Mmi 304 Exam 2

Front 1

how is growth defined in the context of bacteria

Back 1

an increase in cell number

Front 2

How do microbes increase their cell number (forms of replication 3)

Back 2

binary fission, budding, filamentous growth

Front 3

describe binary fission

Back 3

separation of the mother cell into two daughter cells of aproximately equal size

Front 4

describe budding

Back 4

asymmetric creation of a bud on the mother cell that eventually is severed, demonstrated by yeast and some bacteria like caulobacter

Front 5

describe filamentous growth

Back 5

formation of long, branching, non divided filaments containing multiple chromosomes, sometimes have cross walls, some filaments become form spores

Front 6

what is plotted on each axis of a growth curve graph?

Back 6

x axis- time in hours
y axis- cell number on log scale

Front 7

What are the four phases of growth

Back 7

lag, exponential (aka log or balanced), stationary, death

Front 8

describe the lag phase

Back 8

MO adapts to new nutrients, synthesize RNA, protein, and replicate DNA before starting division, no net increase in cell number

Front 9

describe the exponential growth phase

Back 9

enzymes are available, maximal division rate, can last for hour or days

Front 10

describe the stationary phase

Back 10

no increase in cell number because cells stop divding or rate of division equals the rate of death, limitation or nutrients or accumulation of waste products, can last for hours or days

Front 11

describe the death phase

Back 11

exponential decrease in cell number due to cell death, some MO's don't have a death phase or it is dealyed because they can survive w/o nutreints

Front 12

How can growth be measured?

Back 12

increase in cell number or increase in cellular macromolecule such as DNA or protein

Front 13

How can cell numbers be measured?

Back 13

viable plate count, turbidometric measurement

Front 14

define turbidity

Back 14

the light scattering of a culture, visable as cloudiness, measured with spectrophotometer

Front 15

describe the equation T=I/Io and what it meanse

Back 15

this is the equation to measure transmitence (T), Io is the light that enters the sample and I is the light that reaches the detector, the amount of light scattered correlates to the turbidity and thus cell number of a culture

Front 16

Why is transmittence not a good unit for measuring growth

Back 16

Since T=I/Io, T decreases as the turbidity of the culture i.e. growth increases, also it is logarithmically related to the concentration

Front 17

given that transmitence is not a conveniant way to measure growth, what unit is used instead?

Back 17

Absorbance, A=-log(T)

Front 18

How can the light scattering ability or absorption of a sample be measured effectivley

Back 18

a small portion of the spectrum emitted must be used, if more than one part is used, they will absorb or sactter differntly destroying the correlation of cell number with abs

Front 19

how is a single wavelength selected in a spectrophometer

Back 19

a monochromator contains a filter, a prism, or a diffraction grating slit taht breaks white light into its component wavelengths

Front 20

Which wavelength is typically used to meausre cell number and why?

Back 20

600nm, in general shorter wavelengths are better because they are more sensitive but some cannot be used because cellular components absorb light (proteins @280), the color of the media also matters so for our experiment, 600 works well with clear yellow media

Front 21

describe the cuvette and how to handle it properly

Back 21

it is used to contain the sample, it must be clean and free of aberrations which could scatter light ans mess up results

Front 22

What must you zero the spec?

Back 22

zeroing it sets the transmitence to 100% and makes sure that only turbidity from the bacteria is measured and not the media or other things

Front 23

why is it bad to let a sample dry in a cuvette

Back 23

the proteins and amino acids stick to the sides and mess up readings

Front 24

what is the range of acceptable abs values

Back 24

0.1- 1.0, below 0.1 is below the limit of the spec because it is not that sensitive concentrate with centrifuge, above 1 results in MOs shaddowing one another and they cannot be detected properly dilute with sterile medium

Front 25

what is the equation for growth rate

Back 25

k= (3.32 log10 (N2/N1))/ (t2-t1)

Front 26

what is the equation for generation time

Back 26

Tgen= T/n= 1/k

Front 27

why are growth rate equations useful

Back 27

they can compare growth between two cultures

Front 28

what does the growth rate depend on

Back 28

conditions of incubation and the medium used

Front 29

what is a response to changes in environment often dependent on?

Back 29

the de novo production of enzymes to metabolism the nutrient in uestion aka enzyme induction

Front 30

what does the bgal enzyme do?

Back 30

it adds water to the beta linkage of lactose splitting it into glucose and galactose

Front 31

What 3 genes compose the lac operon

Back 31

lacZ, lacY, and lacA

Front 32

where is the gene for the repressor of the operon, lacI located

Back 32

it is located upstream of the lacZYA genes, before the operon!

Front 33

what does the lacZ gene encode

Back 33

the bgalactosidase enzyme

Front 34

what does the lac Y gene code for

Back 34

galactose permease which transports lactose across the cellular membrane

Front 35

what does the lacA gene encode for?

Back 35

thigalactoside transacetylase, the purpose in lactose metabolism is unknown

Front 36

describe the promoter for LacZ,Y, and A

Back 36

they are transcribed by RNA polymerase from one promoter (plac) located upstream of the LacZ gene

Front 37

what happens to the operon when lactose is not present

Back 37

the repressor binds to the operator sequence which is located between plac and lacZ, this prevents RNA polymerase from biding

Front 38

When lactose reaches the cell, bgal acts on this. How can this occur if bgal is not produced in the absence of lactose

Back 38

a small amount of transcription occurs consituativley, this small amount of Bgal acts on the lactose and converts a small amount into allolactose (also makes glucose and galactose) this allolactose binds to the repressor and casues it to fall off the operator and allows transcription to occur

Front 39

how is the repressor removed from the operator site

Back 39

the small amount of bgal avaiable turns the lactose into allolactose. the allolactose binds to the repressor and causes it to fall off the operator site

Front 40

In addition to the repressor being removed from the operator, what else must occur to get significant transcription from the lac operon

Back 40

CRP must bind near the promoter, this protein is activated by high levels fo cAMP which is high when the cell is starving for energy

Front 41

Describe how CRP becomes active

Back 41

when the cell is starving for energy, cAMP levels are high. cAMP bindsto CRP and activates it. The complex binds to the lac operon and activates it

Front 42

why is beneficial to require CRP to activate the lac operon

Back 42

it prevents the lac operon from being turned on when glucose is present. Glucose is a more efficeint source of energy so it is beneficial not to induce the operon when glucose could be used instead

Front 43

describe an end point assay

Back 43

an enzyme, its substrate, and necessary compounds are mixed together, the enzyme then converts the substrate to product, the increase in product or decrease in substrate is measured over a set period of time. the final amount of product produced or substrate lost in the time period is proportional to the concentration of teh enzyme present

Front 44

What are the 4 features of a good enzyme assay

Back 44

1. absolute specificity
2. high sensitivity
3. accuracy and precision
4. convenience

Front 45

what is ONPG used instead of lactose in the bgal assay

Back 45

it is more conveient to measure because bgal breaks the galactosdie bond and splits it into colorless galactose and yellow and basic ONP which can be easily measured w/ spec or pH

Front 46

How is enzyme activity represented?

Back 46

units defined as the number of micromoles of substrate converted to product in 1 minute usually at 30C

Front 47

why is toluene added to the bgal assay

Back 47

it permeablizes the cell and allows substrates to pass in and out

Front 48

explain how to calculate bgal activity from absorbance (4 steps)

Back 48

1. use beer's law to figue out the concentration of ONP from A 420, concentration= absorbance/ extinction coefficient * length of light path c= Abs/ 1.1x10^3 x1
2. multiply by the total voume of the assay
3. divide by the assay time
4. divide by A600

Front 49

how can MOs be controlled

Back 49

killing or inhbiting their growth

Front 50

define antimicrobial agent

Back 50

any compound capale of killing or inhbiting the growth of an MO

Front 51

what term is used to describe compounds that inhibit MO growth but don't kill

Back 51

static ex "bacteriostatic", bacteriostatics at low [] can be bactericidal at high []

Front 52

define disinfectant

Back 52

bactericidal agents that are also toxic to humans and cannot be applied to living tissue ex Lysol

Front 53

define antispetics

Back 53

kill MOs but are safte to use on skin and mucous membranes (contrast w/ disinfectants which are unsafe on humans) ex lyserine, scope

Front 54

define antibiotic

Back 54

chemical compound that is produced by one MO and kills or inhibits another, for it to be useful it cannot be a toxic to the patient at a concentration that kills MOs, most successful antibiotics attack MO enzymatic systems that are different or not present in humans

Front 55

what determines an MOs sensitivity to antibiotics

Back 55

enzymatic and structural constitution

Front 56

define broad spectrum antibiotic

Back 56

effective against many diverse MOs

Front 57

how does the disk agar diffusion test show the efficacy of an anitibiotic agent

Back 57

if a zone of clearing appears around the disk, the agent is effective, the degree of clearing is compared to definitions of resistent, intermediate, or suceptible

Front 58

an important consideration of an isolation protocol

Back 58

the need for seletice enrichment, depends on population of the desired organism in the source material, if high plate directly onto selective media, if low enrich using seletive enrichment first

Front 59

name the four major considerations of enrichment that take advantage of the microbe's biochemical and physiological properteies that favor growth

Back 59

1. choice of inoculum, the source needs to contain the desired bacteria at a resonable concentration
2. pretreatment- heat shocking, etc to remove other microbes
3. choice of medium- compounds inhibit the growth of unwanted contaminants
4. suitable incubation conditions

Front 60

what does Enteric Enrichment browth contain that inhibits unwanted organisms

Back 60

bile salts (oxgall) and brilliant green

Front 61

describe streptomyces

Back 61

gram positive, spore forming bacteria. chemoorganoheterotrophs, grow best at 25C, pH 8-9, break down complex carbons, important for soil enrichment

Front 62

describe streptomyces colonies

Back 62

tough, leathery, frequently pigmented colonies, filamnetous growth

Front 63

what are geosmins

Back 63

volatile low molecular weight compunds producted by streptomyces that give soil its characteristic smell

Front 64

how does actionmycete isolation agar (AIA) help select for streptomyces?

Back 64

an enrichment step is not necessary because soil has tons of streptomyces, AIA contains starch and sodium caseinate as teh sole carbon and energy sources, only organisms that can break these down like molds and streptomyces can grow. sodium propinoate is also added to inhibit mold

Front 65

describe enterobacteriaceae

Back 65

gram negative, non sporulating, short rods, motile by peritrichous flagella or non motle, simple nutritional requirements, faculatitive anaerobes that ferment glucose to acid under anaerobic conditions

Front 66

Describe how the method used to ferment glucose can differentiate between differnt enteric bacteria

Back 66

mixed acid- ferment glucose anaerbically to lactic, succinic, acetic, and formic acid, ethanol, CO2, and H2,
Butanediol- all of these products plus butanediol, less acid is produced b/c neutral end products

Front 67

what tests can be preformed to distinguish between the two types of fermentation used by enteric bacteria?

Back 67

Vogues-Proskauer test
methyl red test

Front 68

Describe shigella

Back 68

close relative of E coli, pathogenic, diarrheal ilness with colonization and attack of the intestines, fever, cramps, bloody diarrhea w/ mucous, can be caused by ingestion of fewer than 100 organisms, improperly prepared food and contaminated water

Front 69

describe salmonella

Back 69

infects humans, mammals, birds, retpiles, two species S. bongori and S. choleraesuis w/ 7 subgroups and 2500 serovars distinguished by cell wall and flagellar antigens, important serovars include S. enterica, S. enteritidis, and S. heidelberg, cause gastroenteritis of varying degrees w or w/o bacteremia, contaiminated food >106 cells/ml or g, sxs 8-30 hrs after ingestion, nausea, fever, diarrhea, abdominal pain, usually subside post 1-2 days

Front 70

give and example of exceptionally dangerous salmonella

Back 70

host dapted biochemically distinct serovars, S. typhi- typhoid fever, S. cholerae-suis "hog cholera" and S> gallinarum "fowl typhoid"

Front 71

Describe the genus Edwardsiella

Back 71

disease in humans and vertebrates, E. tarda opportunistic pathogen for humans woudn infection and gastroenteritis, E. tarda and E. ictaluri cased incetions of comercially raised catfish

Front 72

Describe the genus Yersinia

Back 72

Y. enterololitica- gastroenteritis, Y. pestis- bubonic plage

Front 73

describe the genera proteus, providencia and morganella

Back 73

nonpathogenic, can deaminate phenylalanine, fish and seafood spoilage, Proteus forms wierd swarming patterns that have concentric ring shape

Front 74

describe citrobacter

Back 74

nonpathogenic enteric, can give false positives to salmonella food tests

Front 75

describe Erwinia

Back 75

pathogenic for plants, cause soft rots and wilts of crops and veggies

Front 76

describe serratia

Back 76

red pigmented colonies

Front 77

define coliform

Back 77

non-sporeforming, facultatively anaerobic gram negative rod which ferments lactose to acid and gas withing 48 hrs at 35C, usually enterobacteriaceae of genera enterobacter, klebsiella, and escherichia

Front 78

describe Klebsiella

Back 78

use N2 as sole nitrogen source, insights into nitrogen fixation, K. pneumoniae can cause pneumonia in hiumans found in water, soil, plant material

Front 79

name some diseases that can be transmitted by fecal contamination in the water supply

Back 79

polio, typhoid, cholera, hepatitis, shigellosis, salmonellosis

Front 80

What are the four properties that an indicator organism must have to be useful for water analysis

Back 80

1. the only natural environemtn of the microbe should be in assocation with feces and it should be always present
2. it should not grow outside of its natural environment
3. the bacterium should survive longer than the most viable pathogen but not so long so that historical events are detected
4. It shoudl be easy to detect

Front 81

Why are enterobacter and klebsiella not good indicators of fecal pollution

Back 81

they are able to survive and multiply in the environment

Front 82

what is the water quality demand for drinking water and swiming pool

Back 82

>1 coliform/100ml

Front 83

which bacteria make up the fecal coliforms that are good indicators of fecal pollution

Back 83

E. coli and K. pneumoniae

Front 84

what coliform levels are unacceptable for treated sewage or natural bathing beaches

Back 84

> 400/ 100ml or monthly geometric average > 200/ml

Front 85

describe the multiple tube fermentation test

Back 85

3 steps,presumptive, confirmed, completed, moderately seletive lactose broth medium with durham tube is used in the presumptive test to grow coliforms in from the sample, if tube has growth and gas the test is positive, confrimed tests makes sure that these were acutally coliforms, use seletive medium to elimate orther organisms if these are positive, completed test is used by streaking the highest dilution tube which gave a postive results onto EMB agar, after incubation colonies are evaluated for enteric properties

Front 86

what is the disadvantage of the multiple tube fermentation test for coliforms

Back 86

takes 3-5 days to complete

Front 87

describe the membrane filter technique to test for coliforms

Back 87

100ml or grate of sample passed through filter that retains bacteria, filter is placed onto seletive medium for coliforms

Front 88

what are the advantages of the membrane filter technique for testing for coliforms

Back 88

shorter time, low cost, higher accuracy in couting, simple

Front 89

what are the disadvantages to the membrane filter technique for testing for coliforms

Back 89

particulate samples can clog the filter, metals and phenols can stick to the filter and inhibit growth, non coliforms in the test sample may interfere wit hthe formation of coliform colonies on the plate

Front 90

Describe the presence-absence (P-A) test for coliforms

Back 90

large water sample (100ml) is mixed with tripple strength LLTB in single culture bottle. Brom cresol purple is added as pH indicator. If present, coliforms will ferment the lactose to acid and gase turning the media from purple to yellow

Front 91

Describe the Colilert defined substrate test for colifroms and e coli

Back 91

A 100ml sample is mixed with medium containing ONPG and MUG as the only nutrients, if coliforms are present, ONPG is metabolised resulting in yellow color, if E coli is present it will degreade MUG to a fluorescent product that can be detected by observation under long wave length UV light.

Front 92

What do the Colilert and P-A tests have in common

Back 92

they are preliminary, any positive results warrent further analysis of the sample

Front 93

breifly, how is the bacterial concentration determined in the MPN test

Back 93

we innoculate several tubes of ten fold dilutions. If the 10^-5 tube has growth but the 10^-6 doesn't, we know that the sample has more than 10^5 but less than 10^6 bacteria, by using multiple replicates of each diultion and statistical analysis, we can estimate the conentration

Front 94

How is the number of organisms assed in the MPN test

Back 94

counting the number of positives in the last three diltions showing growth

Front 95

Describe MAGC agar

Back 95

it's the same thing as MAC agar but uses glucose instead of lactose, inhibits gram positives, bacteria that form red colonies are probably enterics because the acid from fermentation leads to the precipitation of bile salts and absportion fo the red indicator

Front 96

describe EMB agar

Back 96

use to isolate enterics and gram negative bacteria, lactose fermenters will look dark purple because they absor eosin-methylene blue complex that forms in the presence of acid, E coli are blue black with green metallic sheen, media strongly inhibits gram positive bacteria

Front 97

describe KIA agar

Back 97

kligler's iron agar,used to differntiate entrics, organisms which ferment glucose but not lactose will produce acids indicated by yellow color, amount of acid from glucose fermentation is not sufficient to conteract teh alkaline reaction from teh aerbic deamination of amino acits in the medium, the slant region will appear red (alkaline) and the butt yellow, organisms which ferment lactose will produce enough acid to produce a yellow color, need to make obs in 18-24 hours, gas formation detected by bubbles or cracks in agar, hydrogen sulfide detected by black coloration, FeSO4 provides ferrous ions which react with the sulfide to pruduce the black indicator compound FeS

Front 98

What does it mean If a KIA slant has a red or orange butt

Back 98

the tube is negative for glucose fermentation because no net acid production occured

Front 99

what does it mean if a KIA slant has a yellow butt

Back 99

fermentation occured because the net acid produced was able to turn the KIA yellow

Front 100

What does it mean if a KIA slant has a red slant

Back 100

the isolate was only able to ferment the small amount of glucose in the medium, if it were able to ferment lactose, enough acid would have been produced to overwhelm the amino acid deamination in the slant causing a yellow color througout the medium

Front 101

what does it mead if a KIA media turns black

Back 101

the isolate producted H2S from tiosulfate, the H2S reacts with the Fe in the medium to form FeS which is black

Front 102

describe the ornithine decarboxylation test

Back 102

ornithine can be decarboxylated by some enterics, in a negative test, the glucose in the medium is fermented to cause a net acidic reaction and a yellow color. in a positive reaction, ornithine is decarboxylated anaerobically to produce a net alkaline reaction which turns the media purple or blue

Front 103

describe the lysine decraboxylase test

Back 103

two tubes are used, one tube contains DCB which has everything but lysine the other, LDB, has lysine. The bacteria is put in both broths. In the DCB tube, acid produced from glucose fermentation turns the medium yellow, all enterics should show this reaction, this tube is compared to the LDB tube. If lysine is decarboxylated the LDB tube will be darker than the DCB because the decarboxylation is an alkaline reaction. A positive result is if the DCB tube is aidic (yellow) and the LDB tube is alkaline (purple, grey, or less yellow than LDB)

Front 104

in DCB, all enterics will...

Back 104

show a net acidic reaction (turn media yellow) because of glucose fermentation

Front 105

A positve result for Lysine decarboxylation is...

Back 105

the LDB tube is alkaline (purple, grey, or less yellow than DCB) and the DCB is yellow (acidic)

Front 106

describe the phenylalanine deamination test

Back 106

a product of Phenylalaine deamination is phenylpyruvic acid. Phenylpruvic acid will react with Fe3+ to form a green complex. Add a few drops of FeCl3 to the phenyalaline agar and look for an immediate green color

Front 107

describe the citrate agar test

Back 107

if citrate is used as a carbon and energy source, a net alkaline reaction will occur and the medium will turn blue because the citrate is removed

Front 108

describe the methyl red test

Back 108

use to distinguish between the two types of fermentation. Butanediol fermentation after 1-2 days results in production of alkaline products and a rise in pH. Methyl red is an indiactor dye. If mixed acid fermentation occured the medium will be acidic and the dye will turn red or pink (pH 4.4), if butanediol fermentation occured the medium will be more alkaline producing a yellow color (pH 6.2) orange indicates an equivocal reaction

Front 109

Describe the Voges-Proskauer test

Back 109

detects the production of acetoin, a intermediate of the butanediol pathway, alpha naphthol and KOH are added to bacteria in an MRVP broth. a positve reaction will turn red which indicates the presence of the neutral end product acetoin

Front 110

how do you pick which three tubes to use when doing MPN

Back 110

you pick the last three (most dilute) tubes showing growth and then you count the number of positives at these dilutions

Front 111

describe APT + azide media

Back 111

For optimal enrichment, isolation, and growth of lactic acid bacteria. Oleic acid in the Tween 80 has divalnet cations and thiamine are required by nutritionally fastidious lactic acid bacteria