely free of all extraneous matter cell suspension must be dense Advantages: reliable reproducible Wet weight - packed, centrifuged cells Advantage: cells can still be used for enzyme isolation or lipid analysis Disadvantage: not accurate and not reliable b. Indirect methods turbidimetric measurements Light → bacteria in suspension → light is scattered instrument: photometer = employs a simple filter to generate light of narrow wavelength (λ) spectrophotometer = employs a prism to generate light in a very narrow band of λ  Advantages: quick readily repeatable does not adversely affect cells Limitations: not suitable for cells growing in clumps can not be used for cultures with < 107 cells/ml can not be used for deeply colored solutions and those containing suspended matter other than the desired organism Commonly used wavelengths : 540 nm (green) 600 nm (orange) 660 nm (red)