Unit 2 Cultivating microorganisms Flashcards
Cells need a lot of this type of nutrients
Cells need a little of this type of nutrients
Macronutrietns
Micronutrients
What are the macronutrients?
What do they build
Carbon, Nitrogen, Phosphorus, Sulfur, Oxygen
Macromolecules
What are some micronutrients?
What do they do?
Na+, Mg2+, Mn2+ (metal ions)
Assist in protein structure/stability
These assimilate carbon from inorganic sources
These assimilate carbon in preexisting organic form
Autotrophs
Heterotrophs
These capture light energy to product ATP
These capture energy from oxidation of reduced organic or inorganic compounds.
Phototrophs
Chemotrophs
These acquire electrons from organic molecules
These (rock eaters) acquire electrons from inorganic sources (H2 gas and elemental sulfur)
Organotrophs
Lithotrophs
What do organotrophs commonly use as a source of carbon?
Do chemotrophs develop ATP?
CO2
Yes
This is an organic source of nitrogen that usually results in ammonia
These are inorganic sources of nitrogen
Decomposition
nitrite, nitrate, N2
Nitrogen is critical to making macromolecules, which ones in particular?
Nucleic Acid
Amino Acid
What is the N acceptor molecule and what form does it usually accept nitrogen in?
What does it form?
α-ketoglutarate
Glutamate
These microbes can synthesize all need macromolecular precursors froma single carbon source
Prototroph
These cannot synthesize all the needed precursors from a single source
Auxotrophs
What can grow an minimal media, prototrophs or auxotrophs?
Most fall into which category?
Prototrophs
If an auxotroph requires something for survival, it is called
Essential
Growth rate depends upon the amount of ___ in the environment
Nutrients
One key nutrient, available in the lowest amount, will dictate how much growth can occur over time. What is it called
Limiting factor
What is the limiting factor for a lot of microbes?
What does it do?
Iron
It passes around electrons efficiently in the ETC
These grow in the presence of oxygen
These require the presence of oxygen
These grow best when there is less oxygen than normal
Aerobes
Obligate Aerobes
Microaerophiles
A lot o microaerophiles are ______, they live beneath the surface but not under completely anaerobic conditions. These microbes have a lot of _____ to know their position
aquatic
Magnetosomes
These type of microbes grow without the presence of oxygen
These aren’t harmed by oxygen but don’t use it
These can’t grow when oxygen is present
These can use oxygen but can also grow in the absence of oxygen
Anaerobes
Aerotolerant anaerobes
Obligate anaerobes
Facultative anaerobes
Do facultative anaerobes grow faster or slower in the presence of oxygen?
Faster with oxygen, slower without
This toxic oxygen product comes from photochemical/photosynthetic microbes, and is a product of peroxidase enzymes.
What defends against it?
Singlet Oxygen O2
Antioxidants like carotenoid pigments
These three toxic oxygen products are by products of metabolism, aka by products of the reduction of O2 during respiration and other biochemical redox reactions
Superoxide anion O2-
Hydroxyl ion OH-
Hydrogen peroxide H2O2
This is the least reactive of the three metabolic toxic oxygen products, which makes it the most lethal
This is the most reactive and least lethal
This is in the middle
Superoxide anion
Hydroxyl ion
Hydrogen peroxide
What does the superoxide anion become when its broken down via enzymes?
H2O2
H2O2 enzymes produce ____ bubbles
O2 bubbles
Our cells have this enzyme which produces O2, a toxin that kills off strict anaerobes. You need enzymes like these if you use oxygen or grow in its presence.
Catalase
Peroxidase is another enzyme
Certain ______ help keep the cellular environment at a required pH to limit competition
Extremophiles
pH affects ______ and ______
macromolecule structures and transmembrane electrochemical gradients
Each microbe has an optimal pH range for growth.
These prefer pH> 8.5
These pH 5.5-8.5
These pH < 5.5
Alkalophiles
Neutrophiles
Acidophiles
Naturally, preferred pH is closer to __ most often for microbes
There are exceptions, especially
They are used for
The enzyme it uses is
7
Alkalophiles
Stain removal, which breaks down proteins that make stains
Protease
Different _____ concentration can result in influx of water into or efflux from the cell. It can cause stress to the cell by swelling or shrinking
What do bacteria have that prevents it from bursting?
Solute
Cell wall
Water must also be available for biochemical reactions. This is measured in terms of
Honey has an aw of 0.6, and it is so hypertonic that microbes can’t grow in it. What is the solute?
What is the aw of water?
Water availability (aw)
Sugar
1.0
This type of bacteria can handle very cold temps, even below freezing and up to 10 degrees celsius. Found in glaciers, high in mountains. THey grow very slowly
Psychophiles
This type of bacteria can handle moderate temps, like our body temperature, and therefore comprises most of the disease causing bacteria. They grow in soil, warm water, places that aren’t too hot
Mesophiles
These bacteria grow at not quite boiling temperatures, grow in hot springs
These bacteria can grow past boiling temps, found in deep sea vent type environments
Thermophiles
Hyperthermophiles
Which type of cell is most distributed throughout all of the categories of temperature loving bacteria?
Archea.
In terms of growing microorganisms, this is the food source that has macro and micronutrients
Media
Microbes can be grown on two types of media..what are they
Which type allows us to examine for things like color, texture and shape to help identify microbes
Solid (agar plates) and liquid (broths)
Agar plates
To create agar, powdered agar is added to a flask containing liquid ______ to a final concentration of 1.5g/100mL (1.5%)
The medium is heated to above ___ to melt the agar
The medium is allowed to cool to around ______ then poured into a plate and allowed to solidify
media
85 degrees celsius
45 degrees celsius
What are the two composition types of media?
This is of unknown chemical composition, even if there is only 1 unknown ingredient
This is of precisely defined chemical composition
Complex or defined
Complex
Defined
Infusion and extract are signs of a ___ composition
Complex
This type of specialized media allows for isolation of microbes with specific properties, like salt tolerance on mannitol sugar agar plates, antibiotics, or dye. It allows some microbes to grow and blocks others
Selective media
Which type of bacteria do dyes block, gram positive or gram negative?
Gram positive
This type of specialized media allows certain microbes to be recognized based on visual reaction in the medium, like lactose fermentation of E. coli on MacConkey agar. pH indicators or difference in colors from dyes can be used. ALL bacteria grow in this.
Differential media
This type of specialized can be used to increase a particular population of microbes with a specific property from a mixture of cell types. Sabouraud dextrose agar can be used to check for mold in air by enriching mold spores or bacteria to form fungi
Enrichment Media
EBM media has 2 dies, + can’t grow and gram negatives can. It also can cause different colors to form enabling differentiation
If only gram negatives are grown on the plate, is this selection media, differentiation media, or both?
If both gram positive and negative are grown, what does it become then?
Differentiation
Both selecive and differenation media
In the blood agar example, what are the zones of hemolysis?
What is the virus going after?
Areas where red blood cells have been lysed
Iron in hemoglobin of red cells.
One of the benefits of solid medium is that cells are held in place on the surface and can be isolated. This can lead to separating a mxture of cells into a pure population.
What are the 3 methods?
Is one strain or one species desired?
Streak Plate
Spread Plate
Pour Plate
One strain, more specific
This technique uses inoculating lube to wipe bacteria on the surface of a plate
______ streaks pick up small amount of bacteria from a previous streak and drag it in a new ______, diluting cells.
What is done to verify that the cells in a colony are the same?
Streak Plate
Quadrant streaks, quadrant
Gram stain
These techniques both started with a diluted sample
This one doesn’t use molten agar, and forms colonies on the surface of the plate
This one uses molten agar creating bacterial colonies on the surface and within the agar,
Spread and Pour Plate
Spread plate
Pour plate
In E. coli, a facultative anaerobe, what happens if the pour plate method is used, specifically, how do colonies form?
Colonies at the surface where oxygen is, they grow small colonies anaerobically within the agar
For unculturable bacteria, DNA can be amplified and sequenced by
Sequences can be used to produce fluorescent probes that will bind to complimentary DNA in this technique, allowing identification. The fluorescent probes bind to known sequences of known microbes.
PCR
Fluorescent in situ hybridization or FISH
During fish, do you actually kill the sample? Does it destroy the sample?
It kills the sample but keeps them intact using a chemical fixative.
The method of identifying unculturable bacteria when DNA that is left behind by microbes is isolated from an environmental sample and sequenced using PCR techniques. The DNA can then be analyzed via computer programs. You DON’T actually analyze the cells just the bacteria.
Not always exact match, can tell you similarities/related microbes. Helps to better formulate growth recipes.
Metagenomics
One reason why growing microbes in the lab is difficult. Some microbes may be accustomed to growing with their friends and neighbors to be isolated
Microbial Consortia
What are the three ways of measuring and counting microbes?
Direct Counts
Viable Cell Counting
Turbidity Measurements
This method of counting microbes uses a special slide with an etched grid and a fixative with a stain.
A known volume (liquid) of cells is loaded onto the grid and cells are counted under a microscope.
What are the pros?
The cons?
Direct Counts
Cheap fast and easy
Cannot differentiate living and dead cells. Can’t say how many were alive or dead at the start of the experiment. Also accuracy depends upon person doing it.
This type of cell count counts living cells.
N=DV
What does N stand for?
D?
V?
Viable Cell Counts
Colony forming units (cells) per millileter
Cumulative dilution factor
Volume put on plate
The step dilution factor equation 1/(1+Vd/Vs)
Vd=?
Vs=?
To get cumulative dilution after successive dilutions, what do you do to the values generated from each dilution?
Volume diluted
Volume of sample
Multiply them
For viable cell counts, serial dilutions and CFUs, the dilutions are plated and what is often used before incubation
What happens after incubation
Spread Plate
Counting
How do you calculate the CFUs per milliliter of initial culture?
What is the max number of colonies that is countable?
What is the minimum?
Multiplying the number of colonies be the inverse inversion factor
300
3
A bacterial culture is diluted to 2*10^-6
0.5ml sample of diluted culture is plated out. 267 colonies grew. The viable cell density in the culture would be how many cfu/ml?
N=267/[(210^-6)0.5ml)
N=2.67*10^8 cfu/ml
This method of counting cells uses a spectrophotometer than sends light through a culture.
If the tube is cloudy, light won’t get through the tube and strike the unit’s sensor (high absorbance). It can give a rough measure of cell density in the tube.
Absorbance is also called?
What is transmittance units?
Turbidity
Optical Density
%
More cells in turbidity measurement is more or less transmittance?
What is a con to turbidity measurement?
Less transmittance
Can’t distinguish between dead and living cells (some cells intact, debris from cells that aren’t intact, etc)
One reason that cell density is measured, it determines the four basic phases of a microbe’s _________ in a closed culture over time
Microbial Growth Curve
During this phase of microbial growth, microbes are gearing up for steady growth
During this phase, microbes are replicating at a constant and steady exponential rate
During this phase replication has halted due to lack of nutrients or excessive wastes, or the replication rate is now equal to the death rate
During this phase, nutrients are depleted and waste levels are high, cells die at a steady exponential rate (also log phase)
Lag Phase
Log Phase
Stationary Phase
Death Phase
What does it mean when a microbe grows in a close culture (or batch)?
No more food put in, no waste removed
What are the two logarithmic phases of the microbial growth curve?
Log phase
Death Phase
Is it possible to see cells during the lag phase during the viable cell count?
What about for turbidity?
No, they aren’t dividing yet so we can’t see them
Gets cloud with cells in there so yes.
There is a difference in slopes between turbidity and viable cell count. Which slope is sharper?
Why?
The viable cell count is sharper, population appears to decline more rapidly
Occurs because debris of dead cells causes cloudiness that is still measured during turbidity
These type of microbes don’t lyse right away when they’re dead, causing the turbidity to appear as a straight line during stationary phase much longer than the viable cell count
Nonautolytic bacteria
When analyzing the growth curve, one can determine:
The time to double the population in the exponential phase
The number of generations per unit of time (inverse of the generation time)
The maximum population density and/or amount of cellular material produced by the culture
Generation Time
Growth Rate
Growth Yield
If you know generation time, you also know
Growth Rate (and vice versa) because they are inverse values of one another
In terms of population growth, a relationship exists between the initial number of cells present in a culture and the number present after a period of exponential growth
N(t)=N(o)*2^n
N(t) is the
N(o) is the
n is the
Final cell number
Initial cell number
Number of generations during the period of exponential growth
You have 10 cells and they go through 5 generations, how many cells produced?
10*2^5
=320 cells produced
The equation g = t/n is the equation for the amount of time it takes the population to double, the entire time period of cell growth and cell division
t=
n=
Generation time
duration of exponential growth
number of generations during the period of exponential growth
Another equation for solving for the generation time g=t/(3.32(logN-logN)
this equation is equal to t/n
!
What is the generation time of a bacterial population that increases from 10,000 cells to 10,000,000 cells in four hours of growth
g=4/(3.32*log10,000,000 - log10,000)
g=4/(3.32*3)
g=0.4 hours
convert to minutes 0.4 * 60 minutes = 24 minutes
This phrase means no vegetative (metabollically active) cels or endospores
Endospores are resistant to bad environments, hard to get rid of. Heat stable. Chemicals don’t hurt. UV doesn’t kill them.
Sterile
This method of removing microbes is done by physical removal, purifies liquids.Uses sand/charcoal, etc for liquid to seep through.
Nowadays what is used as a medium
If you want to keep out vegetative cells, what size should the pores be?
What about viruses?
Filtration
Teflon filters
0.2-0.45 micrometers
10-100 nanometers
Heat labile media is commonly put under what purification/removal system?
Filtration. No heat.
For manipulation/removal of microbes via heat, what affect can it have on proteins and nucleic acids?
Heating cells to what tmeperature kills most microbes quickly? Which microbes are these?
Which type of microbes doesn’t it remove?
Denatures them
100 degrees celsius. these are vegetative cells, not endospores or some viruses
Hyperthermomophiles, endospores, materials that can’t be heated/break down (heat labile media)
If you need to use heat to achieve sterility, what do you use? It’s a giant pressure cooker
What temperature and for how long do samples have to be heated for sterility?
Autoclave
121 degrees celsius for 15-20 minutes
What is temperature manipulation at low heat to reduce microbe numbers? Generally about 70 degrees celsius for a half hour. It increases self life and makes things safer for consumption. (Dairy products)
Does it sterilize?
Pasteurization
No.
Unpasteurized dairy is associated with…
Tuberculosis
What else is pasteurized?
Why?
Bottled juice
E. coli outbreak in apple cider via contamination of apples from livestock
Another method of temperature manipulation, lowers temperature
Does it achieve sterility?
How can it damage cells?
What is it good for?
Freezing
No.
Ice crystals
Stop biochemical reactions
Long term preservation (shelf life)
This form of radiation is used to control microbes in 260 to 280 nm wavelengths to damage DNA and form thymine dimers. It can be exploited on non-living surfaces and water
Does it sterilize?
When does it work best?
UV light
No
Long exposure to it
This is crosslinking of adjacent (next to eachother) thymines, causes DNA replication to be erroneous, increases mutation rates causing negative effects on cell.
Thymine Dimers
Where is UV light commonly used to control microbes?
What are the drawbacks?
It is good for drying your clothes outside. (direct contact)
Fumehoods,
Microbes can hide in nooks and crannies, UV is not very penetrating of surfaces (like bottles of water). Must be direct contact (surface of water at water treatment plants)
Bottles of water are more affected by heating.
Wash your hands after transferring clothes from washer to dryer.
!
Many chemicals can kill microbes, or inhibit their growth
These chemicals are used on non-living surfaces to kill potentially infectious microbes
Does sanitizer (disinfectant) sterilize?
Chemicals that can be used on living tissue to kill potentially infections microbes (only used topically)
Disinfectants
99.9% effective, not against endospores.
No.
Antiseptitcs
To truly disinfect, you have to ___ a surface with disinfectant and let it sit 10-20 minutes
Saturate
Can a chemical be used as an antiseptic and disinfectant?
Yes.
What makes a good microbe killer?
Should kill wide range of microbes Shouldn't be corrosive or toxic Shouldn't leave a residue (especially in kitchens) Shouldn't emit fumes or smell too bad Should be cheap Should be temperature stable
This common disinfectant is routinely used in laboratory settings and is also present in most hand santizers, and to clean injection sites. Optimal concentration is 70%
Hence, it can also be an antiseptic
What do they disrupt/destabilize?
It can denature ______ if used for extended periods of time
Alcohols (ethanol, isopropanol)
Membranes
Proteins
This common disinfectant is added to numerous products, including some soaps, deodorants, and cosmetics.
Has more resistance than alcohols because its being used so much. Should be saved for select occasions, soap and water are better to use.
What does it disrupt?
Phenolic Compounds (triclosan
Membranes and enzymes involved in fatty acid (lipid) synthesis
This common disinfectant is commonly added to swimming pools and hot tubs to inhibit microbial growth. Irritating to tissues, used in well ventilated areas. Optimal concentration is 10%
Can it be used as an antiseptic?
Never mix it with
Oxidizing Agents (sodium hypchlorite - aka bleach)
No. Too irritating to tissues.
Ammonia
How do you decide which method to use to destroy microbes?
Know which microbes you're killing Know how many What kind of object needs to be treated? How long/intense should it be How powerful must the chemical method be and how long should it be applied Is toxicity a problem?
This value is defined as the time required to kill 90% of the target organism/microbe under specific conditions
What type of scale is it?
Decimal Reduction Time
Log scale (factors of 10 on the y scale)
