micro bio exam 2

explain the rationale of fluorescence microscopy

Transmission electron microscopy vs scanning electron microscopy

Transmission

• electrons pass through ultrathin sections of a specimen

• internal structures can be seen

Scanning

• electrons scan surface of entire specimen

• external structures and topography can be seen

Prokaryotic cells vs eukaryotic cells (be able to explain)

Eukaryotic: DNA contained with a nucleus

• larger in size

• often MULTIPLE LINEAR chromosomes, in pairds

• DNA wrapped around histones

• divides by mitosis

• organelles (ER, golgi, mitochondria, chlorplasts)

• polysaccharide cell walls, when present

Prokaryotic- lack nucleus

• smaller in size

• single circular chromosome

• no histones

• divides by binary fission

• no organelles

• bacteria: peptidoglycan cell walls

• archaea: pseudomurein cell walls

list common features found in ALL bacterial cells

• cytoplasm

• 70S ribosomes

• plasma membrane

• nucleoid containing DNA

list common features found in SOME bacterial cells

Describe the structure of the cytoplasmic membrane

• semipermeable membrane that encloses the cytoplasm

• made primarily of phospholipids (two leaflets)

how do molecules move across the cytoplasmic membrane through passive mechanisms

• high to low concentration

• no energy expended!!
  
  

• Simple diffusion: small uncharged molecules pass through

• Facilitated diffusion: charged ions and large molecules pass through a transporter protein

how do molecules move across the cytoplasmic membrane through active mechanisms

low to high concentration

requires a transporter protein and ATP

describe the structure and function of ribosomes

• complex that synthesizes proteins

• made of protein and ribosomal RNA (rRNA)

• 70S (50S + 30S subunits)

chromosomes vs plasmids (compare and contrast)

chromosomes

• found in all bacterial cells

• genetic blueprint for cell

• large

• codes ofr cellular respiration, protein synthesis

plasnid

• very samll, small circular rings of DNA

• some bacteria

• codes for antibiotic resistance

identify the components of peptidoglycan and describe its structure

Chain of alternating sugar residues:

• N-acetylglucosamine acid (NAG)

• N-acetylmuramic acid (NAM)

• Chains are linked by polypeptide bridges

describe the structure of the cell wall in gram positive and gram negative bacteria

gram positive

• thick, multiple layers of peptidoglycan

• stains purple

• located outside cell membrane

• held together by teichoic acid and anchored to cell membrane by lipoteichoic acid

gram negative

• thin single layer of peptidoglcan

• contained within periplasm- space between cell membrane and outer membrane

• porins- proteins embedded within the outer membrane that form channels and control entry/exit

provide a molecular explanation for why gram stain isn’t used to stain acid-fast bacteria such as Mycobacterium spp.

their cell walls contain a highly waxy, hydrophobic lipid layer composed primarily of mycolic acids that prevents water-soluble dyes from soaking

HOW and WHY is the bacterial cells wall targeted by components of our immune system as well as antimicrobial drugs

WHY: the cell wall is essential for bacterial living and composed of peptiodoglycan which is unique to bacteria making it an easy target for killing backteria

how: lysozyme- antimicrobial enzyme in tears and saliva which breaks the bonds between NAG and NAM residues in peptidoglycan

beta-lactum antibiotics inhibits the enzyme that forms peptide bridges in peptidoglycan

describe glycocalyx and provide examples

substance found external to the cell wall

• sticky and gelatinous

• made of polysaccharides

• two types:

  • Capsule: common and protects bacteria from phagocytosis

  • slime layer: produced by bacteria in nature

describe the structure of the flagella and explain how it contributes to bacterial motility

allows for movement (locomotion) towards nutrients or away from danger (taxis)

driven by a motor that propels the bacterial cell through the environment

describe the role of fimbria and pili

fimbriae: hairlike appendages that allow for attachment (adherence)

• important for biofilm formation, colonization within host

• many on cell surface composed of the protein pilin

Pili: composed of protein pilin

• one per cell

• motility (gliding and twitching)

• conjugation pilus is a special type of pilus bringing two cells together

what’s the purpose of endospores

They’re resting cells that are produced when nutrients are depleted

• resistant to desiccation, heat, chemicals, radiation

• ensure the survival of a bacterium through periods of extreme environmental stress,

explain the role of prokaryotes in the evolution of mitochondria and chloroplasts

Endosymbiotic theory- largter microbical cells engulfed smaller bacterial cells, developing into the first eukaryotes

• ingested photosynthetic bacteria and became chloroplasts

• ingested aerobic bacteria and became mitochondria

define metabolism

all chemical reactions within an organism

distinguish between catabolism and anabolism

Catabolism- release energy (break down complex molecules)

Anabolism- require energy (building of complex molecules)

describe the role of ATP and enzymes in metabolism

energy is stored in the form of ATP during metabolism

catalyzed (sped up) by enzymes

• position substrates in a way that favors their conversion into products

how do enzymes convert substrate(s) into products

they bind specifically to active sites and lower the energy needed to start chemical reactions

why do enzymes exhibit specificity

they have a unique structure so their active site only matches a specific target substrate because of its physical shape and chemical properties

how does temperature impact microbial growth at the molecular level vs cellular level

Molecular: affects rate of catalysis

• faster at higher temperature (increase interaction between substrate/enzyme)

• slower at lower temperatures

Cellular:

• low temp: cellular processes slow/stop it inhibits growth and division

• high temp: operate at peak rates maximizing rate of cell division and generation time

• rly high: can cause cell death and cause denaturation

how does heat denature enzymes (use appropriate terminology)

•A protein is a chain of amino acids held together by strong covalent bonds (peptide bonds)

•A protein folds into a distinct conformation (3D structure) due to hydrogen bonds between non-adjacent amino acids

•Heat can break the weak hydrogen bonds, causing the protein to misfold

•A misfolded protein is no longer functional

how does pH impact microbial growth at a molecular and cellular level

molecular: alters the H+ ions by brreaking or altering chemical bonds

how does an inhibitor affect enzyme activity

affects the function of an enzyme

distinguish between a competitive inhibitor and allosteric inhibitor

Competitive: inhibitor binds the active site, preventing the substance from binding

Allosteric: (non competitive) inhibitor binds at another location causing a conformational change and loss of the active site

how does oxygen utilization distinguish between cellular respiration and fermentation

Respiration- requires O2 as an electron acceptor (only aerobic bateria)

Fermentation: no O2 requirement

distinguish between cellular respiration and fermentation based on oxygen utilization

compare and contrast the processes of cellular respiration and fermentation

• Starts with glycolysis?

• Involves the Krebs cycle?

• Involves the ETC?

• Uses an organic molecule as final electron acceptor?

• Energy yield/efficiency?

Cellular Respiration:

• start with glycolysis

• Krebs cycle comes after

  • Citric acid cycle, TCA

• then ETC

• Requires a final electron acceptor; usually oxygen (aerobic)

Fermentation:

• Start with glycolysis

• does not use O2

• No Krebs cycle or ETC

• uses an organic molecule as a final electron acceptor

• **compared to respiration, it produces much less energy per molecule of glucose, BUT it’s produced faster in fermentation

• produces acids (lactic acid), ethanol, gases (CO2)

list the products commonly produced during fermentation

1. acids (lactic acids)

2. alcohols (ethanol)

3. gases (CO2)

using the fermentation test as an example, explain how biochemical tests are used to differentiate between bacteria

1. Inoculate media that contains a single carbohydrate (glucose) to see if the microbe has the enzyme to break it down

2. the media also contains a pH indicator that changes color if pH drops (starts as red/orange and turns YELLOW is produced by fermentation

   1. this means that theirs a drop in pH because acids are being produced

3. Gas may be produced and captured in a Durham tube (CO2 or H2 produced during fermentation)

classify bacteria based on their carbon and energy requirements

Photoheterotrophs: light energy, organic compounds

Photoautotrophs: light energy, CO2 carbon source

Chemoheterotrophs: chemical energy, organic compounds

Chemoautotrophs: chemical energy, CO2 carbon source

what is meant by ‘microbial growth’

growth of a bacterial population (number of cells) not the size of cells

• have physical and chemical requirements

  • Physical: temperature, pH, osmotic pressure

  • Chemical: nutritional requirements (carbon, oxygen)

classify bacteria based on their temperature requirements

Cold: psychrophiles

Moderate (room temp): mesophiles

hot: thermophiles

classify bacteria based on their pH requirements

low pH: acidophiles

neutral pH: neutrophiles

high pH: alkaliphiles

***most bacteria especially human are neutrophiles

how do external solute concentrations impact the flow of water into/out of a cell

water flow is dictated externally by osmosis where water naturally moves from a low to high solute concentration. this direction and impact depends on the outside environment compared to inside

Hypotonic: lower solute concentration outside the cell; water rushes INTO the cell; cell swells and bursts

Hypertonic: higher solute concentration outside the cell; water rushes OUT of the cell; cytoplasm shrivels and colllapses

describe the optimal environmental condition for a halophile

microbe that thrives in high salt environment

• even as high as 30% (for comparison ocean water is 3%)

why do some bacteria require oxygen while other bacteria cannot tolerate it

Required: they rely exclusively on aerobic respiration to generate ATP; without oxygen their entire energy production line shuts down completely causing them to starve

Can’t tolerate: O2 is highly reactive and will damage macromolecules in the cell.

• ROS- reactive oxygen species: group of compounds derived from O2 that damage cells. they tear apart DNA, proteins and cell membranes

how does aerobic bacteria handle reactive oxygen species (ROS)

they produced specialized neutralizing enzymes to regulate stress

classify bacteria based on their oxygen requirements

Obligate aerobes- requires oxygen, rely soley on respiration for energy

Faculative anerobes- can grown in the presence or absence of oxygen

• perform respiration when oxygen is present

• perform fermentation (or aerobic respiration) when oxygen is absent

Anaerobes- unable to use oxygen oxygen and most are harmed by it

• perform fermentation or anerobic respiration

Microaerophiles- require oxygen concentration lower than air

describe the term media and use it appropriately

(singular = medium) also called growth media or culture media, collection of nutrients used to grow bacteria in the lab

when working with a unknown sample, microbiologists streak it onto multiple layers of culture media to encourage bacterial growth

describe the term inoculate and use it appropriately

The process of sterilizing and transferring microbes into media

“I collected bacteria with a sterile loop and inoculated the media”

describe the term incubate and use it appropriately

Placing inoculated media at an appropriate temperature for growth by incubating it

“After inoculating, the media is incubated at 37C overnight”

describe the term culture and use it appropriately

practice of growing microbes in the lab

“today in lab, we cultured E. coli on agar plates”

what is the purpose of solid media vs liquid media

solid: bacterial cells can be separated from another so isolated colonies can develop after incubation

liquid: cells in the culture experience the same environment conditions

why is apgar added to media

acts as a solidifying agent to transform liquid nutrient broths into solid surfaces

distinguish between complex media and defined media

Complex: supports growth of many types of bacteria

• contains ingredients like yeast extract, meat extract, protein digests

• varies slightly

defined: contains only ingredients whose chemical composition is defined

• glucose, ammonium phosphate, sodium chloride

• consistent from batch to batch

explain the purpose of selective and differential media

explain the basis of MSA media as an example of selective and differential media

Selective: contains a high concentration NaCl, which inhibits the growth of most bacteria

Differential: contains mannitol (sugar alcohol) that some Staphloccus spp ferement while other don’t

• products of mannitol fermentaion are acids, which drop the pH of the media

• low pH turns the pH indicator from red to yellow

how is a streak plate used to obtain a pure culture

spreading the bacterial sample thinner and thinner across four quadrants and eventually individual colonies will form

describe how bacteria reproduce

they reproduce through a process called binary fission, where a single parent cell splits into two identical daughter cells

• allows them to grow expodentially

describe how bacterial population size increases over time

increases exponentially over time, doubling with every generation through binary fission.

calculate bacterial population size based on initial population size and number of generations

Nt = N0 × 2^n

Nt = final population size

N0 = initial population size

n= number of generations (how many times the population doubled)

plot a typical bacterial growth curve, identify the 4 stages, explain what is occurring during each stage

1. Lag phase: intense activity preparing for population growth but no increase in population

2. Log phase: logirithmic, exponential increase in population ; maximum population growth bc they have abundant nutrients. due to reproduction by binary fission or mitosis

3. Stationary phase: period of equilibrium; microbial deaths balance production of new cells; environment is running out of space and nutrients

4. death phase: population is decreasing at a logarithmic rate

explain the concepts behind the following methods for quantifying bacterial numbers

• direct microscopic count

• most probably number

• quantitative plate count

• turbidity

1. liquid bacterial culture is placed onto special calibrated glass slide ethched with grids. scientist view grid and count the cells in several squares, then calculates concentration based on microscopic volume of those squares

   

   2. most propable number: sample is diluted across multiple sets of replicate liquid broth tubes. positive tubes show gas bubbles in the chamber and each level comes with a code. code matches to a standardized probability table

   3. transfer the sample to an apgar plate and spread it across the surface. each cell after incubation forms a colony and the number of colonies on the plate represent the numbers in OG sample

   4. Turbity: cloudiness of bacterial culture is proportional to the number of cells in the culture. can use a spectrophotometer to quantity turbiditybeam of light is passed through bacterial suspension to light sensitive detector

define and distinguish between sterilization, disinfection, and antisepsis

sterilization: removing/destroying all microbial life

disinfection: destroying harmful microorganisms; applied surfaces

antisepsis: destroying harfmul microorganisms from living tissue

• mild forms of disinfectants

• achieved using chemicals

  • alteration of membrane permeability

  • damage to proteins (enzymes)

  • damage to nucleic acids

explain the purpose of the following techniques and how each works:

• autoclave

• pasteurization

• filtration

• radiation

autoclave: steam under pressure; kills all organisms and endospores

pasteurization: reduces number of microbes

• high temperature short time thermotolerant organisms survive

filtration: passage of substance through a screenlike material

• vacuum line speeds up the process

• smaller pore sizes are needed to filter out viruses and small bacteria

Radiation: causes mutations (changes in DNA sequence)

• nonionizing radiatino (UV) doesn’t penetrate surfaces

• Ionizing radiation (x-rays, gamma rays, electron beams)

  • high energy

how does low temperature impact microbial growth

slow down or completely stop microbial growth

how does microbial populations decline

when the rate of cell death exceeds the rate of cell reproduction

how is the disk diffusion method used to determine effectiveness of chemical agents

filter paper disks are soaked in a chemical and placed on the culture and marks the zone of inhibition around disks

define food spoilage

process in which food becomes unsuitable for consumption, goes bad, due to microbial activity

proProvide ways of preserving foods, either by reducing the number of microbes present or
reducing the opportunity for microbial growth

reudce microbes present

• pasteurization (short exposure high heat)

• canning (high temp then backagin)

• ionizing radiation (degrade biological molecules)

• chemicals (spices have antimicrobial properties

reduce opportunity

• dehydration and lyophilization (freeze dry)

• refridgeration and freezing (slows growth)

• vacuum seal

• fermentation

Define food poisoning

illness that results from eating food contaminated with food-borne pathogens

Explain why it isn’t obvious if consumption of a given food will cause food poisoning

contamination is invisitble, symptoms delayed immune responses vary

Explain the cause of botulism and why heat isn’t sufficient for preventing botulism

caused by consumption of botulism toxin secreted by bacterium Clostridium botulinum

• its able to form endospores which are heat tolerant packagaing swells due to gas production by it

Describe the appearance of can that is contaminated with C. botulinum

Define fermentation and explain why it is an effective method for preserving food

microorganisms break down carbs in absence of oxygen to produce energy

creates an anerobic environment bc of tightly packed food creates n acidic environment thats harmful to spoilage bacteria and foodborne pathogens

Explain the benefits of fermentation

results in conditions that very few microbes can tolerage, improves digestibility, adds nutrients and flavors

List some common end products of fermentation

• acids

• bases

• alcohol

• 
  

Explain where fermenting microbes come from

indigenous (wild) cultures naturally found on food ex wine and cheeses aged in caves

starter cultures microbes added to foods

Explain why fermented foods are unlikely to be contaminated by human pathogens

due to multi-layered barrier system

List some foods that result from acidic fermentation (and some microbes typically
involved in their fermentation

cheeese

yogurt

sour crream

kefir

Lactobacillus and Streptococcus

soybeans

Describe the three stages and the microbes involved in chocolate production

1. Yeast produces ethanol via alcoholic fermentation or glycolysis\

2. Lactobaccillus lactic acid bacteria produce lactic via lactic acid fermentationo

3. acetic acid bacteria Acetobacter produces via acetic acid fermentation

List some foods that result from ethanolic fermentation (and microbe most often
involved in their fermentation

beer- acolholic fermentaiton of grains

wine- alc ferm of grapes

saccharyomyces cereviase (bewers yeast)