General Microbiology Laboratory Bacterial Generation Time

Mohammed laqqan Microbial Growth  Binary fission  Generation time  Growth curve  Enumeration of bacteria

Mohammed laqqan  Reproduction and cell division  HOW DO BACTERIA REPRODUCE? Binary fission Budding Spore formation

Mohammed laqqan Microbial Growth  Growth= an increase in the number of cells, not an increase in size  Bacterial species only maintained if population continues to grow  Generation=growth by binary fission  Growth rate = cell number/time or cell mass/time  Generation time= time it takes for a cell to divide and the population to double; most are 1-3 hours.  Generation times vary markedly with the species of microorganism and environmental conditions; they can range from 10 minutes for a few bacteria to several days with some eucaryotic microorganisms.

Mohammed laqqan Generation Times BacteriumMedium Generation Time (minutes) Escherichia coliGlucose-salts17 Bacillus megateriumSucrose-salts25 Streptococcus lactisMilk26 Streptococcus lactisLactose broth48 Staphylococcus aureus Heart infusion broth27-30 Lactobacillus acidophilus Milk66-87 Rhizobium japonicum Mannitol-salts-yeast extract Mycobacterium tuberculosis Synthetic Treponema pallidumRabbit testes1980

Mohammed laqqan What occurs during binary fission?

Mohammed laqqan The Growth Cycle  The population growth is studied by analyzing the growth curve of a microbial culture.  The standard bacterial growth curve describes various stages of growth a pure culture of bacteria will go through, beginning with the addition of cells to sterile media and ending with the death of all of the cells present.  Usually analyzed in a closed system called a batch culture; plotted as the logarithm of cell number versus the incubation time.  Bacteria added to fresh media typically go through four more-or-less distinct phases of growth: lag, exponential, stationary, and death.

Mohammed laqqan Typical growth curve for a bacterial population

Mohammed laqqan Phases of Growth

Mohammed laqqan Lag phase The period of apparent inactivity in which the cells are adapting to a new environment and preparing for reproductive growth. Cells are usually synthesizing new components. In practice, bacteria from one medium to another, where there are chemical differences between the two media, typically results in a lag in cell division. This lag in division is associated with a physiological adaptation to the new environment. Cells may increase in size during this time, but simply do not divide (by binary fission). Lag phase varies considerably in length depending upon the condition of the microorganisms and the nature of the medium.

Mohammed laqqan Log (exponential) phase The period in which the organisms are growing at the maximal rate possible given their genetic potential, the nature of the medium, and the conditions under which they are growing. Generation time can be easily obtained from the exponential phase of a growth curve The population is most uniform in terms of chemical and physical properties during this period.

Mohammed laqqan Stationary phase Eventually population growth decrease, and the growth curve becomes horizontal. steady-state equilibriumsteady-state equilibrium rate of cell growth = rate of cell deathrate of cell growth = rate of cell death Cell death may result from Nutrient limitation & Toxic waste accumulation (e.g. acid buildup from fermentation); as well as O2 depletion, critical population level reached.

Mohammed laqqan Death phase Stationary phase, in a standard bacterial growth curve, is followed by a die-off of cells, called Death phase. It is the period in which the cells are dying at an exponential rate. Some of the reasons are: continued accumulation of wastes, loss of cell's ability to detoxify toxins, etc.

Mohammed laqqan Balanced and Unbalanced Growth Balanced (exponential) growth occurs when all cellular components are synthesized at constant rates relative to one another. Unbalanced growth occurs when the rates of synthesis of some components change relative to the rates of synthesis of other components. This usually occurs when the environmental conditions or nutrient levels change.

Mohammed laqqan Measurement of Bacterial Growth  Cell numbers  Cell mass  Metabolic rate

Mohammed laqqan Cell numbers 1- Direct microscopic count accomplished by direct microscopic observation on specially etched slides (such as Petroff-Hausser chambers or hemocytometers) or by using electronic counters. 2- Viable count (colony forming units)

Mohammed laqqan Cell mass  Dry weight versus wet weight  Volume of cells after centrifugation  Measurement of total N or protein

Mohammed laqqan Cell mass / numbers  Turbidity or optical density it is one of the optical methods for counting cells; can estimate cell numbers accurately by measuring visible turbidity. Light scattered is proportional to number of cells. Use a spectrophotometer to accurately measure absorbance, usually at wavelengths around nm. Accurate measure of cells when concentration not too high. Easy and quick to measure (can measure a sample in less than a minute). This technique measures the total mass of organisms and does not distinguish between dead and viable organisms.

Mohammed laqqan  Metabolic rate 0 2 uptake, C0 2 and ATP production 0 2 uptake, C0 2 and ATP production

Mohammed laqqan Turbidity measurements of microbial growth  Optical Density (Counting by Spectrophotometer)

Mohammed laqqanMaterials  TSB of Nonpathogenic Escherichia coli  Inoculating loop  Spectrophotometer  Black markers  Test tube rack  2 tubes of TSB  gloves  lab coat or apron  37C Incubator  Bunson burner  Surface disinfectant  Paper towels

Mohammed laqqanProcedure 1.Wash hands and put on gloves. 2.Assemble equipment and materials and prepare work area. 3.Set the spectrophotometer at 450nm and let it warm up 30 minutes before performing the readings. The meter on the spectrophotometer should read “0% Transmittance”. 4.Obtain 2 tubes of TSB. Label one C (uninoculated) for control and one S (inoculated) for sample. 5.Place tube C into the sample holder of the spectrophotometer. Turn the light control knob until the meter needle is on “100% Transmittance”. Remove the control tube. The spectrophotometer is now standardized. 6.Flame and cool the loop. 7.Mix the culture of E. coli and inoculate tube S with 5 loopfuls of the broth.

Mohammed laqqan 8.Flame and cool the loop. 9.Start the turbidity readings by placing the S tube into the sample well and read the OD (absorbance). Record the results in the data section. 10.Take OD reading over the time period specified in the data section and return the TSB tube to the 37 C incubator between each reading. 11.Turn off the spectrophotometer upon completion of the readings. 12.Plot the readings on the graph paper as absorbance vs. time. 13.Clean work area with disinfectant. 14.Remove gloves and wash hands with disinfectant.

Mohammed laqqanData  Record the absorbance value for each sample reading starting at 0 time.

Mohammed laqqan  Plot the absorbance values vs. time on graph paper. Label the x and y axis. Each of the readings will represent the growth of the E. coli in the culture.  Label each part of the growth curve: Lag phase, Exponential growth phase, and the Stationery phase.

Mohammed laqqan End of lecture