BIO 3054 Microbial Growth PDF

BIO 3054 Chapter 4 Microbial Growth Spring 2025 University of Oklahoma Dr. Jyotisna Saxena Binary Fission (1 of 2) Growth: increase in the number of cells, not size Prokaryotic cells divide by binary fission - cell division following enlargement of a cell to twice origin...

BIO 3054 Chapter 4 Microbial Growth Spring 2025 University of Oklahoma Dr. Jyotisna Saxena Binary Fission (1 of 2) Growth: increase in the number of cells, not size Prokaryotic cells divide by binary fission - cell division following enlargement of a cell to twice original size - septum: partition between dividing cells, pinches off between two daughter cells Cell growth is exponential - Population doubles with each cell division - One cell divides into two, two into four etc. Binary Fission (2 of 2) Generation time: time it takes for microbial cells to double in number a.k.a. doubling time Varies among species and also depends on growth conditions example: Escherichia coli = 20 minutes Mycobacterium = days Total # cells = 2 # of generations During cell division, each daughter cell receives a chromosome and sufficient copies of all other cell constituents to exist as an independent cell.  Alternatives to Binary Fission Budding cell division: Unequal cell growth forming different daughter cells. Hyphae: long, thin filaments of actinomycetes (Gram- positive filamentous bacteria, e.g., Streptomyces) – Hyphal growth occurs only at filament tip – Cell growth is not linked directly to division (no septa) Mycelia: weaved hyphae Arthrospores: survival structures formed from mycelia ▪ Multiple fission: hyphal filament forms many septa simultaneously  Microbial Growth System Batch culture: a closed-system microbial culture of fixed volume - A few bacteria are inoculated into a liquid growth medium - The population of bacteria is counted at intervals - The environment is constantly changing due to nutrient consumption and waste production Continuous culture (Chemostat): an open-system microbial culture - A known volume of fresh medium is added at a constant rate, while an equal vol. of spent culture medium is removed at the same rate - Once equilibrium is reached, the growth vessel volume, cell #, nutrients/waste status remain constant - This state is k/n as Steady State  Bacterial Growth Curve (1 of 5) A typical growth curve for population of cells grown in a closed system (batch culture) is characterized by four distinct phases. 1. Lag phase 2. Exponential or log phase 3. Stationary phase 4. Death phase  Bacterial Growth Curve (2 of 5) 1. Lag phase: interval between inoculation of a culture and beginning of growth – # of cells does not increase – Cells prepare for growth Intense metabolic activity, “tooling up” for rapid growth ▪ new conditions require altering metabolic state ▪ time needed for biosynthesis of new enzymes and to produce required metabolites before growth can begin  Bacterial Growth Curve (3 of 5) 2. Log phase – Period of exponential growth Doubling of population with each generation Growth at a constant rate Cells are metabolically identical Produce “primary metabolites” Compounds required for growth continues until conditions can no longer sustain growth – Cells enter Late log phase Synthesize “secondary metabolites” – Used to enhance survival – Can make antibiotics in this phase  Bacterial Growth Curve (4 of 5) 3. Stationary phase Overall population remains relatively stable Diminished nutrients, accumulating wastes Cell growth = cell death – Dying cell supply metabolites for replicating cells Secondary metabolites synthesized 4. Death phase Total number of viable cells decrease at constant rate Death is exponential (logarithmic) Much slower rate than growth  Bacterial Growth Curve (5 of 5) Phase of prolonged decline – Once nearly 99% of all cells dead, remaining cells enter prolonged decline – Marked by very gradual decrease in viable population – Phase may last months or years – Most fit cells survive Each new cell more fit than previous  Bacterial Growth in Nature Conditions in nature have a profound effect on microbial growth – Cells sense changing environment Synthesize compounds useful for growth S. aureus growing on the Inside of a patient’s medical Cells produce multicellular catheter associations to increase survivability – Example » Biofilms » Slime layers Biofilm layer  Biofilm Growth (1 of 2) Planktonic Biofilm formation begins when growth planktonic bacteria attach to surfaces Sessile - Other bacteria attach and grow on initial growth layer - Different properties than planktonic cells - Have characteristic architecture - Contains open channels for movement of nutrients and waste ▪ Microbial communities o Share nutrients o Shelter bacteria from harmful environmental factors ▪ Bacteria communicate cell-to-cell via quorum sensing - Bacteria secrete an inducer (signaling chemical) to attract other bacterial cells Biofilm Growth (2 of 2) ▪ Medical and industrial applications Cells within biofilms can cause disease o Biofilms are involved in 70% of infections o Catheters, heart valves, contact lenses o Treatment becomes difficult o Architecture resists immune response and antimicrobials o 1000x resistant to microbicides o Streptococcus mutans: cavities & gum disease Found in sewage treatment systems; fuel tanks and on ship hulls can clog/corrode pipes Bioremediation is beneficial use of biofilm Microbial mats: multilayered sheets with different organisms in each layer (e.g., hot springs, intertidal regions) Pseudomonas aeruginosa Biofilm Development  Laboratory Conditions are Different From Those Found in Nature Prokaryotes in nature live in mixed communities Many interactions are cooperative – Waste of one organism nutrient for another Some cells compete for nutrients – Synthesize toxic substance to inhibit growth of competitors In a laboratory setting it is very hard to reproduce many of these close associations and biofilms that are found in nature Cells in a laboratory are grown in closed or batch system – No new input of nutrient and no release of waste Population of cells increase in predictable fashion following the growth curve  Environmental Factors on Microbial Growth As a group, prokaryotes inhabit nearly all environments – Some live in “comfortable” habitats – Some live in harsh environments Most of these are termed extremophiles and belong to domain Archaea Major environmental conditions that influence growth 93 °C (200 °F) to 174 °C (345 °F). – Temperature – pH – Water availability – Oxygen/other gases  The Requirements for Growth Physical requirements – Temperature – pH – Osmotic pressure – Oxygen Chemical requirements – Carbon – Nitrogen, sulfur, and phosphorous – Trace elements – Oxygen – Organic growth factors  I. Physical Requirements: Temperature Cardinal Temperatures – Minimum, optimum, and maximum temperatures at which an organism grows – Characteristic of any given microorganism – Differ dramatically between species – At optimum, all/most cellular components are functioning at maximum rate Physiological changes at the cardinal temperatures Temperature Classes of Microorganisms Each species has well- defined temperature range Within range lies optimum growth temperature Prokaryotes divided into 5 categories Copyright © 2021, 2018, 2015 Pearson Education, Inc. All Rights Reserved 1. Psychrophile (cold loving) Temperature -5°C to 15°C Requirements Found in Arctic and Antarctic regions 2. Psychrotroph 0°C to 30°C Important in food spoilage in refrigerator 3. Mesophile (moderate temperature loving) 10°C to 45°C More common Disease causing Extremophiles: Organisms Normal microbiota that grow under very hot or very 4. Thermophiles cold conditions 45°C to 70°C Common in hot springs 5. Hyperthermophiles (extreme thermophiles) 70°C to 110°C (even upto 122°C) Usually members of Archaea Found in hydrothermal vents & organic compost piles Microbial Life in the Cold (1 of 2) Cold Environments – Much of Earth’s surface is cold – Oceans 5ºC – Arctic and Antarctic are permanently frozen or rarely unfrozen Constantly v s seasonally cold ersu  Microbial Life in the Cold (2 of 2) Psychrophilic and Psychrotolerant Microorganisms Psychrophiles: optimal growth temperature  15C, o maximum < 20°C, o minimum  0C ▪ Constantly cold environments ▪ Found in polar regions, permanent snowfields, glaciers Psychrotolerant: can grow at 0°C but have optima of 20°C to 40°C Snow Algae ▪ More widely distributed in nature than psychrophiles ▪ Isolated from soils and water in temperate climates and food at 4°C  Adaptations to Life @ Cold Temperatures production of enzymes that function optimally in the cold ▪ more  -helices than  -sheets → greater flexibility for catalysis at cold temperatures ▪ more polar and fewer hydrophobic amino acids ▪ fewer weak bonds (e.g., hydrogen and ionic bonds) Cytoplasmic membranes function at low temperatures. ▪ higher unsaturated and shorter-chain fatty acid content ▪ some polyunsaturated fatty acids, which remain flexible at very low temperatures – Cold shock proteins (chaperones) – Cryoprotectants (e.g., antifreeze proteins, certain solutes) prevent formation of ice crystals. – Exopolysaccharide cell surface slime  Microbial Life at High Temperatures Hyperthermophiles ▪ inhabit boiling hot springs, seafloor hydrothermal vents, that can experience temperatures in excess of 100 °C ▪ Optimum > 80°C ▪ chemoorganotrophic and chemolithotrophic species present ▪ generation times (g) as low as one hour common ▪ high prokaryotic diversity (both Archaea and Bacteria represented) ▪ Methanopyrus: most thermophilic, up to Growth of Hyperthermophiles in 122°C Boiling Water Thermophiles ▪ moderately or intermittently hot environments (45°C to 80°C Above 65°C only prokaryotic life forms thrive, but extensive diversity is present Group Upper temperature limits (C) degrees centigrade Blank Macroorganisms Blank Animals Fish and other aquatic vertebrates 38 Insects 45–50 Ostracods (crustaceans) 49–50 Blank Plants Vascular plants 45 (60 for one species) Mosses 50 Blank Microorganisms Blank Eukaryotic microorganisms Protozoa 56 Algae 55–60 Fungi 60–62 Blank Bacteria and Archaea Blank Bacteria Cyanobacteria 73 Anoxygenic phototrophs 70–73 Chemoorganotrophs/chemolithotrophs 95 Blank Archaea Chemoorganotrophs/chemolithotrophs 122  Adaptations to Life @ High Temperatures Hyperthermophiles and Thermophiles Protein and Membrane Stability at High Temperatures Enzymes and proteins: - Heat stability from subtle amino acid substitutions resist denaturation - Increased ionic bonding and highly hydrophobic interiors - Production of solutes (e.g., di-inositol phosphate, diglycerol phosphate, mannosylglycerate) helps stabilize proteins. - Thermophilic and hyperthermophilic enzymes commercially useful e.g., Taq polymerase for polymerase chain reaction [PCR]) Cytoplasmic membranes must be heat stable ▪ Bacteria have lipids rich in long-chain and saturated fatty acids, fewer unsaturated fatty acids. ▪ Most hyperthermophiles (Archaea) have C40 hydrocarbons made of repeating isoprene units bonded by ethers to glycerol phosphate, and membrane forms lipid monolayer rather than bilayer. II. Physical Requirements: pH pH expresses acidity or alkalinity of a solution. pH 7 = neutral; acidic pH < 7; and alkaline pH > 7 Each microbe has a pH range ~2–3 pH units within which growth is possible. Most natural environments are pH 3–9 Most bacteria grow best between pH 6.5 and 7.5 Molds and yeasts grow between pH 5 and 6 Food preservation may involve bacterial fermentation that produces acids (sauerkraut, pickles, some cheeses) Growth media in the laboratory may include buffers to minimize pH changes The pH Scale  Relationships of Microorganisms to pH Physiological class Approximate Example organisma pH optimum (optima range) for growth Neutrophile (pH > 5.5 and < 8) 7 Escherichia coli Acidophile (pH < 5.5) 5 Rhodopila globiformis 3 Acidithiobacills ferrooxidans 1 Picrophilus oshimae (archaea) Alkaliphile (pH ≥ 8) 8 Chloroflexus aurantiacus 9 Bacillus firmus Picrophilus and Natronobacterium 10 Natronobacterium gregoryi (archaea) are Archaea; all others are Bacteria ▪ Governed by stability of cytoplasmic membrane At neutral pH, membranes of strong acidophiles lyse; protons required for stability  Effects of pH on Microbial Growth Cytoplasmic pH and Buffers – Optimal pH refers to extracellular only – The intracellular pH must stay relatively close to neutral (pH 5–9), consistent with macromolecule stability Extreme acidophiles and alkaliphiles maintain cytoplasmic pH near neutrality. – Microbial culture media typically contain buffers to maintain constant pH. Copyright © 2021, 2018, 2015 Pearson Education, Inc. All Rights Reserved III. Physical Requirements: Water availability Water availability important; depends on environmental moisture/dryness and concentration of solutes Osmosis: Water diffuses from it’s high to low concentrations. Typically, the cytoplasm has a higher solute concentration than the surrounding environment; thus, the tendency is for water to move into the cell (positive water balance). When a cell is in an environment with a higher external solute concentration, water will flow out unless the cell has a mechanism to prevent this.  Osmotic Pressure Hypertonic environments (higher in solutes than inside the cell) cause plasmolysis (shrinkage of the cell’s cytoplasm) as water moves out of the cell by osmosis Microorganisms in high salt environments Bacteria increase internal solute concentration Synthesize small organic molecules – Bacteria that require high salt for cell growth termed halophiles (2-5% NaCl) Seawater contains ~3% NaCl – Extreme or obligate halophiles require high salt concentration (as high as 15-30% NaCl), often unable to grow at lower concentrations – Facultative halophiles/ Halotolerant tolerate high salt concentrations (2–10% NaCl) but generally grow best in the absence of added solute  Osmolarity and Microbial Growth Halophiles and Related Organisms – Osmophiles: live in environments high in sugar Xerophiles: able to grow in very dry environments – Lowest aw = 0.61 for life; physiochemical constraints on obtaining water at lower aw Compatible Solutes – to maintain positive water balance, microbes pump solutes from environment into cell or synthesizing cytoplasmic solutes – compatible solutes do not inhibit biochemical processes  IV. Oxygen and Microbial Growth (1 of 5) Not all organisms need/can use oxygen! 1. Obligate aerobes: absolutely require oxygen – grow at full O2 tension (~21%) and respire O2 2. Facultative anaerobes: able to grow with or without oxygen – grow via fermentation or anaerobic respiration when oxygen is NOT available 3. Obligate anaerobes: can’t use oxygen and most are harmed by it 4. Aerotolerant: tolerate oxygen and grow in its presence but cannot use oxygen 5. Microaerophiles: can use O2 only at levels reduced from that in air (microoxic) due to limited respiration or oxygen sensitivity Anoxic (oxygen free) habitats: mud, bogs, marshes, animal intestines, other diverse habitats  Anaerobic Growth Media and Methods Reducing media (reducing agent added) – Contain chemicals (e.g. sodium thioglycolate) that combine with O2 to deplete it – Heated to drive off O2 Growth Versus O2 Concentration Thioglycollate medium Copyright © 2021, 2018, 2015 Pearson Education, Inc. All Rights Reserved  Oxygen and Microbial Growth Why is oxygen toxic? – Molecular oxygen (O2) is not toxic. – Exposure to oxygen yields toxic byproducts. These products are formed inside phagolysosome in phagocytes Organisms that can tolerate oxygen (aerobes, facultative anaerobes, and aerotolerant anaerobes) have strategies to detoxify some of the harmful forms of oxygen  Toxic forms of oxygen: detoxifying enzymes Organisms that can tolerate oxygen (aerobes, facultative anaerobes, and aerotolerant anaerobes) have strategies to detoxify some of the harmful forms of oxygen Superoxide dismutase (SOD) catalyzes the following reaction:.O2 +.O2 + 2H+ ⎯⎯ → H2O2 + O2 Catalase and peroxidase converts hydrogen peroxide to water and oxygen H2O2 ⎯⎯ → 2 H2O + O2  Method for Testing a Microbial Culture for the Presence of Catalase The Effect of Oxygen on the Growth of Various Types of Bacteria  The Requirements for Growth Physical requirements – Temperature – pH – Osmotic pressure – Oxygen Chemical requirements – Carbon – Nitrogen, sulfur, and phosphorous – Trace elements – Oxygen – Organic growth factors Elemental and Macromolecular Composition of a Bacterial Cell Nucleic acids: Mostly RNA, not DNA WHY? Feeding the Microbe: Chemical Makeup of a Cell – Handful of elements dominate living systems – C, O, N, H, P, S are ~96% of dry weight of bacterial cell (Figure 4.1) and required by all life – K, Na, Ca, Mg, Cl, Fe ~3.7% of dry weight – Macromolecules (proteins, lipids, polysaccharides, lipopolysaccharides, nucleic acids) ~96% of dry weight – 6 basic nutritional requirements: Carbon, Energy, Nitrogen, Minerals, Vitamins/growth factors, water Feeding the Microbe: Macro- and Micronutrients Carbon: Heterotrophs: use organic carbon as carbon source. Autotrophs: use inorganic carbon (CO2) as carbon source to synthesize organics (i.e. glucose) Nitrogen (N): ▪ Component of proteins, DNA, and ATP ▪ ammonia (NH3), nitrate (NO3−), or nitrogen gas (N2). ▪ Nearly all microbes can use NH3. ▪ many use nitrate (NO3−) ▪ some use organics (e.g., amino acids) or N2 (nitrogen- fixation) Oxygen (O) & Hydrogen (H): - Oxygen (O) and hydrogen (H): from water Feeding the Microbe: Macro- and Micronutrients Phosphorus (P) ▪ nucleic acids, ATP and phospholipids ▪ usually inorganic phosphate (PO43−) Sulfur (S) ▪ sulfur-containing amino acids (cysteine and methionine) ▪ vitamins (e.g., thiamine, biotin, lipoic acid) ▪ microbes assimilate sulfate (SO4−2), sulfide (H2S) ▪ Some bacteria use sulfate (SO42-) or H2S Feeding the Microbe: Macro- and Micronutrients Potassium (K) ▪ required by several enzymes Magnesium (Mg) ▪ stabilizes ribosomes, membranes, and nucleic acids ▪ also required by many enzymes (co-factor) Calcium (Ca) and sodium (Na) ▪ required by some microbes (e.g., marine microbes) ▪ Spore formers Feeding the Microbe: Trace Metals and Growth Factors Trace metals: required in small/trace amounts – Many enzymes require metal ion or small organic as a cofactor for catalysis e.g. Fe, Cu, Mo, Zn – Iron (Fe) ▪ cellular respiration, related oxidation-reduction reactions Growth factors: organic micronutrients ▪ vitamins – Most function as coenzymes. – Most frequently required growth factors ▪ others: amino acids, purines, pyrimidines, other organics Classification of bacteria based on carbon and energy Chemoorganotrophs: organic source Chemolithotrophs: oxidize inorganic ions (e.g. nitrate, iron, sulfur etc.) to obtain energy to fix CO2 Photoautotrophs: photosynthetic pigments (chlorophyll or bacteriochlorophyll) use solar energy and CO2 via photosynthesis to make food Photoheterotrophs: few photosynthetic bacteria use solar energy for energy (via photosynthesis) but depend on some organic molecules e.g. succinate or glutamate for carbon needs. Growth Media and Laboratory Culture (1 of 3) Culture medium: Nutrient solutions used to grow microbes in the laboratory -Sterile: no living microbes -Typically sterilized in an autoclave Sterilization: removing and destroying all microbial life, e.g. Autoclaving, Incineration, membrane filtration, irradiation Inoculum: introduction of microbes into a medium Culture: microbes growing in or on a culture medium  Growth Media and Laboratory Culture (2 of 3) Culture media can be liquid or solid – Liquid is broth media Used for growing large numbers of bacteria – Solid media is broth media with addition of agar – Agar Complex polysaccharide Agar marine algae extract Liquefies at temperatures above 95°C Solidifies at 45°C Generally not metabolized, nor degraded by microbes Laboratory Culture Growth Media and Laboratory Culture (3 of 3) ▪ Bacteria grow in colonies on solid media surface ▪ All cells in colony descend from single cell ▪ Approximately 1 million cells produce 1 visible colony ▪ Colony morphology (visible characteristics) ▪ Sometimes used to identify microorganisms ▪ Routinely used to determine if a culture is pure (one microbe), contaminated, or mixed  Culture/Growth Media Classes of Culture Media – Defined media: exact chemical composition known – Complex media: composed of digests of microbial, animal, or plant products (e.g., yeast and meat extracts) – Selective medium ▪ contains compounds that selectively inhibit growth of some microbes but not others e.g. MacConkey agar: For isolation of Gram-negative bacteria – Differential medium ▪ contains an indicator, usually a dye, that detects particular metabolic reactions during growth  Examples of Culture Media for Microorganisms with Simple and Demanding Nutritional Requirementsa aThe photos are tubes of (a) the defined medium for Escherichia coli, and (b) the complex medium described. Note how the complex medium is colored from the various organic extracts and digests that it contains. Photo credits: Cheryl L. Broadie and John Vercillo, Southern Illinois University at Carbondale.  Culture/Growth Media Classes of Culture Media Blood Agar MacConkey Agar – Differential medium ▪ contains an indicator, usually a dye, that detects particular metabolic reactions during growth Some media have both selective and – Example differential characteristics Blood agar – Certain bacteria produce hemolysin to break down RBC » Hemolysis MacConkey agar – Contains pH indicator to identify bacteria that produce acid  Pure Culture & Aseptic Technique Pure culture: defined as population of cells derived from a single cell – All cells genetically identical Cells grown in pure culture allow to study the activities of a specific species Pure culture obtained using Aseptic technique Transfer without contamination (Figure 4.3) Pure cultures containing a single microbe usually require streak plate technique with inoculating loop Cells grown in culture media can be broth (liquid) or solid form (colony) Aseptic Technique & Quadrant Streak Streak Plate Method & Colony Forming Unit The streak plate method: is the simplest and most commonly used in bacterial isolation from mixed cultures Goal is to reduce number of cells being spread Solid surface dilution Each successive spread decreases number of cells per streak Bacteria grow in colonies on solid media surface – A colony is a population of cells arising from a single cell – Approximately 1 million cells produce 1 visible colony – A cell is often called a colony-forming unit (CFU) 1 cell = colony forming unit (cfu)  Special Culture Techniques Biosafety levels – BSL-1: no special precautions; basic teaching labs – BSL-2: lab coat, gloves, eye protection, biosafety cabinets – BSL-3: intended for working with highly infectious airborne pathogens ▪ biosafety cabinets to prevent airborne transmission ▪ negatively pressurized, equipped with air filters – BSL-4: sealed, negative pressure; “hot zone” ▪ exhaust air is filtered twice through HEPA filters ▪ workers wear “space suits” with an air supply ▪ only four in the United States BSL-3 BSL-4  Measurement of Microbial Growth Direct measurements – Indirect measurements- count microbial cells – doesn’t count microbial ▪ Direct microscopic cells count ▪ Turbidity ▪ Plate count ▪ Filtration ▪ Metabolic activity ▪ Most probable number ▪ Dry weight (MPN) method Microscopic Counts of Microbial Cell #s (1 of 2) Petroff-Hausser cell counter – Direct Microscopic cell count: observing and enumerating cells present – Volume of a bacterial suspension placed on a slide – Counting chambers with squares etched on a slide used for liquid samples – Average number of bacteria per viewing field is calculated Several limitations- what are they? Number of cells counted Number of bacteria/ml = Volume of area counted Microscopic Counts of Microbial Cell #s (2 of 2) Microscopic cell counts in microbial ecology – Often used on natural samples – Use stains to visualize and provide phylogenetic or metabolic properties – e.g., DAPI binds DNA. – Other fluorescent stains differentiate live and dead cells. – Phylogenetic stains can determine proportions of Bacteria and Archaea in a sample. Viable Counting of Microbial Cell #s (1 of 4) Viable (alive/living) counts: measurement of living, reproducing population – Two main ways to perform viable (plate) counts (Fig 4.5) ▪ Spread-plate method: bacteria mixed into a dish with agar ▪ Pour-plate method: bacteria spread on the surface of an agar plate – Count colonies on plates with 30–300 colonies – Reporting in colony-forming units (CFU) instead of number of viable cells accounts for clumps. Figure 4.5 Two Methods for the Viable Count Spread-plate method Pour-plate method Viable Counting of Microbial Cell #s (2 of 4) Serial Dilutions of the Sample Diluting a Sample – Samples often contain thousands, millions, billions of viable cells – Ten-fold serial dilutions commonly used – Serial (successive) dilutions needed for dense cultures Viable Counting of Microbial Cell #s (3 of 4) Applications of the Plate Count – Advantage-quick and easy – Disadvantage- materials/labor needed – used in food, dairy, medical, and aquatic microbiology – high sensitivity – can target particular species in mixed samples (e.g., Staphylococcus) – common in wastewater and other water analyses Viable Counting of Microbial Cell #s (4 of 4) “The great plate count anomaly”: Direct microscopic counts of natural samples reveal far more organisms than those recoverable on plates. Why is this? – Less than 1 % of microbes can be cultured in lab. – Different organisms may have vastly different requirements for growth. – May underestimate actual by one to several orders of magnitude  Counting bacteria by membrane filtration Solution passed through a filter that collects bacteria Filter is transferred to a Petri dish containing nutrients and colonies grow on the surface The Most Probable Number (MPN) Method Multiple tube test Compare with a statistical table Count positive tubes The Most Probable Number (MPN) Method Compare with a statistical table Turbidimetric Measures of Microbial Cell Numbers (1 of 3) Indirect measurement Cell suspensions are turbid (cloudy) because cells scatter light. More cells, more light scattered, more turbid suspension Turbidity—measurement of cloudiness with a spectrophotometer Turbidity measurements are rapid, widely used for estimates  Turbidimetric Measures of Microbial Cell Numbers (2 of 3) Optical Density and Its Relationship to Cell Numbers – Measured with a spectrophotometer – Unit is optical density (OD) at specified wavelength (e.g., OD540 for measurements at 540 nm [green light]). – For unicellular organisms, OD is proportional to cell number within limits. – To relate a direct cell count to a turbidity value, a standard curve must first be established. Turbidimetric Measures of Microbial Cell Numbers (3 of 3) Other Issues with Turbidimetric Growth Estimates – quick and easy to perform but not distinguish live vs. dead – typically do not require destruction or significant disturbance of sample – Same sample can be checked repeatedly. – sometimes problematic (e.g., microbes that form clumps or biofilms in liquid medium)