Ch. 5-6 Microbial Growth PDF

Ch.6 - Microbial Growth strawberries infected with “grey mold” fungus  Ch.6 – Microbial Growth o bacterial growth – exponential o bacterial division and population growth curve o physical requirements for growth o chemical requirements for growth o biofilms o selective vs. differential...

Ch.6 - Microbial Growth strawberries infected with “grey mold” fungus  Ch.6 – Microbial Growth o bacterial growth – exponential o bacterial division and population growth curve o physical requirements for growth o chemical requirements for growth o biofilms o selective vs. differential media o pure cultures – isolating and preserving o direct measurements of growth o indirect measurements of growth  Learning Objectives: o Explain how nutrition and the environment can impact microbial growth and differentiation. o Discuss microbial classifications based on nutritional needs and environmental limits. o Describe how understanding microbial growth helps identify disease-causing pathogens. o Discuss biofilms and their importance to infectious diseases. What do bacteria do, if the conditions are right, better than anything else? o divide and grow: bacterial growth is exponential Linear vs. exponential growth: Linear growth: constant growth rate regardless of prior size Exponential growth: growth rate changes in proportion to the prior size population size doubles with each round of division Doubling down: exponential growth bacteria grow to certain size then divide by Binary Fission linear growth exponential growth  How fast can bacteria ? Exponential growth = very, very, very fast!!! Generation time = doubling time time it takes for bacterial population to Theoretical: How many bacteria in 2 days? double Start with 1 E.coli cell, generation time = 20 minutes Most bacteria have generation times of Grow for 48 hours, unrestricted = 144 generations 1-2 hrs. Exponential growth = (starting #) * 2n (n=# generations) Generation times can range from 20 Theoretical population = 1 * 2144 = 2.23 x 1043 cells!!! min. to over 24 hrs. Escherichia coli - 20 min. Mycobacterium Mycobacterium tuberculosis: 24 hrs leprosae: 3 wks  Generation time - Time required for a cell to divide 20 minutes to 24 hours Binary fission - doubles the number of cells each generation total number of cells = 2number of generations Growth curves are represented logarithmically logarithmic conversion for bacterial growth curve graphically analyzing bacterial growth logarithmic representation of standard growth curve  Ch.6 – Microbial Growth o bacterial growth – exponential o bacterial division and population growth curve o physical requirements for growth o chemical requirements for growth o biofilms o selective vs. differential media o pure cultures – isolating and preserving o direct measurements of growth o indirect measurements of growth  The Growth of Bacterial Cultures Colony size due to bacterial division: Increase in number of cells, not cell size o Binary fission - means “division in half” Parent cell splits into two halves , producing two new cells o Budding - Form of asexual reproduction new organism develops from an outgrowth or bud remaining attached as it grows separating from the parent organism when mature Binary Fission: “division in half” Binary Fission: “division in half” Budding Yeast Cells  Prokaryotic Cell Division Binary Fission: Asexual reproduction of bacteria and archaea 1 circular chromosome attached to plasma membrane – no nucleus New chromosome attaches to DIFFERENT part of plasma membrane Grow new cell wall and plasma membrane down center of cell  The Population Growth Curve growth curve - in laboratory studies, populations typically display a predictable pattern over time standard laboratory growth curve Stages in a normal growth curve: 1. Lag phase – “flat” period of adjustment, enlargement; little growth 2. Exponential growth phase – a period of maximum growth will continue as long as cells have adequate nutrients and a favorable environment 3. Stationary phase – rate of cell growth equals rate of cell death caused by depleted nutrients, O2, T excretion of organic acids and pollutants 10 4. Death phase – as limiting factors intensify cell death will start occur  Ch.6 – Microbial Growth o bacterial growth – exponential o bacterial division and population growth curve o physical requirement for growth o chemical requirements for growth o biofilms o selective and differential media o pure cultures – isolating and preserving o direct measurements of growth o indirect measurements of growth  o Bacteria can be found nearly everywhere Niche: totality of adaptations organisms make to their habitat Thermal Geyser, Yellowstone deep sea thermal vents Requirements for Growth - physical and chemical: Environmental factors: affect the function of metabolic enzymes, and enzymes can only function in a particular range of conditions Chemical requirements: Physical requirements: Carbon, Nitrogen, sulfur, and phosphorous Temperature Trace elements pH Oxygen Osmotic pressure Organic growth factors  Physical Requirements: Temperature temperature adaptation classifications: o Psychrophiles: cold-loving grow between -20C and 10C cause food spoilage o Mesophiles: moderate-temperature-loving optimum temp. between 20C and 40C o Thermophiles: heat-loving optimum growth temperature >45C prokaryotic temperature classifications found in hot springs and organic compost Three growth rate temperatures: o Hyperthermophiles: o Minimum growth temperature optimum growth temperature >70C! o Optimum growth temperature almost exclusively Archaea o Maximum growth temperature extreme thermophiles around a hydrothermal vent  Temperature: Bacterial Growth and Food Spoilage Temperature vs. Growth: -30⁰C to 0⁰C - no significant growth 0⁰C to 10⁰C - refrigerated temp: slow growth / few pathogens 10⁰C to 50⁰C - rapid growth / toxins 50⁰C -60⁰C - very slow growth >60⁰C - destroys most microbes Bacillus cereus temperature vs. growth rate scale temperature growth rate in rice  Physical Requirements: pH - Potential of Hydrogen pH scale pH adaptation classifications: o Acidophiles grow in acidic environments (pH down to 1) Low pH: High H+ / Low H+ “absorber” (OH-) oAlkaliphiles grow in basic environments (pH up to 11) High pH: Low H+ / High H+ “absorber” (OH-) Standard pH growth ranges: Most bacteria grow between pH 6.5 and 7.5 Molds and yeasts grow between pH 5 and 6 Morning Glory Pool – Yellowstone pH: 5.5; temp: 72⁰C  Physical Requirements: Osmotic Pressure difference in solute concentration inside vs. outside cell o Hypotonic – higher INSIDE cell H2O enters cell o Isotonic – same inside/outside no net H2O movement either direction o Hypertonic – higher OUTSIDE cell H2O leaves cell affect of different osmotic conditions on a cell Osmosis Rule: H2O ALWAYS follows high solute!!!! osmotic adaptation classifications: Halophiles – live in salty environments o extreme (obligate) halophiles require high salt (high osmotic pressure) halophiles on Nevada Salt Flats o halotolerant (facultative halophiles) - tolerate high salt  Extreme halophiles – obligate halophile Nevada Salt Flats  Osmotic Pressure: difference in solute concentration inside vs. outside cell hypertonic environments: higher in solutes than inside the cell plasmolysis due to loss of water bacterial plasmolysis: H2O leaves cell – growth inhibited  Barometric adaptation classifications: o Barophiles – can survive under extreme pressure will rupture if exposed to normal atmospheric pressure Organisms at the bottom of the Mariana Trench live under 1000x normal atmospheric pressure and will burst if exposed to our environment Mariana Trench – deepest part of worlds ocean  Ch.6 – Microbial Growth o bacterial growth – exponential o bacterial division and population growth curve o physical requirement for growth o chemical requirements for growth o biofilms o selective and differential media o pure cultures – isolating and preserving o direct measurements of growth o indirect measurements of growth  Chemical Requirements: what bacteria need to grow Carbon: basic building block of life o structural backbone of organic molecules amino acid o heterotrophs - use organic molecules as energy sugar, protein, lipids o autotrophs - use CO2 to make own organic molecules Nitrogen: component of amino acids, DNA, RNA and ATP most bacteria decompose protein material for their nitrogen source some bacteria use NH4+ or NO3– from organic material a few bacteria use N2 in nitrogen fixation nucleotides  Chemical Requirements: Sulfur: used in certain amino acids most bacteria decompose protein for their sulfur source some bacteria use SO42– or H2S DNA Phosphorus: sulfur containing amino acids o used in DNA, RNA, and ATP DNA/RNA sugar-phosphate backbone o found in membranes phospholipid bilayer ATP o Trace elements: zinc, copper, iron, etc. o Organic growth factors: vitamins! Most bacteria make their own Some bacteria depend on finding vitamins in the environment o Water (H2O): Source of O and H, + so much more! O2 Oxygen Requirements: oxygen adaptation classifications oxygen: only some bacteria need it……and some can’t stand it! o obligate aerobes — require oxygen o facultative anaerobes — grow via aerobic respiration, fermentation or anaerobic respiration o obligate anaerobes — unable to use oxygen and are harmed by it Bacterial oxygen requirement growth patterns o aerotolerant anaerobes — tolerate but cannot use oxygen o microaerophiles —require oxygen concentration lower than air  Why is oxygen toxic? o Superoxide free radicals = O2- highly reactive molecule that forms from O2 Superoxide dismutase (SOD) cleans it up: hydrogen peroxide – also toxic! SOD O2- + O2- + 2H+ H2O2 + O2 SOD is found in all organisms that can grow in the presence of oxygen but hydrogen peroxide is also toxic... How to deal with H2O2: Catalase: 2 H2O2 2 H2O + O2 produces O2 gas no gas produced Peroxidase: H2O2 + 2 H+ 2 H2O  Ch.6 – Microbial Growth o bacterial growth – exponential o bacterial division and population growth curve o physical requirement for growth o chemical requirements for growth o biofilms o selective and differential media o pure cultures – isolating and preserving o direct measurements of growth o indirect measurements of growth  Biofilms aggregate of bacteria in which cells are embedded within a self-produced matrix of Extracellular Polymeric Substance (EPS) and adhere to each other and/or to a surface SEM of biofilm in hydrogel medical tubing (slime biofilm) o form slime or hydrogels that adhere to surfaces bacteria communicate cell-to-cell via quorum sensing quorum sensing: a system of stimuli and response correlated to population density o share nutrients o shelter bacteria from harmful environmental factors  Biofilms o found in digestive system and sewage treatment systems; can clog pipes o 1000x more resistant to microbicides o involved in ~70% of medical infections catheters, heart valves, contact lenses, dental caries SEM of biofilm on medical mesh biofilm in old sewer pipe  Ch.6 – Metabolism o bacterial growth – exponential o bacterial division and population growth curve o physical requirement for growth o chemical requirements for growth o biofilms o selective and differential media o pure cultures – isolating and preserving o direct measurements of growth o indirect measurements of growth  Bacterial Growth Media: Terms o Sterile: no living microbes o Inoculum: introduction of microbes into a medium o Culture: microbes growing in or on a culture medium o Culture Media: Nutrients prepared for microbial growth o Agar: complex polysaccharide from Red Algae used as a solidifying agent for culture media in Petri plates, slants, and deeps generally not metabolized by microbes liquefies at 100C solidifies o Defined media: exact chemical composition is known each component needed for growth added separately fastidious organisms are those that require many growth factors provided in chemically defined media o Complex media: chemical composition varies batch to batch ground-up extracts and digests of yeasts, bacteria, meat, or plants; o both defined and complex media can be nutrient broth or nutrient agar  Selective and Differential media can be complex or defined media Selective media: media that can support the growth of some microbes; but not others suppress unwanted microbes and encourage growth of desired microbes contain inhibitors to suppress growth EMB plate “selects” for certain “broad” types of bacteria (eosin methylene blue) Selective Media Types: Antibiotics (+/- resistance) Chemicals Nutrients (+/- nutritional ability) Salt (+/- halophiles) Mannitol Salt Agar (MSA) plate selective media pH (+/- acidiphiles or alkaliphiles) “selects” for gm+ selective media “selects” for gm- Oxygen (+/- obligate aerobes/anaerobes) salts inhibit gm- dyes inhibit gm+  Differential media: media that causes certain microbes to “stand out” (color) allow distinguishing of colonies of different microbes on the same plate breakdown of a substance on plate; production of a substance (dye etc.) or pH change allows “differentiation” between particular species of bacteria differential pH: pH test strips MSA Plate – differentiates for mannitol fermentation color indicates pH range differential pH: yellow color indicates low pH  Differential medias: EMB (eosin methylene blue) RBC hemolysis (lysis) Listeria selective agar o differential media: o differential media: lactose fermentation (pink) cephalosporin antibiotic Blood Agar Plate: o Differential media: Lactose sugar fermentation RBC hemolysis alpha, beta, gamma Antibiotic sensitivity “going for” Fe2+ in hemoglobin of RBC Listeria  Anaerobic Growth Media and Methods Reducing media: used for the cultivation of anaerobic bacteria contain chemicals (sodium thioglycolate) that combine O2 to deplete it heated to drive off O2 anaerobic laboratory growth chamber Brewers Jar for anaerobic growth capnophiles: microbes that require high CO2 conditions CO2 packet / candle jar  Enrichment Media: Encourages growth of desired microbe by increasing very small numbers of a desired organism to detectable levels Usually a liquid Culture Media Summary:  Take home message: Bacteria can grow exponentially, but are often limited by their surroundings Bacteria in general can grow almost anywhere… but particular bacteria may have very specific growth conditions We can recreate growth conditions for many types of bacteria with different kinds of media gut microbiota  Ch.6 – Microbial Growth o bacterial growth – exponential o bacterial division and population growth curve o physical requirement for growth o chemical requirements for growth o biofilms o selective and differential media o pure cultures – isolating and preserving o direct measurements of growth o indirect measurements of growth  Obtaining Pure Cultures isolate single colonies by spreading culture on agar plate o Pure culture - contains only one species or strain o Colony - population of cells arising from a single cell or from a group of attached cells colony-forming unit (CFU) – cell or group of cells that a colony originates from o Mixed Colony - more than 1 species within colony o Pure Colony - colony is single species Streak plate method used to isolate pure cultures  Two ways to obtain pure cultures: 1. Streaking for Isolation o streaking for isolation CRITICAL for isolating bacterial species! 2. Serial Dilution / Spread Plate Technique o dilution process for isolating pure cultures and determining concentration of bacteria in a culture Streaking for Isolation: Serial dilution / Spread plate technique  Preserving Bacterial Cultures o Deep-freezing: –50 to –95C o Lyophilization (freeze-drying): frozen (–54 to –72C) and dehydrated in a vacuum water is removed after culture is frozen and placed under a vacuum allowing ice to change directly from solid to vapor without passing through a liquid phase Lyophilization: freeze-drying Deep-freezing  Ch.6 – Microbial Growth o bacterial growth – exponential o bacterial division and population growth curve o physical requirement for growth o chemical requirements for growth o biofilms o selective and differential media o pure cultures – isolating and preserving o direct measurements of growth o indirect measurements of growth  Direct Measurements of Microbial Growth – counting microbial cells spread plate method pour plate method 1. Plate count 2. Filtration 3. Most probable number (MPN) method 4. Direct microscopic count 1. Plate count Count colonies on plates that have 30 to 300 colonies (CFUs) To ensure the right number of colonies, the original inoculum must be diluted via serial dilution Two bacterial dilution techniques: 1. spread plate method – bacteria diluted in liquid broth and spread directly on surface of agar plate 2. pour plate method - bacteria diluted into a soft agar and poured onto agar plate.  2. Filtration Solution passed through a filter that collects bacteria Filter is transferred to a Petri dish and grows as colonies on the surface bacteria trapped by filter filter with bacteria placed on endo agar  3. Most Probable Number (MPN) Method Multiple tube test: Inoculate tubes with different sample volumes Count positive tubes Compare with a statistical table Most Probable Number (MPN) technique MPN result reference table  4. Direct Microscopic Count Volume of a bacterial suspension placed on a slide with grid Average number of bacteria per viewing field is calculated Uses a special Petroff-Hausser cell counter gridded cell counting slide number of cells counted o number of bacteria/ml = volume of area counted  Ch.6 – Microbial Growth o bacterial growth – exponential o bacterial division and population growth curve o physical requirement for growth o chemical requirements for growth o biofilms o selective and differential media o pure cultures – isolating and preserving o direct measurements of growth o indirect measurements of growth  Estimating Bacterial Numbers by Indirect Methods Turbidity measurement of cloudiness with a spectrophotometer Metabolic activity amount of metabolic product (color change) is proportional to the number of bacteria Dry weight bacteria are filtered, dried, and weighed used for filamentous organisms transmission or absorbance Spectrophotometer: measures turbidity The optimum pH for MOST bacteria is near… A. 1. B. 5. C. 7. D. 9. A psychrophile has an optimal growth temperature of about… A. 15°C. B. 25°C. C. 37°C. D. 100°C. Organisms that require high salt concentrations for growth are called… A. thermophiles. B. obligate halophiles. C. acidophiles. D. anaerobes.  What is/are the energy source(s) utilized by primary producers in the hydrothermal vents on the ocean floor? A. light B. hydrogen sulfide C. organic molecules D. carbon dioxide Addition of salts preserves foods because they… A. lower pH. B. increase osmotic pressure. C. create an isotonic environment. D. lower osmotic pressure.  Ch. 6 Learning Objectives After this lecture, you should be able to… Differentiate between linear and exponential growth Explain generation time; theoretical growth calculation Bacterial reproduction: budding and binary fission; bacterial growth cycle; population growth curve Physical growth requirements; o Classify microbes into five groups on the basis of preferred temperature range. o Classify microbes by pH and osmotic and atmospheric pressure conditions  Identify how and why the pH of culture media is controlled.  Explain the importance of osmotic pressure to microbial growth. Chemical o Provide a name and use the four main elements needed in large amounts for microbial growth. o Explain how microbes are classified on the basis of oxygen requirements. o Identify ways in which aerobes avoid damage by toxic forms of oxygen. o Describe biofilms and their potential for causing infection and their use of quorum sensing Learning objectives: 1. Linear vs exponential growth: linear growth - occurs when the population increases by the same amount over each time interval, such as in cells that divide at a set rate regardless of the population size. This growth is rare in nature as it suggests a constant resource availability which is not typical. Exponential growth - characterized by the population doubling at regular intervals, each new generation is proportional to the current population resulting in a rapid increase. This pattern is common in bacteria and other organisms under ideal conditions with abundant resources. 2. Generation time and theoretical growth calculation: generation time: the time takes for a bacterial population to double in size. It varies among species and under different environmental conditions. Theoretical growth calculation: often modeled by the equation N(t) = N0 x 2(t/G), where N(t) is the number of cells at time t, N0 is the initial number of cells, G is the generation time, and t is the time elapsed. This formula assumes exponential growth. This formula assumes exponential growth. 3. Bacterial reproduction and growth cycle: budding - a form of asexual reproduction seen in some bacteria and yeast where a new cell grows out of the body of a parent. Binary fission - most common bacterial reproduction method where the cell divides into two genetically identical cells. Growth cycle - includes the lag phase (adjustment period), log phase (exponential growth), stationary phase (growth ceases as resources are consumed), and death phase (decline in viable cells). 4. Physical growth requirement: temperature classification: - psychrophiles: optimal growth below 15 deg C - mesophiles: optimal growth between 20 deg C and 45 deg C - thermphiles: optimal growth between 45 deg C and 80 deg C - hyperthermophiles: optimal growth above 80 deg C pH and osmotic pressure: - pH: microbes are classified as acidophiles, neutrophils, or alkaliphiles based on their optimal pH range. - osmotic pressure: critical for maintaining cell integrity and function. Microbes adapt to various osmotic conditions through mechanisms like compatible solute accumulation. 5. Chemical requirements: four main elements - carbon, nitrogen, phosphorus, and sulfur: essential for constructing cellular components like protein, nucleic acids, and lipids. oxygen requirements: - obligate aerobes: require oxygen to survive. - obligate anaerobes: oxygen is toxic; survive in oxygen-free environments. - facultative anaerobes: can live with or without oxygen. - microaerophiles: require oxygen but at lower concentrations than are present in the air. - aerotolerant anaerobes: indifferent to oxygen, do not use it but are not harmed by it. 6. Aerobic protection and biofilms: protection from oxygen toxicity - aerobes produce enzymes like superoxide dismutase and catalase to neutralize reactive oxygen species (ROS) such as superoxide ions and hydrogen peroxide. biofilms and quorum sensing: - biofilms: structured communities of microorganisms attached to surfaces and protected by an extracellular matrix. They are difficult to eradicate and can lead to persistent infections. - quorums sensing: cell to cell communication mechanism used by bacteria to regulate gene expression based on the density of their population. This behavior is crucial in biofilm formulation and virulence factor production, affecting infection dynamics and treatment strategies.  Ch. 6 Learning Objectives Be familiar with the major terms related to microbial growth. Define and distinguish chemically defined and complex media; provide examples of each. Define and distinguish selective and differential media; provide examples of each. Describe what is used to grow anaerobic bacteria; what is a capnophile? Justify the use of each of the following: anaerobic techniques, selective and differential media, enrichment medium. Define colony, pure culture, mixed culture and streak for isolation. Describe how pure cultures can be isolated by using the streak plate method. Describe how pure cultures can be isolated with the dilution/spread plate method; describe an advantage the spread plate method has over streaking for isolation. Explain how microorganisms are preserved by deep-freezing and lyophilization (freeze-drying). Differentiate direct and indirect methods of measuring cell growth. Explain four direct methods of measuring cell growth. Explain three indirect methods of measuring cell growth.  Bacterial Growth Media: Terms o Sterile: no living microbes o Inoculum: introduction of microbes into a medium o Culture: microbes growing in or on a culture medium o Culture Media: Nutrients prepared for microbial growth o Agar: complex polysaccharide from Red Algae used as a solidifying agent for culture media in Petri plates, slants, and deeps generally not metabolized by microbes liquefies at 100C solidifies o Defined media: exact chemical composition is known each component needed for growth added separately fastidious organisms are those that require many growth factors provided in chemically defined media o Complex media: chemical composition varies batch to batch ground-up extracts and digests of yeasts, bacteria, meat, or plants; o both defined and complex media can be nutrient broth or nutrient agar Learning objectives 1. Chemically defined vs complex media: chemically defined media: these media have a known specific chemical composition. Each ingredient and its concentration are known, which allows for precise control and reproducibility in experiments. Example: glucose salt broth, where exact amounts of glucose, salts, and other components are added explicitly. Complex media: these media contain some components that are not chemically definable. Such media often include extracts and digests of yeasts, meat, or plants and provide a rich mixture of nutrients for microbes that have nutrients for microbes that have complex nutritional needs. Example: nutrient broth or tropic soy broth, which contain peptones, vitamins, and minerals in undefined proportions. 2. Selective and differential media: selective media: designed to suppress the growth of unwanted bacteria and encourage the growth of the desired organisms. Example: MacConkey agar which inhibits the growth of Gram-positive bacteria while allowing gram- negative bacteria to grow. Differential media: allow multiple types of organisms to grow but are designed to display visible differences among those microorganisms. Example: blood agar, which differentiates bacteria based on their hemolytic properties. 3. Growth of anaerobic bacteria and capnophiles: anaerobic bacteria: grown in environments devoid of of oxygen. Techniques include using an anaerobic jar or chamber that contains chemical packets that remove oxygen or by heating to drive off oxygen. Capnophile: microorganisms that thrive in the pretense of high concentrations of CO2. Often grown in candle jars or CO2 incubators that increase the CO2 concentration significantly. 4. Justification for techniques: anaerobic techniques: essential for cultivating anaerobes which can be killed or inhibited by oxygen, important for medical diagnostics. Selective and differential media: critical for isolating and differentiating microorganisms in a mixed culture, which aids in identification and further analysis. Enrichment medium: used to favor the growth of a particular microorganism over others, enriching a sample for specific bacteria that might be present in small numbers. 5. Colony, pure culture, mixed culture, streak for isolation: colony: a visible mass of microorganisms all originating from a single mother cell genetically identical.  pure culture: contains only one species or strain of an organism growing in the same space. Mixed culture: contains more than one type of organism growing in the same space. Streak for isolation a method used to isolate a single species from a mixed culture by streaking it across the surface of a plated agar medium. 6. Isolation techniques: streak plate method: a loop is used to streak a microbial sample over the agar surface to separate individual cells to create isolated colonies. Dilution/spread plate method: serial dilutions of a sample are spread over the surface of the agar to reduce the density of organisms, facilitating isolated colony growth. - advantage over streaking: more quantitative as it allows for the estimation of the concentration of organisms in a sample. 7. Preservation methods deep freezing: microorganisms are stored at temperatures between -50 deg C and -95 deg C which preserves them for many years. Lyophilization (freeze drying): microorganisms are quickly frozen and then the water is removed by sublimation in a vacuum allowing them to be stored at room temperature for long periods. 8. Direct and indirect methods of measuring cell growth: direct method: involve counting the cells themselves. Examples: plate counts, filtration followed by plate counts, direct microscopic count. Indirect method: measures the growth by indirect effects like turbidity. Examples: turbidometric measurements (spectrophotometry), metabolic activity, dry weight measurement. 9. Four direct methods: - plate counts: count colonies on plates after incubation. - membrane filtration: cells filtered, rinsed, and grown on a membrane in a culture plate. - direct microscopic counts: using a counting chamber to count cells under a microscope - electronic counters: such as a coulter counter which measures changes in electrical resistance caused by a particle passing through a small opening. - metabolic activity: assay of a metabolic product to estimate microbial growth.