Lesson 4 Bacterial Culture, Growth And Environmental Influence/Control Of Growth Flashcards
What are the basic requirements for microbial growth?
- physical requirements- (pH, light, temperature, water, salinity, osmotic pressure etc.)
- Chemical requirements- (nutrients) essential nutrients/growth factors and nonessential nutrients
- macronutrients (C,O,H,N,P,S)
- cations necessary for proteins function (K,Na,Mg,Ca,Fe)
- Micronutrients- trace elements necessary for enzyme function (Co,Cu,Mn,Zn)
Carbon accounts for ___% of the cells dry weight
50
What are the major elements in cell macromolecules?
C, O, N, H, P, S
Cats only nap heavily past seven
self-feeders, fix CO2 and assemble into organic molecules (mainly sugars)
Autotrophs
Uses preformed organic molecules (organic forms of carbon)
Heterotrophs
Use chemical reactions triggered by light for their energy source
Phototrophs
Oxidation Dash reduction reactions, organic/inorganic molecules
Use potential energy that is stored in bonds as energy source
Chemotrophs
Uses inorganic molecules as a source of electrons. (Ex: hydrogen, hydrogen sulfide, ammonium, nitrate)
Lithotrophs
Organic molecules as a source of electrons
Oxidize organic compounds such as sugars to obtain energy
Organotrophs
How are microbes classified based on their carbon and energy acquisition?
- Autotrophs-use inorganic CO2 as a carbon source
- Photoautotrophs-use light for energy, includes plants, algae, cyanobacteria and green and purple sulfur bacteria
- chemolithoautotrophs (lithotrophs) use chemical compounds for energy (hydrogen, sulfur, in nitrifying bacteria, and some archaea) - heterotrophs-use organic compounds as their carbon source
- photoheterotrophs-use light for energy (Green and purple nonsulfur bacteria and some archaea)
- chemohetrotrophs (organotrophs)-use chemical compounds for energy (aerobic respiration and anaerobic, fermentation, most animals, fungi, protozoa, and most bacteria)
Can grow both anaerobically as a photoheterotroph or aerobically as a chemoheterotroph
Chloroflexus aggregans
Other essential nutritional factors include….
- nitrogen (needed by cells to make up amino acids, nucleic acid)
- Sulfur (needed to synthesize sulfur containing amino acids and certain vitamins)
- phosphorus (for DNA, RNA, ATP and membranes(phospholipids))
- trace elements (inorganic elements that usually function as cofactors or electron donors/acceptors) and vitamins
N2 makes up nearly ____% of earths atmosphere
79%
Nitrogen must be fixed and converted to ________ ions (- form that can be used for bio synthesis)
Nitrogen fixtures possess ________.
Ammonium ions
Nitrogenase
In organic nitrogen to organic nitrogen is a ________ process.
Bacterial
These bacteria have the enzyme called nitrogenase
Symbionts in legumes plants like soy beans, chickpeas, and clover
Forms bacteria filled nodules in and on the roots
Rhizobium
- in organic elements required in small amounts (K,Mg,Ca,Fe,B vitamins, etc.)
- enzyme cofactors/coenzymes
Trace elements and vitamins
What are the three ways nutrients are taken in?
- substrate-specific carrier proteins, or permeases
- nutrient-binding proteins
- Protane channels or pores
Type of diffusion that does not use energy, and cannot go against its gradient
- passive diffusion
- facilitated diffusion (ex: glycerol transporter of E. coli)
This transport goes against the gradient, and requires energy
Ion goes down it’s gradient, moves a solute up it’s gradient)
Active transport
Coupled transport (antiport)
What is the largest transporter that is powered by ATP and found in all three domains of life?
ABC transporters
What are the two main types of ABC transporters?
- uptake ABC transporters-critical for transporting nutrients
- efflux ABC transporters-Multi drug efflux pumps, push out antibiotics and toxins
- uses energy to chemically alter the substrate during its transport
- phosphotransferase system (PTS)-Energy from phosphoenolpyruvate (PEP) to attach a phosphate to specific sugars (in many bacteria)
Group translocation
Have complex growth requirements. Difficult to grow in the lab
Fastidious organisms
Why can some organisms not be grown in the lab?
Some species depend on factors provided by other species
An example of an obligate intracellular bacteria that is unculturable. It grows in eukaryotic cells)
Rickettsia Prowazekii
What is a culture growing method for anaerobic bacteria?
Pour plate method
Bacteria is diluted in agar and then the tube of auger is poured into a petri plate so the bacteria are growing within the agar (not exposed to oxygen)
Blood agar can be ________ or ________ for hemolysins
General or differential
Nutrient rich* but poorly defined culture media. Contain things like milk, yeast, or soy.
Ex: BHI, TSA (used also as supportive or general purpose media)
Complex media
Culture media that contains only the nutrients that are essential for growth of the given microbe
Minimal defined media
Complex media, specific blood components are added. General media used for fastidious organisms and also use as differential media.
Enriched media
Ex: blood and chocolate (blood auger that has been caramelized) agar plates
Culture media that Fehler the growth of one organism over another. Compounds that prevent some microbes from growing
Selective media
Ex: PEA agar for gram-positive organisms
Culture media that Exploit differences between species that grow equally well
Differential media
Ex: blood agar for hemolysins
True or false, some types of media fall into more than one category.
True
Ex: MacCobkey Agar (selective and differential)
MacCobkey Agar …
________ agents are bile salts and crystal violet, which prevent growth of bacteria other than gram-negative enteric bacteria.
Selective
MacCobkey Agar …
________ agent is lactose, which some bacteria ferment (pink) and others do not.
Differential
Techniques for counting bacteria
- Petroff-Hauser counter
- fluorescent-activated cell sorter (FACS) or flow cytometer
- visible counting methods- spread and pour plate techniques
- bacterial growth:plate count
- Direct counts on membrane filters
- viable counting methods- most probable number (MPN)
- Spectrophotometric Analysis
Also known as hemocytometer, has a grid to help count
-useful for counting both prokaryotes and eukaryotes
-easy quick and inexpensive
-doesn’t tell you if cells are dead or alive
Trypan blue can be used, dead cells absorb the dye, live do not
Petroff-Hauser counter
Fluorescent cells are passed by a laser
Detectors measure light scatter in the four direction and to the side
Fluorescent-activated cell sorter (FACS) or flow cytometer
- Serial deluded samples plated
- Count # colonies (25-300)
- determine CFU/mL
- CFU/ml of sample = #colonies X delusion factors
Plate count
When doing a plate count, what range is statistically significant?
25-300 colonies
Can we used when sample has a really low bacterial count
An example would be 10 microbes in 10 gallons
Direct counts on membrane filters
- dilutions are made and added to suitable media
- “delusion to extinction”
- not for precise count, but for a rough range
Viable counting methods- most probable number (MPN)
- County method that is based on turbidity and indirectly measures biomass (dead or alive)
- measures amount of light which passes through cuvette
- transmittance or optical density is measured (turbid or transparent)
Spectrophotometric analysis
Refers to population growth rather than growth of individual cells
Microbial growth
Bacterial growth is measured at the _________ level
Population
The number of cells in a population over time
Growth rate
Most bacteria divided by….
Binary fission
The time it takes for microbes to double
Generation time
Binary fission may be symmetrical or asymmetrical.
True or false?
True
Unique method of reproduction where live offspring emerge from body of dead mother cell
Viviparity
Ex: Epulopiscium (Symbiant of the surgeonfish)
- usually plotted as logarithm of cell number versus time
- has four distinct phases
- observe when micro organisms are cultivated in batch cultures
Growth curve
What are the four phases of a growth curve?
- Lag phase- cells added, synthesizing needed components, adapting, replenishing (flat/ no curve)
- Exponential (log) phase- Constant and maximal rate, chemically and physically most uniform at this phase (steep upward slope)
- Stationary phase- Constant total number of viable cells, reproduction and death rate are balanced (flat Plateau)
- Death phase- cells may be viable but not culturable, might be dormant, or apoptosis(cell death) (downward slope)
What might account for the stationary phase in a growth curve?
- due to starvation and other stressful conditions activates survival strategy
- nutrients depleting, limited oxygen, or toxic waste accumulation
- might start forming endospores
All cells in population achieve a study state, allows for detailed study of bacterial physiology
(Chemostat) ex:human GI tract
Continuous culture conditions
How to calculate cell numbers in regards to time?
Number of cells in population at time (zero)
Multiplied by
2^(The number of divisions in the given time frame)
What are the two types of biofilms?
Sessile-grow attach to surfaces
Planktonic- free floating
Bacteria develop an extracellular matrix which enables them to attach to each other and surfaces
Biofilms
Ubiquitous in nature and in water environments, complex slime enclosed communities
This helps increase resistance to antibiotics
Requires a molecule called autoinducer
Cell to cell communication within the microbial populations
Quorum sensing system
How are biofilms formed?
———-
An example of cell differentiation…
Endospores of gram-positive bacteria
What bacteria produce endospores?
Gram positive and Colstridium spp.
What’s the difference between endospores and vegetative cells?
Endospores-metabolically inactive dormant form
Vegetative-metabolically active cell
How are endospores formed?
- Septum forms near one pole, DNA extends into axial filaments
- The septum separates forspore from mother cell
- Mother cell engulfs the forspore, surrounding it with a second membrane
- Chromosome of mother cell is destroyed
- forspore develops cortex of peptidoglycan. Coat proteins are deposited on outer membrane
- dipocolinic acid is synthesized, and calcium is incorporated into the spores core
- Mother cell release spore
- replication of DNA into axial filament; separation, -engulfment of forspore
- cortex formation
- coat proteins deposition
- dipicolinic acid productions
- spire release
Diseases caused by spore forming bacteria…
- anthrax
- tetanus
- pseudomembranous colitis
- gas gangrene
- botulism
Anabaena Differentiates into specialized cells called ________.
Heterocysts
-allows it to fix nitrogen anaerobically while maintaining oxygenic photosynthesis
Cell differentiation:
Starvation triggers the aggression of 100,000 cells which form a fruiting body
Fruiting bodies of Myxococcus xanthus
Cell differentiation of Streptomyces (fungi)?
Aerial hyphae and Anthrospores
Organisms exhibit distinct cardinal growth temperatures
Growth doubles for every increase of ____ degrees centigrade
10
Temp. For psychrophiles?
Mesophiles-
Thermophiles-
Hyperthermophiles-
psychrophiles- 0-20 degree C
Mesophiles-15-45 (37 body temp) most bacteria*
Thermophiles-40-80
Hyperthermophiles-65-121 (most are archaeans)
Organisms that live under extreme pressure
Ex: Mariana Trench, over 1,000 atm
Barophiles (piezophilic)
Organisms that can survive at high pressures but can survive and less extreme environments as well
Barotolerant organisms
When animal cells shrivel due to hero tonic solutions?
When the plasma membrane pulls from outer cell wall due to extreme hypertonic environment?
Crenation
Plasmolysis
Microbes that live in 10% salinity? Tolerant to high osmotic pressure
Microbes that live in 20% salinity? (most are archeans and found in Salt Lake or the Dead Sea)
Facultative halophile (osmotolerant)
Extreme or obligate halophiles
Extreme pH affects the structure of all ___________
Macromolecules
Organisms that grow best at pH
5-8 (includes most pathogens)
Organism that grow best at a pH of 0 to 5, often chemoautotrophs
Organisms that grow in a pH of 9 to 11 (in soli a d water)
Neutrophiles
Acidophiles
Alkalophiles
Fungi, molds, and yeast grow in a PH around 5 to 6, in between Neutrophiles and acidophiles
Coping mechanisms of alkalophiles?
- acidic cytoplasm
- PH resistant enzymes
- Gram-negative bacteria that causes this disease
- facultative anaerobe
- transmission: fecal-oral route
- causes sudden onset of dysentery “ Rice water stools”
- High mortality if untreated
- treatment include supportive, antibiotics, vaccine (not very effective)
Vibrio cholera
Secretes cholera toxin (2 subunits) structurally similar to AB toxins, AKA enterotoxin
Saline soda lakes contains alkalophiles such as….
- archaeon- Halobacterium salinarum
- Cyanobacterium- spirulina (A food staple for the pink flamingo that contributes to its pink color)
Strict anaerobes lack or have very low quantities of _______ and ________.
SOD and catalase
This tube is used for growing bacteria, has strong reducing properties, autoclaving the broth flushes out most of the oxygen
Thioglycolate tube
Organisms that need oxygen, have electron transport chain, cannot undergo fermentation
This includes all animals, most fungi, many bacteria
Obligate aerobes
Growth will be at the top of the tube only
Organisms that can tolerate little or no oxygen, killed by oxygen, anaerobic respiration or fermentation
Obligate anaerobes
Growth seen only at the bottom of the tube
Organisms that’s thrive in oxygen but also capable of living without it, this includes most bacteria
Facultative anaerobes
Growth seen mostly at top and some in the middle
Organisms that are indifferent to the presence of oxygen, usually a fermentative metabolism
Aerotolerant anaerobes
Growth seen equally throughout the tube
Organisms that only need small amounts of O2, “think godly locks culture”, only grows if the amount of O2 is just right
Microaerophiles
Growth seen a little bit below the surface
How are anaerobes cultured in the lab?
- special reducing agent (thioglycolate or Oxyrase)
- anaerobe jar (candle or palladium) oxygen is removed by a gas pack in which reaction catalyzes palladium removes oxygen from the environment
- anaerobic chamber with glove ports (oxygen is replaced with CO2 and nitrogen gas)
Organisms that thrive in high concentrations of carbon dioxide
Capnophiles
Ex:Campylobacter
-The sudden infusion of large colonies of a former limiting nutrient, can lead to a bloom or overgrowth of microbes, can threaten other species
Eutrophication
Run-off from fertilizers commonly cause this
The killing or removal of pathogen‘s from in animate objects, reducing the number of viable organisms that are present. Physical or chemical agents applied to an object to kill microbes
Disinfection
Reducing microbial populations to safe levels, often used in food prep
Sanitation
An agent that inhibits bacterial growth, but does not kill. Keeps the organisms in stationary phase
Bacteriostatic
The length of time it takes an anti-microbial agent to kill 90% of the population
Decimal reduction time (D-value)
Mechanisms of action of antimicrobials?
- protein denaturation
- Membrane disruption
- nucleic acid damage
- inhibition of metabolism
What’s the difference between using moist heat and dry heat to kill microbes?
Moist heat- damages vital proteins by denaturing them (this will kill most microbes, and much faster)
Dry heat- causes oxidative damage to the proteins
At what temperature pressure and time is a steam auto clave used?
It can kill endospores
121C (250F) at 15 psi for about 20 minutes
A way to reduce microbial content that is similar to freeze drying
Lyophilization
What temperature is commonly use for food preservation
Around 4-8 C
Used with micropore filters of pore size of around .2 µm can remove most bacteria in microbial cells, but not small enough to prevent viruses through. This prevents altering the medium high heat. May not be sterile due to tiny microbes like viruses in tiny bacteria like mycoplasma
Filtration
This forces air through HEPA filters which remove more than 99.9% of airborne particles that are .3 µm in size or larger. Used for organisms that are a BSL OF 3 or higher
Laminar flow biological safety cabinets
Why are many forms of electromagnetic radiation harmful to microbes?
Causes DNA damage
compares efficiency of disinfectants
This is a benchmark to which all other disinfectants are measured
Is based on the highest delusion or lowest concentration of a disinfectant that will kill all the bacteria after 10 minutes of exposure. Known as the maximum effective delusion. Dividing the reciprocal of the maximum effective dilution for the test agent by the reciprocal of the maximum effective solution for phenol gives us the phenol coefficient
Phenol coefficient
^ in coefficient, ^ The efficiency of disinfectant
Produced by fungi and fungal like bacteria that exert antibacterial actions
Natural agents
The use of one micro to control the growth of another
Biocontrol
Ex:probiotics, phage therapy
Used to treat bacterial infection diseases with a virus that is targeted to that bacterium. Bacteriophages are viruses that infect bacteria. A possible alternative to anabiotic‘s in the face of rising antibiotic resistance
Phage therapy
