Microbial Growth Flashcards
How is microbial growth defined?
Measure in increase in number of cells aka growth in population.
What is Binary Fission?
Cell division following enlargement of a cell to twice its minimum size
What is generation time/doubling time? What is it dependent on?
Time required for microbial cells to receive a chromosome and sufficient copies of all other cell constituents to exist as an independent cell. Dependent on growth medium, carbon source, pH, temperature, etc.
What is exponential growth of a microbial organism?
Cell numbers double at a constant and specific time interval.
Nt = N0 x 2^n
Nt: final cell number
N0: Initial cell number
n: number of generations during the period of exponential growth
How is cell number growth graphed?
Since raw data displays an exponential slope since cells increase exponentially, number is graphed as log(y) so the plot is linear
What is growth rate (k)?
Rate of increase in population or biomass. In binary fission, it represents doublings per hour.
What is the mathematical relationship between growth rate (k) and Generation time (g)
k = 1/g
What is the difference between generation time and growth rate?
Generation time (g): TIME per GENERATION
Growth rate (k): GENERATIONS per unit of TIME
How do you calculate specific growth rate?
k = (logNt - logN0) / 0.301(delta T)
What is a specific growth rate?
Fastest growth rate at optimal conditions (temp, growth medium)
What are the parts of a growth curve of a closed system?
- Lag phase: interval between inoculation and beginning of growth (adjustment phase)
- Exponential phase: cells are in the healthiest state and are dividing
- Stationary phase: cells metabolically active but growth rate is 0 (e.g. essential nutrient is used up, waste accumulates in medium)
- Death phase: cells eventually die, but some form spores/cysts or dormant
What is a continuous culture?
Open system microbial culture of fixed volume.
What is a chemostat?
Continuous culture device that independently and simultaneously controls the growth rate and population density of the culture through the control of a limiting nutrient.
What is a counting chamber?
Sample is put on a grid and each square corresponds to a calibrated volume to count.
What are the limits of microscopic counts?
- cannot distinguish between live and dead cells without special stains
- small cells are overlooked
- precision is difficult to achieve
- Cell suspensions of low density are hard to count
- motile cells need to be immobilized
- cells may move, form clumps based on random distribution and dispersal of cells.
What is flow cytometry?
Uses laser beams, fluorescent dyes and electronics to count the total number of cells
What is the viable cell count?
Measures only living cells that are capable of growing to form a population. You can use the spread plate method or the pour-plate method.
What is the issues with viable cell counts?
- Requires lots of prep (dilution tubes, agar plates, incubation time)
- Unreliable when assessing total cell numbers of natural samples
- Selective culture media growth target only particular species and cannot grow every microbe.
What is the great plate anomaly?
miscroscopic counts of natural samples reveal far more organisms than those recoverable on plates, so it is suggested that only 1-10% of microbial diversity is culturable from most environmental samples.
How does spectrophotometry count bacteria?
Incident light shines through sample which scatter light. The higher the turbidity (optical density), the larger the number of particles, the greater the absorbance, the lower the light transmission.
What is the disadvantage of spectrophotometric counts?
- Absorbance does not distinguish dead cells from living cells.
- has a finite linear range of measurement
- only works if the cells are evenly distributed throughout the medium (no clumps or biofilms)
What is required to relate a direct cell count to a turbidity value?
Standard curve must be established to another counting method (e.g. viable cell count, weight of biomass/dry weight)
How to count total mass of cells (dry cell weight)?
Specific aliquot (volume) of cells are concentrated, washed to remove media components, concentrated again and then dried.
What are the cardinal temperatures?
Minimum: lowest temperature at which the organism can survive and replicate.
Optimum: temperature at which enzymatic reactions occur at the maximal possible rate
Maximum: The highest temperature at which growth can occur before its proteins begin to denature.
What are the groups of optima microbes are classified into?
Psychrophile: optima at low temp
Mesophile: optima at midrange temp
Thermophile: optima at high temp
Hyperthermophile: optima at very high temp
What is a Mesophile, where is it found, and an example?
microorganisms that have midrange temperature optima, typically found in warm-blooded animals, terrestrial and aquatic environments, temperate and tropical latitudes (e.g. E. Coli)
What are Extremophiles?
A general term to describe organisms that grow under either extremely hot or extremely cold conditions.
What are psychophiles and where are they found?
Organisms with cold temperature optima (<20ºC) that inhabit permanently cold environments such as the deep ocean, arctic, and antarctic environments.
What are psychrotolerant organisms?
Organisms that can grow at 0ºC, but have optima of 20ºC-40ºC and are more widely distributed in nature than true psychrophiles
What are the adaptations that support psychrophily?
Production of enzymes that function optimally in the cold, and modified cytoplasmic membranes with high unsaturated fatty acid content.
What are thermophiles and where are they found?
Organisms with growth temperature optima between 45ºC and 80ºC found in terrestrial hot springs and very active compost.
What are hyperthermophiles and where are they found?
Organisms with optima greater than 80ºC found in boiling hot springs, seafloor hydrothermal vents (100ºC+)
What is the current temperature maximum record?
Archaeon Methanopyrus kandleri, grows at 122ºC (cannot be killed off, hotter than autoclave)
What are molecular adaptations to thermophily?
Modifications to provide thermal stability to enzymes, proteins, and cytoplasmic membranes (saturated fatty acids, lipid monolayer)
What enzymes produced in hyperthermophiles are used in industrial microbiology?
Taq polymerase: used to automate repetitive steps in PCR
Hydrolytic enzymes such as proteases, cellulases, and lipases.
What are enzymes of thermophiles favoured in industry over mesophiles?
Enzymes are more stable and tend to have higher activity than those of mesophiles.
What are the upper temperature limits for life currently known?
Laboratory experiments suggest 140-150ºC
What is the evolution of Hyperthermophily?
Hyperthermophilic Archaea and Bacteria are found on the deepest, shortest branches of hte phylogenetic tree and may be the closest relative to the last universal common ancestor (LUCA)
What are the groups of pH microbes are classified into?
Neutrophiles: organisms grown best between pH 6-8
Acidophiles: Organisms that grow best at low pH (<6)
Alkaliphiles: organisms that grow best at high pH (>9)
What is the difference between internal and external pH of microbes that thrive in differing pH?
Internal pH must stay relatively close to neutral despite highly basic/acidic external pH
Why do culture media contain buffers to maintain constant pH?
Some bacteria produce acids (Acetic, lactic, sulfuric) as a byproduct of metabolic processes that decrease the pH
Some bacteria grow on amino acids which releases ammonia and increases the pH
What is water activity (a_w)?
Amount of water that is interacting with ions and polar compounds in solution.
What are halophiles + example?
Microbes that grow best at high solute concentrations and reduced water potential (e.g. marine microbes)
What are extreme halophiles + example?
microbes that require high levels of NaCl (15%-30%) for growth (e.g. Dead sea microbes)
What are halotolerant microbes + example?
Microbes that can tolerate some reduction in water activity of environment, but generally grow best at lower solute concentration (e.g staphylococcus aureus)
What are osmophiles?
Organisms that grow with high sugar as solute
What are Xerophiles?
Organisms able to grow in very dry environments.
What mechanisms do microbes have that combat low water activity?
Increasing internal solute concentration by pumping inorgaic ions from environment into the cell and synthesizing/concentrating organic solutes.
How to group microbes based on oxygen use/oxygen tolerance?
Obligate aerobes: require oxygen to live
Strict anaerobes: do not require oxygen and may be killed by exposure
Facultative aerobes: can live with or without oxygen, using it when available
Aerotolerant anaerobes: can tolerate oxygen and grow in its presence even though they cannot use it
Microaerophiles: can use oxygen only when it is present at levels reduced from that in the air
How to distinguish oxygen tolerance?
Complex medium (such as thiglycolate) reacts with oxygen creating an anaerobic environment (anoxic zone) and aerobic environment (oxic zone)
Where are the oxygen tolerance groups found in the tube of thioglycolate?
obligate aerobe: grows ONLY in oxic zone at top of tube
strict anaerobe: grows ONLY in anoxic zone
Facultative anaerobe: grows throughout the tube
Microaerophile: grows in the narrow band between oxic and anoxic zones
Aerotolerant anaerobe: grows well throughout tube
How do you grow anaerobic mircrobes?
- Add reducing agents to culture media to reduce oxygen
- Remove air and replace it with an inert gas (N or Ar)
What are the three toxic forms of oxygen that are formed spontaneously in the cell?
- Superoxide anion (O2^-)
- Hydrogen peroxide (H2O2)
- Hyrdoxyl radical (OH)
What enzymes are present to neutralize most of the toxic oxygen species spontaneously formed in the cell?
- Catalase
- Peroxidase
- Superoxide dismutase
- Superoxide reductase
What is the difference between sterilization, inhibition, decontamination, and disinfection?
Sterilization: killing/removing all viable organisms within a growth medium
Inhibition: effectively limiting microbial growth without killing
Decontamination: treatment of an object to make it safe to handle
Disinfection: Targets removal of all pathogens, but not necessarily all microorganisms
What is heat sterilization?
Most widely used method of controlling microbial growth by heating to a temperature hot enough to denature macromolecules.
What is the decimal reduction time (D)?
Amount of time required to reduce viability tenfold
What is pasteurization?
Process of using precisely controlled heat to reduce (but not sterilize) the microbial load in heat-sensitive liquids.
What is the difference between LTLT and HTST pasteurization?
LTLT: 63ºC / 30 min
HTST: 72ºC / 15 sec
What is the autoclave?
A sealed device that uses steam under pressure to get the temperature of water above 100ºC. Organisms are heated at 121ºC for 15 minutes at 15 pounds per square inch of pressure.
How is radiation used to regulate growth?
Microwaves, UV, X-rays, gamma rays, and electrons used to reduce microbial growth.
Why is UV helpful to use in the lab to control growth?
UV has sufficient energy to cause modifications and breaks in DNA and useful for decontaminating surfaces, but cannot penetrate solid, opaque, or light absorbing surfaces.
What is ionizing radiation?
Electromagnetic radiation with enough energy to remove tightly bound electrons from the orbit of an atom, producing electrons, hydroxyl radicals, and hydride radicals.
What are sources of radiation?
Cathode ray tubes, X-rays, and radioactive nuclides.
What is used for sterilization in the medical field and food industry?
Radiation is approved by the WHO and is used in the USA for decontaminating foods susceptible to microbial contamination.
What is filtration?
Control of growth while avoiding the use of heat on sensitive liquids and gases.
What is the difference between a depth filter and a membrane filter?
Depth filter: uses “depth” of multiple layers of fibre to trap and hold particles within the filter media (e.g. HEPA filter)
Membrane filters: trap contaminants larger than the pore size on the surface of the membrane
How is filtration accomplished with membrane filters?
Through a syringe, pump, or vaccuum.
What is a nucleopore filter?
A type of membrane filter with pores measured in micrometers. The typical 0.2 micrometer pore prevents most bacteria from passing.
How are antimicrobial agents classified?
Bacteriostatic: prevents cell growth as long as agent is present
Bacteriocidal: kills the cells without lysing them (counted in total cell count)
Bacteriolytic: kills and lyses the cells (not counted in total cell count)
What is the minimum inhibitory concentration (MIC)? How does it vary?
Smallest amount of antibiotic needed to inhibit the growth of a microorganism. Lowest concentration in liquid media with NO VISIBLE GROWTH. Varies with organism used, inoculum size, temperature, and pH.
What is the minimal lethal concentration (MLC)
Smallest amount of antibiotic needed to kill off all organisms. Lowest concentration in solid media with NO COLONIES FORMED.
How does the Disc diffusion assay work?
Plate is inoculated with a liquid culture of a test organism. Filter paper soaked with antimicrobial egent is placed onto the plate, which formed a zone of inhibition (no growth) around the disk.
What categories are microbial growth divided into?
- products used to control microorganisms in commercial and industrial applications (e.g. food, air conditioning, textiles, fuel)
- Products designed to prevent growth of human pathogens in inanimate environments and external body surfaces (e.g. Sterilant, disinfectant, sanitizer, antiseptic)
What is the difference between sterilant, disinfectant, sanitizer, and antiseptic?
Sterilant: destroys all microorganisms including endospores
Disinfectant: kills microorganisms but not all endospores
Sanitizer: reduced numbers of microorganisms on surfaces
Anticeptic: kills/inhibits the growth of microorganisms (non-toxic enough to be applied on living tissue)
