Module 6B

Question 1

what are 3 methods to quantify microbes

Answer 1

• direct counts
• viable cell counts
• turbidity measurements

Question 2

which quantifying method allow you to differentiate between viable cells and dead cells

Answer 2

viable cell counts

Question 3

what are the steps to using a Petroff-Hauser counting chamber

Answer 3

1. add a drop or two of bacterial suspension to the center of the chamber
2. lower a cover slip over the grid, suspension spreads out evenly and excess flows into the side channels
3. use a microscope to count the cells in the grid

Question 4

how can you count the number of viable cells with direct counts

Answer 4

use a viability stain (e.g. acridine orange; binds to nucleic acids within the cells)

Question 5

what do viable cell counts count

Answer 5

colony-forming units (CFUs)

Question 6

how is it ensured that the number of colonies is countable

Answer 6

serial dilutions

Question 7

how are pure culture obtained for viable cell counts

Answer 7

spread plate or pour plate method

Question 8

how are CFUs/mL calculated

Answer 8

multiplying the number of colonies by the inverse of the dilution factor

Question 9

how is turbidity measured

Answer 9

using a spectrophotometer

Question 10

what do the turbidity values mean

Answer 10

light absorbance is proportional to the abundance of cells within a growth medium: more light absorbance => more cells

Question 11

what are the phases of growth curves

Answer 11

• lag phase
• exponential phase
• stationary phase
• death phase

Question 12

what adjustments are needed in the lag phase of growth curves

Answer 12

can include producing new proteins for transporting and metabolizing newly available carbon and nutrients

Question 13

define: generation time

Answer 13

time it takes a species of bacteria (or archaea) to divide in the exponential phase [same as the population doubling time]

Question 14

define: growth rate

Answer 14

inverse of generation time

Question 15

define: growth yield

Answer 15

maximum population density, or the amount of biomass that can be recovered in early stationary phase

Question 16

what phase does a continuous culture maintain

Answer 16

exponential growth

Question 17

how is a continuous culture made

Answer 17

fresh media is continuously provided to the culture at a limited rate

Question 18

define: chemostat

Answer 18

system that brings in fresh medium and removes old medium/microbes

Question 19

what are the types of filters

Answer 19

• pre-filter
• polymer membrane filter
• nucleopore membrane filter

Question 20

how are viruses filtered

Answer 20

with ultrafiltration methods (pore size: 10-100nm)

Question 21

what are examples of pre-filters

Answer 21

• depth filter
• cheesecloth
• Whatman filter paper

Question 22

which filter removes the largest particles

Answer 22

pre-filters

Question 23

what are polymer membrane filters made of

Answer 23

cellulose acetate or cellulose nitrate

Question 24

what are polymer membrane filters used for

Answer 24

routine sterilization

Question 25

what are nucleopore membrane filters made of

Answer 25

polycarbonate

Question 26

how thick are the pores of nucleopore membrane filters

Answer 26

10 um

Question 27

how are nucleopore membrane filters made

Answer 27

formed by exposing polycarbonate films to radiation, tiny cracks are enlarged by a chemical etching process

Question 28

what are nucleopore membrane filters useful for

Answer 28

microscopy

Question 29

describe the consistency of pore size of polymer membrane filters and nucleopore membrane filters

Answer 29

polymer membrane filter - pores somewhat variable in diameter nucleopore membrane filter - consistent pore size

Question 30

how does heat kill microorganisms

Answer 30

by denaturing proteins and nucleic acids

Question 31

what types of microorganisms are harder to destroy with heat

Answer 31

hypothermophiles and endospores

Question 32

what do autoclaves use to sterilize

Answer 32

pressure and heat

Question 33

why do autoclaves use pressure

Answer 33

it prevents fluids from evaporating or boiling

Question 34

what standard values are used in autoclaves

Answer 34

pressure: 15 psi temperature: 121 C time: 20 mins

Question 35

what is the use of pasteurization

Answer 35

- to decrease microbial "load" - the total number of microorganisms in a food product - destroys all pathogens

Question 36

what are 3 commonly used processes with pasteurization

Answer 36

- high temperatures for short times (HTST) [72 C for 15 sec] - ultra-high temperature (UHT) [135 C for < 1 sec] - extended shelf life [ESL] - filtering the material and then using a low-temp pasteurization

Question 37

why is refrigeration/freezing effective for preservation

Answer 37

it reduces microbial growth and spoilage, the formation of ice crystals can be harmful to the membranes and cell walls of microorganisms

Question 38

how are cultures rapidly frozen

Answer 38

- for cultures that we want to preserve, add a cryoprotectant like dimethylsulfoxide (DMSO) or glycerol - lower the culture directly into liquid nitrogen

Question 39

what is the advantage of flash freezing

Answer 39

it preserves viability

Question 40

what is electromagnetic radiation used for

Answer 40

eliminates microorganisms from surfaces or any materials that allow light to penetrate

Question 41

why is UV light damaging to DNA

Answer 41

it forms thymine dimers in DNA (two T bases become covalently linked)

Question 42

what surfaces are electromagnetic radiation good for

Answer 42

non-living surfaces, NOT used for tissues

Question 43

what advantage does ionizing radiation have over electromagnetic radiation

Answer 43

it penetrates materials well

Question 44

what protein & DNA damage does ionizing radiation induce

Answer 44

- double-stranded DNA breaks - stray electrons - hydroxide ions - hydride radicals

Question 45

what are some examples where ionizing radiation is used

Answer 45

- eliminating insects from materials and supplies - exposing tissue or cartilage used for grafting in medical procedures

Question 46

what are nonselective antimicrobial agents

Answer 46

they are toxic to any cells, any protein is targeted (target specific groups on proteins and amino acids)

Question 47

what are selective antimicrobial agents

Answer 47

they target specific microbial groups

Question 48

what does Penicillin act on

Answer 48

Gram-positive cell walls

Question 49

what are the three ways that antimicrobial agents kill cells

Answer 49

- bacteriostatic - bacteriocidal - bacteriolytic

Question 50

what is the purpose of bacteriostatic antimicrobial agents

Answer 50

they inhibit growth (don't kill), buy time for the immune response to clear the infection

Question 51

what is the difference between bacteriocidal and bacteriolytic

Answer 51

bacteriocidal: - result in cell death - viability decreases - cells are still visible bacteriolytic: - result in cell lysis - number of visible cells decrease

Question 52

what is the difference between disinfectants and antiseptics

Answer 52

disinfectants: used on inanimate surfaces antiseptics: used on living tissue

Question 53

how do alcohols disinfect

Answer 53

they dissolve the membranes of bacteria

Question 54

where are alcohols used for disinfection

Answer 54

- used in laboratory settings - present in most hand sanitizers

Question 55

how do phenolic compounds disinfect

Answer 55

affect the membrane of bacteria

Question 56

where are phenolic compounds used for disinfection

Answer 56

added to products like soaps, deodorants, and cosmetics

Question 57

how do oxidizing agents disinfect

Answer 57

they remove electrons indiscriminately from proteins and DNA

Question 58

where are oxidizing agents used for disinfection

Answer 58

commonly added to swimming pools and hot tubs

Question 59

how do quaternary ammonium compounds disinfect (Benzalkonium chloride)

Answer 59

affect the bacterial membranes

Question 60

how does glutaraldehyde disinfect

Answer 60

it crosslinks proteins, used as a preservation agent

Question 61

how is glutaraldehyde used for disinfection

Answer 61

often used to prepare biological specimens

Question 62

what are antibiotics

Answer 62

antimicrobial agents that are produced by microorganisms

Question 63

how do antibiotics work

Answer 63

they bind to proteins or organelles and interrupt the energy management of the cell

Question 64

what is an example of a broad-spectrum antibiotic

Answer 64

tetracycline

Question 65

what are 2 examples of narrow-spectrum antibiotics

Answer 65

- polymixin B - penicillin

Question 66

how is an antibiotic sensitivity assay prepared

Answer 66

1. microorganisms is spread over the surface of a plate 2. paper disks are impregnated with different antibiotics are placed on the surface of the plate 3. the plate is incubated overnight 4. the amount of clearing provides an indication of how sensitive your microorganism is to an antibiotic

Question 67

what is the decimal reduction time (D value)

Answer 67

time required to kill 90% of the target organisms under specific conditions

Question 68

what is the decimal reduction dose (D10)

Answer 68

radiation dose required to kill 90% of the target organisms under specific conditions