Growth and Reproduction in Bacteria PDF

X. Growth and Reproduction in Bacteria MTN Cabasan 1 Objectives 1. Classify the microorganisms according to preferred temperature range 2 Identify how and why pH of culture media is...

X. Growth and Reproduction in Bacteria MTN Cabasan 1 Objectives 1. Classify the microorganisms according to preferred temperature range 2 Identify how and why pH of culture media is controlled 3. Explain the importance of osmotic pressure to microbial growth 4. Name a use for each of the four elements (carbon, nitrogen, sulfur, and phosphorus) needed in large amounts for microbial growth 5. Explain how microorganisms are classified on the basis of oxygen requirements 2 MTN Cabasan 1  Objectives 6. Identify ways in which aerobes avoid damage by toxic forms of oxygen 7. Define microbial growth, including binary fission 8. Compare the phases of microbial growth, and describe their relation to generation time 9. Differentiate direct and indirect methods of measuring cell growth 10. Explain direct and indirect methods of measuring cell growth 11. Calculate mathematical equations for microbial growth. 3 Requirements for Growth: 1) Temperature  Most microorganisms grow well at the temperatures that humans favor.  Certain bacteria are capable of growing at extremes of temperature that would certainly hinder the survival of almost all eukaryotic organisms.  Microorganisms are classified into three primary groups on the basis of their preferred range of temperature: - psychrophiles (cold-loving microbes), - mesophiles (moderate-temperature– loving microbes), and - thermophiles (heat-loving microbes). 4 MTN Cabasan 2  Requirements for Growth: Temperature  bacteria grow only within a limited range of temperatures  their maximum and minimum growth temperatures are only about 30°C apart.  They grow poorly at the high and low temperature extremes within their range.  minimum growth temperature - the lowest temperature at which the species will grow.  optimum growth temperature - the temperature at which the species grows best.  maximum growth temperature - the highest temperature at which growth is possible. 5 6 MTN Cabasan 3  Psychrophiles - capable of growing at 0°C. can grow at 0°C but has an optimum growth temperature of about 15°C. Most are so sensitive to higher temperatures- cannot grow in a warm room (25°C). Found mostly in the oceans’ depths or in polar regions; seldom cause problems in food preservation. Or can grow at 0°C has higher optimum temperatures, usually 20–30°C and cannot grow above about 40°C. most likely to be encountered in low-temperature food spoilage - grow fairly well at refrigerator temperatures= psychrotrophs; spoilage microorganisms 7 Refrigeration - the most common method of preserving household food supplies.  Principle: microbial reproductive rates decrease at low temperatures.  Microbes usually survive even subfreezing temperatures (they might become entirely dormant), they gradually decline in number. Some species decline faster than others.  Psychrotrophs do not grow well at low temperatures, however, they are able to slowly degrade food.  Spoilage – in the form of mold mycelium, slime on food surfaces, or off-tastes or off-colors in foods.  The temperature inside a properly set refrigerator will greatly slow the growth of most spoilage organisms and will entirely prevent the growth of all but a few pathogenic bacteria. 8 MTN Cabasan 4   The effect of the amount of food on its cooling rate in a refrigerator and its chance of spoilage. Notice that in this example, the pan of rice with a depth of 5 cm (2 in) cooled through the incubation temperature range of the Bacillus cereus in about 1 hour, whereas the pan of rice with a depth of 15 cm (6 in) remained in this temperature range for about 5 hours. 9 Mesophiles- most common type of microbe; optimum growth temperature of 25–40°C.  Organisms that have adapted to live in the bodies of animals usually have an optimum temperature close to that of their hosts.  optimum temperature for many pathogenic bacteria = about 37°C (temp set for clinical cultures) most of the common spoilage and disease organisms 10 MTN Cabasan 5  Thermophiles - capable of growth at high temperatures. have an optimum growth temperature of 50–60°C  in sunlit soil and in thermal waters - hot springs. cannot grow at temperatures below about 45°C. Endospores formed by thermophilic bacteria are unusually heat resistant and may survive the usual heat treatment for canned goods. important in organic compost piles (temperature = 50–60°C). 11 Archaea an optimum growth temperature of 80°C or higher. hyperthermophiles or extreme thermophiles. Most live in hot springs associated with volcanic activity, and sulfur is usually important in their metabolic activity. The known record for bacterial growth and replication at high temperatures = about 121°C (near deep sea hydrothermal vents). 12 MTN Cabasan 6  pH  acidity or alkalinity of a solution.  Most bacteria grow best = narrow pH range near neutrality, between pH 6.5 and 7.5.  Very few bacteria grow at an acidic pH below about pH 4.  sauerkraut, pickles, and cheeses -preserved from spoilage by acids produced by bacterial fermentation.  some bacteria, acidophiles- tolerant of acidity.  One type of chemoautotrophic bacteria, found in the drainage water from coal mines and oxidizes sulfur, can survive at a pH 1.  Molds and yeasts will grow over a greater pH range than bacteria, but the optimum pH is generally below that of bacteria, usually about pH 5 to 6.  Alkalinity also inhibits microbial growth but is rarely used to preserve foods. 13 2) pH When bacteria are cultured in the laboratory, they often produce acids that eventually interfere with their own growth. To neutralize the acids and maintain the proper pH, chemical buffers are included in the growth medium. peptones and amino acids - in some media act as buffers phosphate salts in many media have the advantage of exhibiting their buffering effect in the pH growth range - buffers are nontoxic; provide phosphorus, an essential nutrient. 14 MTN Cabasan 7   Microorganisms - require water for growth, and their 3) Osmotic Pressure composition is 80–90% water.  High osmotic pressures have the effect of removing necessary water from a cell.  When a microbial cell is in a solution; solutes is higher than in the cell (the environment is hypertonic to the cell), the cellular water passes out through the plasma membrane to the high solute concentration.  osmotic loss of water causes plasmolysis, or shrinkage of the cell’s cytoplasm 15 extreme halophiles - adapted so well to high salt concentrations obligate halophiles – require high salt concentrations for growth. Organisms from such saline waters – (Dead Sea) often require nearly 30% salt (the inoculating loop must first be dipped into a saturated salt solution). facultative halophiles - do not require high salt concentrations but are able to grow at salt concentrations up to 2% (concentration that inhibits the growth of many other organisms). A few species of facultative halophiles can tolerate even 15% salt. 16 MTN Cabasan 8  Nitrogen, Sulfur, and Phosphorus  For the synthesis of cellular material protein synthesis- requires considerable amounts of N and S DNA and RNA synthesis- require N and some P ATP synthesis Nitrogen makes up about 14% of the dry weight of a bacterial cell, and sulfur and phosphorus together constitute about another 4%. 17 3) Nitrogen, Sulfur, and Phosphorus  Organisms use nitrogen - to form the amino group of the amino acids of proteins. Bacteria –decompose protein-containing material and reincorporating the amino acids into newly synthesized proteins and other nitrogen-containing compounds. Other bacteria use nitrogen from ammonium ions (NH4+) Other bacteria - able to derive nitrogen from nitrates photosynthesizing cyanobacteria - use gaseous nitrogen (N2) directly from the atmosphere- nitrogen fixation 18 MTN Cabasan 9  5) Trace Elements very small amounts of other mineral elements (iron, copper, molybdenum, and zinc) required by microbes Most are essential for the functions of certain enzymes, usually as cofactors. Although these elements are sometimes added to a laboratory medium, they are usually assumed to be naturally present in tap water and other components of media. Even most distilled waters contain adequate amounts, but tap water is sometimes specified to ensure that these trace minerals will be present in culture media. 19 6) Oxygen 20 MTN Cabasan 10  7) Organic Growth Factors  Essential organic compounds an organism is unable to synthesize  they must be directly obtained from the environment.  One group of organic growth factors for humans is vitamins.  Most vitamins function as coenzymes, the organic cofactors required by certain enzymes in order to function.  Many bacteria can synthesize all their own vitamins and do not depend on outside sources.  some bacteria lack the enzymes needed for the synthesis of certain vitamins, and for them those vitamins are organic growth factors.  Other organic growth factors required by some bacteria are amino acids, purines, and pyrimidines. 21 Binary Fission 22 MTN Cabasan 11  generation time The time required for a cell to divide (and its population to double) 23  Most bacteria have a generation time generation time of 1 to 3 hours; others require more than 24 hours per generation  If a doubling occurred every 20 minutes—E. coli under favorable conditions—after 20 generations a single initial cell would increase to over 1 million cells [a little less than 7 hours].  In 30 generations, or 10 hours, the population would be 1 billion, and in 24 hours it would be a number trailed by 21 zeros 24 MTN Cabasan 12  Logarithmic Representation of Bacterial Populations A growth curve for an exponentially increasing population, plotted logarithmically (dashed line) and arithmetically (solid line). For demonstration purposes, this graph has been drawn so that the arithmetic and logarithmic curves intersect at 1 million cells. This figure demonstrates why it is necessary to graph changes in the immense numbers of bacterial populations by logarithmic plots rather than by arithmetic numbers. For example, note that at ten generations the line representing arithmetic numbers has not even perceptibly left the baseline, whereas the logarithmic plot point for the tenth generation (3.01) is halfway up the graph. 25 Bacterial Growth Curve Bacterial populations follow a sequential series of growth phases: the lag, log, stationary, and death phases. Knowledge of the bacterial growth curve is critical to understanding population dynamics and population control in the course of infectious diseases, in food preservation and spoilage, and as well as in industrial microbiology processes, such as ethanol production. 26 MTN Cabasan 13  Direct Measurement of Microbial Growth: Serial dilutions and plate counts  In serial dilutions, the original inoculum is diluted in a series of dilution tubes.  Each succeeding dilution tube will have only one-tenth the number of microbial cells as the preceding tube.  Samples of the dilution are used to inoculate Petri plates, on which colonies grow and can be counted.  This count is then used to estimate the number of bacteria in the original sample. 27 Plate Counts measures the number of viable cells Disadvantage: it takes some time, usually 24 hours or more, for visible colonies to form Plate counts assume that each live bacterium grows and divides to produce a single colony. (Note: bacteria frequently grow linked in chains or as clumps_ Colony-forming units (CFU) 28 MTN Cabasan 14  Plate Counts U.S. Food and Drug Administration convention is to count only plates with 25 to 250 colonies, many microbiologists prefer plates with 30 to 300 colonies. To ensure that some colony counts will be within this range, the original inoculum is diluted several times = serial dilution 29 Serial Dilutions  Example: a milk sample has 10,000 bacteria per milliliter.  If 1 ml of this sample plated out = 10,000 colonies formed in the Petri plate Countable?  If 1 ml of this sample were transferred to a tube containing 9 ml of sterile water, each ml of fluid in this tube = 1000 bacteria.  If 1 ml of this sample were inoculated into a Petri plate - too many potential colonies to count on a plate.  Do another serial dilution.  1 ml containing 1000 bacteria would be transferred to a second tube of 9 ml of water.  Each ml of this tube - contain only 100 bacteria  if 1 ml of the contents of this tube were plated out = 100 colonies would be formed Countable? 30 MTN Cabasan 15  Pour Plates and Spread Plates  Methods of preparing plates for plate counts. (a) The pour plate method. (b) The spread plate method.  Q In what instances would the pour plate method be more appropriate than the spread plate method? 31 Filtration  at least 100 ml of water are passed through a thin membrane filter ; pores with too small to allow bacteria to pass.  bacteria are filtered out and retained on the surface of the filter.  This filter is then transferred to a Petri dish containing nutrient medium (colonies arise from the bacteria on the filter’s surface).  applied frequently to detect and enumerate coliform bacteria (indicators of fecal contamination of food or water 32 MTN Cabasan 16  Most Probable Number (MPN) Method  statistical estimating technique: the greater the number of bacteria in a sample, the more dilution is needed to reduce the density to the point at which no bacteria are left to grow in the tubes in a dilution series.  most useful when the microbes being counted will not grow on solid media (chemoautotrophic nitrifying bacteria).  It is also useful when the growth of bacteria in a liquid differential medium is used to identify the microbes (coliform bacteria, which selectively ferment lactose to acid, in water testing).  The MPN is only a statement that there is a 95% chance that the bacterial population falls within a certain range and that the MPN is statistically the most probable number. 33 34 MTN Cabasan 17  Direct Microscopic Count  measured volume of a bacterial suspension placed within a defined area on a microscope slide.  often used to count the number of bacteria in milk.  A 0.01-ml sample is spread over a marked square centimeter of slide, stain is added so that the bacteria can be seen, and the sample is viewed under the oil immersion objective lens.  Once the number of bacteria has been counted, the average number of bacteria per viewing field can be calculated.  From these data, the number of bacteria in the square centimeter over which the sample was spread can also be calculated.  Because this area on the slide contained 0.01 ml of sample, the number of bacteria in each ml of the suspension is the number of bacteria in the sample times 100. 35 electronic cell counters or Coulter counters, - automatically count the number of cells in a measured volume of liquid. 36 MTN Cabasan 18  Estimating Bacterial Numbers by Indirect Methods: Turbidity  Measured in a spectrophotometer (or colorimeter).  Ispectrophotometer - a beam of light is transmitted through a bacterial suspension to a light-sensitive detector.  As bacterial numbers increase, less light will reach the detector.  The change of light will register on the instrument’s scale as the percentage of transmission (%T).  instrument’s scale - logarithmic expression called the absorbance (or optical density, or OD).  The absorbance is used to plot bacterial growth.  estimations of bacterial numbers obtained by measuring turbidity 37  Turbidity estimation of bacterial numbers.  The amount of light striking the light- sensitive detector on the spectrophotometer is inversely proportional to the number of bacteria under standardized conditions.  The less light transmitted, the more bacteria in the sample.  The turbidity of the sample could be reported as either 20% transmittance or 0.7 absorbance. Readings in absorbance are a logarithmic function and are sometimes useful in plotting data. 38 MTN Cabasan 19  Estimating Bacterial Numbers by Indirect Methods: Metabolic Activity measure a population’s metabolic activity. method assumes that the amount of a certain metabolic product, such as acid, CO2, ATP, or DNA, is in direct proportion to the number of bacteria present. Example: microbiological assay (in which acid production is used to determine amounts of vitamins) 39 Estimating Bacterial Numbers by Indirect Methods: Dry Weight For filamentous bacteria and molds the fungus is removed from the growth medium, filtered to remove extraneous material, and dried in a desiccator., then weighed. For bacteria, the same basic procedure is followed. 40 MTN Cabasan 20