Module 3: Microbial Growth Flashcards
3 Types of Microbial Growth
- Binary Fission
- Budding Cell Division
- Biofilm
Binary Fission
- Organelles duplicated
- DNA replication
- Elongation; cell organelle migration to opposite poles; cleavage furrow
- Septum Formation
- Production of 2 Daughter Cells
- INTERCALARY GROWTH
Budding Cell Division
- Mother cell produces appendage which forms the daughter cell
- Mother cell retains original size; appendaged is pinched off and deteriorated
- POLAR GROWTH
Biofilm (and 3 Properties); matrix name
- an assemblage of surface-associated microbial cells that are enclosed in an extracellular polymeric substance matrix
- “microbial mat” one species on top of one another
- EPS - exopolysaccharide matrix
3 Properties:
(i) Resistant to antibiotics or harmful chemicals - prevents entry
(ii) Hides them from protist grazer e.g. planktonic organisms (suspended in lipid environments)
(iii) Prevents being washed away by water
Generation Time (g)
Time taken for a cell to double
Solve for g (generation time)
g = t/n
g = 0.301/slope
g: generation time
t: time measurement
n: number of generations
Solve for N (final cell count)
N = N(0) x 2^n
N: final cell count
N(0): initial cell count
n: number of generations
Solve for n (number of generations)
n = 3.3[log(N) - log(N(0))]
n = [(log(N) - log(n(0))] / 0.301
n: number of generations
N: final cell count
N(0): initial cell count
4 Stages of Bacterial Growth Curve
- Lag Phase
- adapting to a new, nutrient-rich environment
- metabolically active but does not increase in cell number - Exponential Phase
- Cell Division occurs at a rapid rate
- limiting factor: ATP production - Stationary Phase
- production of new cells at the same rate of cell death - Death Phase
- nutrients are depleted
- overwhelming amount of toxic waste
- cells undergo involution
- endospore cells > vegetative cells
Duration of Lag Phase depends on
- Nutrients Available
- Species
Toxic Environment of Bacteria Growth Cycle
- Damaging pH
- Depletion of O2
Define Chemostat & 2 factors affecting cell density and growth rate
- a system in which the chemical composition is kept at a controlled level, especially for the culture of microorganisms.
- Dilution Rate
- Concentration of Limiting Reactant
5 Methods of Bacterial Growth Measurement
- Direct Microscopic Count
- counting via Petroff-Hauser counting chambers (both living and dead cells) - Viable Cell Count
- colony counting (spread plate, pour plate) - Most Probable Number
- estimate number of viable cells via gas production or turbidity - Membrane Filtration count
- physical separation of cells by size - Turbidimetric Method
- measuring via optical density and absorbance
Serial Dilution
Purpose, Range, Classifications and Process
- PURPOSE: to lessen bacterial load by relegating it to single colonies
- RANGE: 30-300 colonies
- CLASSIFICATIONS: TFTC (too few to count), TNTC (too numerous to count)
- PROCESS: constantly diluting the original inoculum by 1/10 to form 1:10, 1:100, 1:1000, 1:10000 plates
Dilution Factor DEFINE; EQUATION
- DEFINE: Volume by which factor the sample was diluted
- EQUATION: volume transferred of sample/total volume
Spread Plate
Pour Plate
Streak Plate
- SPREAD: sample is pipetted onto AGAR plate; spread evenly before incubation
- POUR: sample is pipetted onto sterile plate; sterile medium is added and mixed for solidification and incubation
- STREAK: flame looped then applied like butter to agar plate
Colony Forming Units Equation
CFU/(mL or g): (# of counter colonies x dilution factor)/volume of plated sample
The Great Plate Count Anomaly
and EXPLANATION
- Direct cell counting w/ microscope of a cultivated medium reveals more than if sample was from the environment
- Plate count shows lower number of cells
WHY?
- Entire bacterial population environment cannot be isolated in the laboratory
- Some may actually be nonviable (do not grow or divide), others are viable but nonculturable (VBNC)
Solutions to Solve the Great Plate Count Anomaly
- Use of highly selective medium
- Varying incubation conditions
Most Probable Number TYPE OF SAMPLE; 2 FACTORS; PROCESS
- TYPE: Water and Air samples (It is used when samples contain too few bacteria to provide reliable viable cell numbers by classical plate count or if its unculturable in media)
- 2 FACTORS: Gas Production and Turbidity (clear-ness property of solution)
- PROCESS: undiluted mixture, 1:10, 1:100 of a bacterial sample are divided into 3 or 5 test tubes, +phenol red, results (count the # of positive test tubes) then use a table to determine the estimated number of bacteria cells
Membrane Filtration PROCESS
- physically separate
- membrane filter
- cotton plug in vacuum line ensures sterility
- each colony growth captures one species of organism (in agar medium)
Turbidimetric Method 2 FACTORS
- Optical Density
- measures amount of attenutation of light (intensity lost)
- based on light scattered
- the slower the light travels, the higher the optical density - Absorbance
- measures amount of light absorbed
- no basis on light scattered
OD measures HOW MANY cells
ABSORBANCE measures HOW LARGE cells
What are the 4 Physical Properties that affect Microbial Growth?
- pH
- Temperature
- Osmosis pressure
- Oxygen
Three classifications of pH and their pH ranges
What classification is E.coli?
- Neutrophiles
- optimum pH 5.5 - 7.9
- E.coli - Acidophiles
- <5.5 - Alkanophiles
- >=8
Name two kinds of commonly used buffers
- KH2PO4
- CaCO3
What are Acidophiles?
- Acidithiobacillus feroxidase
- Picrophilus oshimae
What are Akaliphilic?
- Agrobacterium sp.
- Some can be Halophilic (salt-rich environments)
4 Classifications of Microorganisms based on Temperature
- Psychrophile
- optimal growth <15C - Mesophilic
- 10-50-ish growth range - Thermophilic
- optimal growth 45-80C - Hyperthermophilic
- Bacteria: up to 90C
- Archaea: >100C
Where are Thermophiles and Hyperthermophiles found?
Thermophiles: hot springs
Hyperthermophiles: geysers + hydrothermal vents
Psychrophiles’ 4 Stability Properties (COLD ADAPTATIONS)
- Cytoplasmic membrane stability
- high concentration of unsaturated, short chain fatty acids
- maintains fluidity - Lipids
- contains polyunsaturated fatty acids - Cold-Shock Proteins
- maintains protein activity under low temperatures
- binds to mRNA to facilitate translation process - Cryoprotectant
- antifreeze proteins
- prevents ice crystal formation that can puncture cell membrane
–> composed of GLYCEROL + DMSO (dimethylsulfoxide)
Hyperthermophiles’ 2 Stability Properties (HOT)
- Proteins
- protein folding
- enzymes are more heat stable - Membrane
- lipids contain saturated fatty acids
- Archaea: lipid monolayer - prevents membrane from peeling apart
What are the Reactive Oxygen Species?
- Triplet & Singlet Oxygen
- Singlet oxygen is highly reactive
- Some organisms contain carotenoids which converts it into nontoxic forms - Superoxide Anion & Hydroxyl Radical
- strong oxidizing agents
- destroyed by enzyme superoxide - Hydrogen Peroxide
- damaging to cellular components
- destroyed by enzyme catalase & peroxidase
Positive Water Balance
Cell cytoplasm has higher solute concentration so water flows into the cell
4 Classifications of Halophiles and which is E.coli
- Nonhalophilic
- E. coli
- Low to no growth rate in presence of NaCl (salt) - Halophilic
- Increasing growth rate (up to a point) in increasing presence of NaCl - Halotolerant
- Growth rate isn’t influenced by salt concentration - Extreme halophilic
- Growth rate doesn’t increase until high concentration of salt is present
Osmophilic
Thrives in high sugar environments
Xenophilic
Thrives in dry environments
What are Compatible Salts?
Solutes that can be used by the cell to protect its macromolecules (by increasing internal solute concentration to prevent the outflow of water)
2 Methods of increasing internal solute concentration
- Pumping solutes into cell
- Synthesizing solutes
3 Types of Aerobes
(i) Obligate
- required O2
- aerobic respiration
- skin/dust
(ii) Facultative
- not required, but better w/ O2
- aerobic respiration, anaerobic respiration, fermentation
- mammalian large intestine
- E. coli
(iii) Microaerophilic
- required but at lower levels of the atmosphere
- aerobic respiration
- lake water
2 Types of Anaerobes
(i) Aerotolerant
- O2 not required; growth constant w/ O2
- fermentation
- upper respiratory tract
(ii) Obligate
- O2 is toxic
- fermentation or anaerobic respiration
2 Types of Compatible Solutes and its corresponding
- KCl, Halobacterium sp.
- Proline, Staphylococcus sp.
