BIO 350: Exam 2

What macromolecules (major units) are needed for life?

There are both environmental and chemical factors needed for growth. All cells need access to large amounts of carbon (largest component), oxygen, phosphorus, sulfur, and oxygen (macronutrients) to build macromolecules.

Various micronutrients are also required by microbes: including several metal ions (Na+, Mg2+, Mn2+, etc.). Often required for protein structure and activity (divalent cations are for instability).

What macromolecule is the largest component needed to build macromolecules?

Carbon

Why are metal ions required for microbes to grow?

They stabilize protein structure/activity (as DNA is very negative).

What does bacterial growth refer to?

Both individual cells getting bigger, as well as populations increasing through binary fission.

Compare and contrast binary fission and mitosis.

Both are forms of asexual cell division that produce two genetically identical daughter cells. Binary fission happens in prokaryotes, while mitosis occurs in eukaryotes. Binary fission is a fast, simple process, while mitosis occurs in 4 distinct stages. They use different factors to ensure chromosomes are separated and do not stay attached during cell division.

What is an example when separation does not occur in prokaryotes following binary fission?

When looking at diplococcus, as structures are supposed to be attached.

Describe binary fission (bacterial growth).

1. Cell elongates and DNA is replicated (simple nucleoid).

2. Plasma membrane begins to constrict and new cell wall is made (dependent on gram-positive/negative).

3. Cross-wall forms, completely separating the two DNA copies.

4. Cells separate.

What are the differences in gram-positive/negative cell walls?

The thickness of the peptidoglycan layer and presence of an outer membrane.

With gram-positive cell walls, they have a thick peptidoglycan layer but are missing an outer membrane. Gram-negative cell walls have a thin peptidoglycan layer but have a present outer membrane (with a lipid bilayer outside peptidoglycan).

How can different types of microorganisms be grown in the laboratory?

Different types of media are utilized for microbial growth due to differences in metabolism and chemical/environmental requirements so need a different nutrient broth to see growth - microbes can be grown on both solid (agar plates) and liquid media (broths).

True or False: Microbes can be grown on both solid (broths) and liquid media (agar plates).

False.

How do you produce agar?

1. Powdered agar is added to a flask containing liquid media.

2. The medium is heated to above 85 degrees C to melt the agar. This allows the media to be stabilized, killing everything that should not grow.

3. The medium is allowed to cool to around 45 degrees C, then poured into a plate and allowed to solidify.

What are the different kinds of culture media?

By chemical composition - defined and complex.

What is defined culture media?

A classification of culture media where the exact nutritional chemical substances that are in the medium are known (extremely stringent and carefully measured out).

What is complex culture media?

A classification of culture media that contains complex ingredients like yeast extract, peptone, etc. where the exact chemical formula is known and varies slightly by batch.

What is a fastidious organism? What does that say about the type of culture media needed to allow for growth of that organism? Name an example.

Any living thing, usually a bacterium or microorganism, that has complex or specific nutritional and environmental requirements to grow. They need a special nutrient agar - enriched, defined media containing specific vitamins, amino acids, etc. to be able to survive and multiply.

An example of this type of organism is Leuconostoc mesenteroides.

What kind of media contains yeast extract? Why?

Complex media because that type of culture media does not need exact measurements, and yeast extract contains dead, ground up yeast that have every macromolecule needed for life.

What kind of media contains peptone? Why?

Complex media because it is soy protein that’s been digested but the amount is dependent, and complex media does not need exact measurements.

What is special about the culture media needed for Thiobacillus thiparus?

It is strictly inorganic, sulfur-based and completely lacks organic carbon sources (like glucose or peptone), meaning it has a defined medium. It is an obligate chemolithoautotroph, so the media needs to include sodium and some type of carbon source.

How do you obtain a pure culture?

You must separate a signle mcirobial cell from a mixed population and allow it to grow an isolated colony. This can be done in two different ways:

1. Spread/pour plate methods

2. Streak plate method

3. Selective/enrichment media

What is the spread method?

A technique to obtain pure culture, which involves a small amount (0.1 mL) of diluted bacterial suspension that is dropped onto a center of a solid agar plate. A sterile rod (hockey stick) is used to spread the liquid evenly across the entire surface. Bacterial colonies will grow on the surface of the agar.

What is the pour method?

A technique to obtain pure culture, which involves a 1 mL diluted, liquid cooled (but not solidified) agar culture that is poured onto an empty, sterile Petri dish. The agar cools and solidifies, trapping the bacteria in place. They grow into isolated colonies on the surface and embedded inside the agar.

What kind of technique is good for obtaining a pure culture of anaerobes: spread or pour method?

Pour

What is the difference between the spread and pour plate method?

The primary difference is where the bacteria end up growing (surface for spread, but embedded and surface for pour) and how the sample is introduced to the agar (dropped onto surface of agar for spread, but mixed with liquefied molten agar before solidification).

What happens if the microbes cannot withstand the heat of the molten agar in the pour plate method of obtaining a pure culture?

The microbes can die due to heat shock.

What is the streak plate method?

A technique to obtain a pure culture (the most common), where a sterile inoculating loop is dipped into a mixed culture and streaked back and forth across one quadrant of an agar plate. The loop is resterilized, dragged through the first quadrant, and then streaked into the second (repeated 3 times). The bacterial density decreases with each quadrant so that individual cells are deposited far apart and grow into isolated pure colonies.

What are microscopy cell counts? How does it work?

A lab method used to estimate the total number of microbial cells in a liquid sample by counting them directly under a microscope. To perform this, a Petroff-Hauser counting chamber is utilized.

1. A bit of sample is poured onto coverslip (grid has 25 large squares).

2. Optional: potential staining before looking into microscope for more contrast

3. Look through microscope and will observe cells in large squares.

4. Physical objects are counted (which is why need good contrast).

5. To calculate the number in the total sample, multiply the number of cells x 25 large squares x mL.

What is utilized to perform microscopy cell counts to obtain number of cells?

Petroff-Hauser counting chamber

How do you obtain a viable cell count?

Using serial dilution

How do you perform a viable cell count using serial dilution?

Sample is diluted, 1 mL sample are poured onto 9 mL of broth and colonies are counted. The observed number is multiplied by the dilution factor.

What are the different ways you can measure total cell number?

Petroff-Hauser counting chamber and turbidity

How do you use turbidity to measure cell number in solution?

This method can only be used in solution (liquid culture). A spectrophotometer uses a specific wavelength that shoots the light through a filter, that then passes through a sample containing cells. The cells would then scatter light because they are “big” physical objects that disrupt the photons, allowing for an estimation of cell concentration to be made without having to wait for colonies to grow. The spectrophotometer would then read the optical density (turbidity). The cloudier the solution, the more cells are present.

What is the problem with measuring turbidity?

Once it saturates or no more light can come through, it doesn’t matter if the cell count changes, the spectrophotometer cannot read any differences between no light and “increased” no light.

What is generation time?

The time it takes for a microbial population to double in size (also known as doubling time).

What is exponential growth?

A phase of rapid development where the cell population doubles at a constant, repeating interval (e.g. 1 cells becomes 2, then 4, 8, 16, 32). This occurs because every new cell generated is capable of dividing again. This requires optimal conditions.

How is turbidity plotted on a graph?

Time (hours) vs. optical density or cell numbers/mass (can use pour/spread to compare) vs. optical density

How does a bacterial population change over time in a closed batch culture?

When bacteria are inoculated into a closed environment (like a flask of broth with a fixed amount of nutrients), they follow a predictable pattern of growth known as bacterial growth curve that consists of four distinct phases:

1. Lag phase - there is no immediate increase in cell number because the cells are adjusting to their new environment and not yet dividing.

2. Exponential or log phase - rapid growth occurs, as cells are dividing at their max possible rate under the given conditions (population doubles - generation time).

3. Stationary phase - saturated culture; the growth rate slows down and number of new cells being produced = the number of cells dying so population stays constant This occurs because nutrients are running out and toxic, metabolic waste products are beginning to accumulate.

   1. Decline phase - the number of dying cells outnumbers the number of new cells and population begins to decrease due to the environment becoming completely toxic and nutrients being depleted; some bacteria can survive these harsh conditions if they form endospores to survive this phase.

What factors impact growth conditions of microbes?

Temperature, pH, osmolarity, nutrients, and oxygen.

What are biofilms?

A highly organized, cooperative community of microorganisms that stick to surfaces and secrete slimy, protective matrix (like polysaccharides, extracellular DNA, signaling molecules) to protect themselves.

True or False: Most prokaryotes in the environment live in biofilms.

True.

What are planktonic bacteria?

Lone, individual cells that float around.

What are microenvironments?

The tiny local environment that a microbe experiences.

How do microenvironment and biofilm relate?

Aerobic and anaerobic microorganisms can live in the same biofilm due to different microbes experiencing different microenvironment.

What are the organisms that can live in extreme cold (0-12 degrees C) called?

Psychrophiles

What are organisms that can live at normal environmental conditions (9-48 degrees C) called?

Mesophiles

What are the organisms that live at temperatures a little above normal (41-68 degrees C) called?

Thermophiles

What are organisms that live at higher temperatures (66-112 degrees C) called?

Hyperthermophiles

True or False: Archaea are the only prokaryotes that can survive temperatures above 90 degrees C.

True.

Where are hyperthermophiles found?

Usually in hot springs or geothermal vents at the bottom of the ocean that evolved to survive extreme environments.

How do saturated and unsaturated fatty acids affect plasma membrane?

Saturated has no double bonds, so fatty acid tails are able to compress more closely together, forming a rigid plasma membrane and solid molecule. Unsaturated have double bonds, forming kinds in tails of fatty acids, which forms a kink/bend allowing for a more flexible plasma membrane so liquid molecules.

At cold temperatures, are there more saturated or unsaturated fatty acids? Why?

There are more unsaturated fatty acids because do not want to form a rigid plasma membrane that would cause the molecule to solidify; want a flexible plasma membrane to maintain liquid structure and prevent expansion that can lead to burst.

At hot temperatures, are there more saturated or unsaturated fatty acids? Why?

There are more saturated fatty acids because want a rigid membrane that can withstand the heat and not boil as a liquid.

How do thermophiles and hyperthermophiles withstand their extreme environments?

They alter their:

1. Amino acid compositions for more ionic bonds to form, as they are stronger and able to withstand heat to prevent denaturing.

2. Utilize solutes like diglycerol phosphate that increases the stability of proteins to protect folding.

3. Membrane lipids to contain longer chains and saturated lipids (as they have higher melting points).

How do psychrophiles withstand their extreme environments?

They alter their:

1. Amino acid compositions to form more alpha-helices and fewer beta-pleated sheets, as the former are more stable; form fewer ionic bonds, allowing the membrane to be more flexible.

2. Membrane lipids to contain shorter chains and unsaturated lipids (have lower melting points) to maintain membrane flexibility as well.

3. Utilize solutes like glycerol to that function as cryoprotectants to protect protein from denaturing in the cold.

4. Excrete a slime layer on the cell surface called a exopolysaccharide (cryoprotect).

What solute is used in thermophiles and hyperthermophiles to withstand their extreme environment?

Diglycerol phosphate

What solutes are used in psychrophiles to withstand their extreme environment?

Glycerol and slime layer on cell surface (exopolysaccharide) as cryoprotectant

What are the extremophile prokaryotes: archaea or bacteria?

Archaea

What are the physiological classes of organisms in terms of the pH they can withstand?

Neutrophiles, acidophiles (pH < 5.5), and alkaliphiles (pH > 8)

How do acidophiles withstand their extreme environments?

Because they live in environments with high proton concentration, the ETC does not work well, as the massive [H+] outside of the cell does not allow for proton motive force. H+ does not want to be let into the cell because it would acidify the cytoplasm, denaturing the proteins, so they utilize …

1. Extra membrane proteins: pump excess protons out

2. Membrane lipids: tetraether lipids (less permeable to H+), as they cross-link the phospholipids from the outer to the inner leaflet, so fewer gaps within the plasma membrane.

How do alkaliphiles withstand their extreme environments?

They do not have lots of H+ in their environment, so there is no generation of a proton motive force that could sustain life, so have to convert H+ pumps and ATP synthetase by …

1. Membrane gradients: use other ions (Na+) instead of protons to generate a Na+ motive force

2. Membrane lipids: negatively charged head groups to balance Na+, so more phosphates on glycerol heads

3. Solutes: trehalose is used as a buffering agent to protect from alkaline stress and prevents denaturing.

What is used as a buffering agent in alkaliphiles to withstand their extreme environments?

Trehalose

What are the types of organisms that live in extreme osmolarity environments?

1. Nonhalotolerant organisms

2. Halotolerant organisms

3. Halophiles

4. Extreme halophiles

What are nonhalotolerant organisms?

Extreme organisms that cannot live at basically any concentration of salt (ex. E. coli).

What are halotolerant organisms?

Organisms that do not require salt but can survive at high amounts (ex. Staphylococcus aureus).

What are halophiles?

Organisms that are obligate salt-lovers and require salt to live (ex. Aliivibrio fischeri - live in the ocean).

They synthesize or important compatible solutes (undergoing a metabolic change) to maintain water balance.

What are extreme halophiles?

They have to live in really salty environments, and if not they would die (ex. Halobacterium salinarum).

What can happen to a cell in a hypotonic solution that has a weak/damaged cell wall?

The cell bursts (osmotic lysis), as water is traveling into the cell and the wall cannot contain the swelling. Cells with peptidoglycan cell walls try to fight the turgor pressure.

What can happen to a cell in a hypertonic solution?

Since water is moving out of the cell, the cytoplasm starts to shrink (plasmolysis), which is detrimental to the cell. The cell wall does not move because is rigid, but the cell might take on a wrinkled structure (seen in halotolerant and halophiles).

What are obligate aerobes? What is their growth pattern in a thioglyclolate tube?

They require oxygen to grow, as use aerobic respiration for energy. They would only grow at the very top of a tube, as it is an area of high oxygen.

What are obligate anaerobes? What is their growth pattern in a thioglyclolate tube?

Oxygen is toxic to them. They use fermentation or anaerobic respiration. They only grow at the very bottom of a tube, where there is no oxygen.

What are facultative aerobes? What is their growth pattern in a thioglyclolate tube?

They do not need oxygen, but grow much faster with it. They can switch between aerobic respiration (high energy) and fermentation (lower energy). They grow throughout the tube, but they are heaviest at the top.

What are microaerophilic aerobes? What is their growth pattern in a thioglyclolate tube?

They use oxygen, but only a tiny amount. They require oxygen for respiration, but high levels of oxygen overwhelm them. They grow in a narrow band just below the surface of the oxic zone.

What are aerotolerant anaerobes? What is their growth pattern in a thioglyclolate tube?

They do not need oxygen (ignore it completely). They strictly use fermentation to grow. They evenly scatter all the way through the tube.

Why do oxygen requirements differ between microorganisms?

Oxygen was released into the atmosphere via the great oxidation event by photosynthetic prokaryotes and since has been produced by the former and other photosynthetic organisms like plants. However, some things cannot survive with oxygen present due to production of toxic oxygen species (superoxide radicals).

How do aerobes survive against toxic oxygen products?

They use enzymes like superoxide dimutase and catalase (protects against peroxide - if add hydrogen peroxide directly to colony and if bubbles appear, the organism is catalse positive) to destroy oxygen products.

True or False: Disinfection is sterility.

False.

What is sterility?

The complete and total destruction of all living microorganissm, including the most resilient biological structures (endospores, etc.).

What is disinfection?

The process of eliminating most or all pathogenic (disease-causing) microorganisms from inanimate objects, but fails to kill highly resistant bacterial endospores.

What is aseptic?

Absence of contamination (for labwork or surgery).

What is bactericidial?

The killing of bacteria. This can be visualized in a turbidity measurement but different in serial dilution plating.

What is bacteriostatic?

Stops the growth of bacteria (not dead, just paused).

What is biocidal?

The killing of living things.

What is biostatic?

The stopping of growth of living things.

What is germocidial?

The killing of infectious agents like pathogens in general.

What is germostatic?

The pause in growth of infectious agents.

What are physical methods of microbial control?

Temperature, filtration, desiccation, osmotic pressure, and radiation.

How is temperature a physical method of microbial control?

Heat can be used to boil something that is not a thermophile, denaturing protein and disintegrating the membrane. This is not enough to kill endospores and some viruses, as they need pressure (autoclave).

Pasteurization uses mild heat to reduce the spoilage organisms (eliminates pathogenic microbes in food).

Refrigeration (4 degrees C) causes bacteriostaticism (limiting growth of E.coli).

What is autoclaving?

A physical method of microbial control that is uses steam under pressure to achieve complete sterility, killing all bacterial cells and endospores.

What is filtration?

A physical method of microbial control that filters out microbes.

What is desiccation?

A physical method of microbial control that removes water (dehydrates) from an environment to inhibit microbial growth.

What is osmotic pressure?

A physical method of microbial control that uses high concentrations of high salt to create a hypertonic environment, drawing water out of the cell to prevent it from lysing from turgor pressure.

What is radiation?

A physical method of microbial control that uses electromagnetic energy to damage or destroy cellular components, like DNA, leaving it unabel to replicate.

What are phenolics and terpenes (plant extracts) used for?

They are a chemical method of microbial control that disrupt the membranes and denature proteins.

What are halogens (chlorine and iodine) used for?

They are a chemical method of microbial control that are powerful oxidizing agents, most common is sodium hypochlorite (bleach), that oxidizes proteins and cellular components. Since ETC requires reduced electron carriers (like NADH and FADH2), if they are constantly oxidized, then they can no longer capture the electron to make energy.

What are alcohols used for?

They are a chemical method of microbial control that unfolds proteins and dissolve membranes, but they do not kill bacterial endospores (so not a great disinfectant unless completely submerged).

What are surfactants (detergents)?

They are a chemical method of microbial control that acts on a surface and mainly involved in disrupting membranes.

What are antibiotics (antimicrobial drugs)?

They are a chemical method of microbial control, where they kill or selectively inhibit the growth of microorganisms without causing harm to host organism.

What is decimal reduction time (D)?

The time it takes to reduce by 1 order of magnitude the survival fraction of microorganisms at a specific temperature.

What is autoclave time?

The duration a load must be held at a specific sterilization temperature and pressure to achieve complete sterility.

What is bacteriolytic?

Bursts bacterial cell. This can be seen in turbidity or optical density.

How can you tell how strong a chemical agent is at microbial control?

Minimum inhibitory concentration (MIC)