lab final

Question 1

exercise 22: oxidase test

Answer 1

detection of cytochrome c oxidase (in most aerobic organisms it accepts electrons from ETC and transfers them to oxygen)

all organisms with oxidase are aerobes, but not all aerobes have oxidase (not found in facultative anaerobes)

oxidase strip is imprinted with the substrate N, N-dimethyl-p-phenylenediamine and a-naphthol. organisms inoculated directly onto the strip. enzyme oxidizes the compounds, results in the production of indophenol blue.

use to separate gram negative unknowns, do if you suspect unknown is an aerobe

Question 2

aerobe

Answer 2

capable of growth in the presence of normal atmospheric levels of oxygen. require oxygen as final electron acceptor.

Question 3

obligate anaerobe

Answer 3

undergo either fermentation or anaerobic respiration, oxygen not required. lack superoxide dismutase and catalase or peroxidase

Question 4

facultative anaerobes

Answer 4

undergo aerobic respiration, fermentation, or anaerobic respiration. grow better in oxygen. contain superoxide dismutase and catalase or peroxidase

Question 5

MacConkey agar

Answer 5

crystal violet and bile salts make it selective for gram negative organisms.

lactose and neutral red are differential for lactose fermentation. if an organism is lactose fermenting, the pH of the medium will drop and the colony will absorb the neutral red indicator and turn pink to brick-red.

used to differentiate gram negative enterics

Question 6

Columbia CNA agar with 5% sheep blood

Answer 6

selective for gram positive organisms due to colistin (C) and nalidixic acid (NA). colistin disrupts the cytoplasmic membrane of gram negative bacteria and nalidixic acid prevents DNA replication.

5% sheep blood allows for differentiation based on hemolysis pattern. b-hemolysis, colonies are surrounded by a clear zone with few or no intact red blood cells. a-hemolysis, colonies are surrounded by a zone of intact but discolored red blood cells. g-hemolysis, no change in medium

Question 7

mannitol salt agar

Answer 7

7.5% sodium chloride makes it selective for halophiles.

differential based on mannitol fermentation, using the indicator phenol red. if an organism ferments mannitol, the pH drops and the colonies will turn yellow. colonies that do not ferment mannitol will remain translucent.

Question 8

carbohydrate fermentation

Answer 8

breaking down carbohydrates provides energy for the cell (respiration + fermentation)

carbohydrates need to get into the cell
- larger sugars have a hard time passing through the membrane
- b-galactose hydrolyzes lactose into glucose and galactose
- sucrase hydrolyzes sucrose glucose and fructose

Question 9

Triple sugar iron (TSI) agar

Answer 9

differentiates between organisms by their abilities to ferment glucose, sucrose, or lactose, and to liberate hydrogen sulfide gas from sodium thiosulfate.

contains 1% solutions of both lactose and sucrose and a 0.1% solution of glucose

results based on pH changes –> fermentation produces acids

cracks mean there is gas production and black means there is H2S

slant and butt both turn yellow it ferments fructose and/or sucrose

slant and butt initially yellow and then alkaline so only yellow in the butt it ferments glucose

Question 10

IMViC series

Answer 10

indole

methyl red-voges proskauer

citrate

Question 11

indole test

Answer 11

• indole is a waste product of tryptophanase
• indole test indirectly detects the presence of tryptophanase by measuring an end-product of the reaction involving this enzyme
• to perform indole test , bacteria are stabbed into a sulfide-indole-motility (SIM) agar deeps
• addition of Kovac’s Reagent reacts with indole, turns red for a positive result
• detects H2S production (blackening)
• spreading from the stab sight means the microbe is motile

Question 12

methyl red-Voges Proskauer (MRVP) test

Answer 12

• two kinds of fermentation: mixed-acid fermentation and 2,3-butanediol fermentation
• methyl red test will detect lowered pH from mixed acid fermentation (red color at pH of 4)
• voges proskauer test will detect acetoin from 2,3-butanediol fermentation (pinkish/rose color)
• test differentiates between organisms by detecting fermentative end-products following growth in a buffered peptone-glucose broth

Question 13

citrate test

Answer 13

• Simmon’s citrate agar detects citrate utilization
• if citrate is utilized, organisms must have citrate permease and citrase.
• citrate permease imports citrate into the cell, citrase oxidizes the substrate
• CO2 and salt forms sodium carbonate, raises pH
• detected with bromothymol blue (indicator is green at a neutral pH but changes to blue if the pH becomes more basic
• also has ammonium as the only nitrogen source

Question 14

urease test

Answer 14

• urea is a waste product of protein degradation
• urease hydrolyzes urea into ammonia
• phenol red in urea agar changes color as pH raises
• salmon color is negative (neutral pH)
• fuchsia color is positive (pH of 8.4)
• occasionally the glucose in the medium is fermented and acidic products accumulate. the acidic pH and phenol red will cause the agar to turn yellow (considered a negative result)
• used only in the differentiation of gram negative, oxidase negative rods or coccobacilli

Question 15

nitrogen fixation

Answer 15

the process by which microorganisms that possess the enzyme nitrogenase convert atmospheric nitrogen into ammonia

can be accomplished by two microbial groups: free-living nitrogen-fixing bacteria and symbiotic nitrogen-fixing bacteria

Question 16

free-living nitrogen-fixing bacteria

Answer 16

these organisms protect or sequester nitrogenase in anaerobic heterocysts

Question 17

symbiotic nitrogen-fixing bacteria

Answer 17

the second and more important type of nitrogen-fixing bacteria

these bacteria infect the roots of leguminous plants (soybeans, beans, peas) and form root nodules (growths on the plant roots that harbor the bacteria). nitrogen is fixed by a symbiotic process between the plant and bacteria.

Question 18

ammonification

Answer 18

amino groups (-NH2) from amino acids are removed and converted into ammonia, which is liberated from soil.

Question 19

nitrification

Answer 19

the nitrogen in ammonia is oxidized to produce nitrate via a two-step process. the ammonia is first oxidized to nitrite ion (NO2-) and then the nitrites are further oxidized to yield nitrate ions (NO3-)

requires molecular oxygen

Question 20

denitrification

Answer 20

the process by which the nitrate ion is converted via multiple steps to more reduced forms of nitrogen, which include nitrite reductase, nitric oxide reductase, and nitrous oxide reductase.

only occur under anaerobic conditions and only if the organism possesses the appropriate enzymes

Question 21

nitrate broth test

Answer 21

• determines the presence of nitrate reductase
• broth contains potassium nitrate as the source of nitrate
• following incubation, the medium is evaluated for the presence of reduced forms of nitrates by the addition of nitrate reagent A (sulfanilic acid) and nitrate reagent B (a-naphthylamine).

red color after addition of nitrate reagents A and B is a positive reaction. no color change after addition of zinc dust if negative after step 1 is also positive

no color change after addition of reagents A and B is negative. red color change after addition of zinc dust if negative after step 1 is also negative.

Question 22

catalase

Answer 22

catalase neutralizes up to 40,000,000 molecules of hydrogen peroxide per second and turns it into water

tells us if aerotolerant organism uses catalase or peroxidase

test: add H2O2 to culture, look for production of oxygen (bubbling). test is instantaneous

perform test on gram + cocci

Question 23

fibrinogen

Answer 23

a glycoprotein that humans produce involved in many cellular processes. comes in 2 forms: insoluble dimeric glycoprotein and soluble disulfide linked dimeric glycoprotein. both forms are involved in wound healing.

prothrombin is also involved in the healing process

Question 24

insoluble dimeric glycoprotein

Answer 24

linker between connective tissues that surrounds and supports cells in mammalian tissues, produced by many different cell types

Question 25

soluble disulfide linked dimeric glycoprotein

Answer 25

synthesized by hepatocytes (liver cells) found in the plasma (clear, yellowish fluid portion of the blood, lymph, or intramuscular fluid which contains WBC and soluble clotting factors)

Question 26

coagulase

Answer 26

extracellular protein, results in clotting by binding to prothrombin and forming staphylococci

found in staphylococci

activates thrombin, which converts fibrinogen to fibrin, leading to clotting

could potentially protect the bacteria from WBC

Question 27

coagulase test

Answer 27

• detects presence of coagulase
• inoculate a loopful of organism into a tube of citrated rabbit plasma
• tubes contain citrate, EDTA, and rabbit plasma as the source of fibrinogen
• citrate and EDTA work to prevent false positive results (plasma clots on its own)
• the citrated rabbit plasma tube is then monitored for the formation of a fibrin clot (positive if one forms)
• important for distinguishing species of staphylococci which are gram + cocci that are catalase positive

Question 28

nuclease (DNase)

Answer 28

the general name for an enzyme that degrades nucleic acid

function: DNase enzymes perform a variety of crucial cellular functions hydrolyzing the phosphodiester backbone of DNA

Question 29

DNases are ubiquitous in bacterial pathogens and serve multiple functions:

Answer 29

• acquiring nucleotide nutrients
• allowing or preventing uptake of foreign DNA
• controlling biofilm formation/dispersion/architecture
• invading the host through tissue damage
• evading immune defense by degrading the DNA matrix of neutrophil extracellular traps (NETS)
  -immunomodulating the host immune system

Question 30

phage restriction

Answer 30

• bacteria defend against phages using restriction enzymes
• cuts DNA in specific places to prevent infection by phage
• bacteria protect their own DNA through methylation (prevents phage restriction from acting on own DNA)
• DNA provides a source of carbon and nitrogen to the cell

Question 31

extracellular nucleases

Answer 31

• bacteria can use extracellular DNA as a source of carbon and nitrogen (DNA can also be broken down into monomers for use in replication)
• during infection, WBCs lyse, leading to the accumulation of live WBCs, live bacteria, and lysed cell debris –> pus
• DNA in pus makes it sticky, harder for bacteria to spread
• DNase breaks down DNA in pus, allowing bacteria to spread more readily

Question 32

DNase test

Answer 32

• DNase agar is a differential medium that measures an organism’s ability to produce the exoenzyme deoxyribonuclease or DNase, which degrades DNA
• DNase agar contains nutrients for bacteria, DNA, and possibly the indicator methyl green
• methyl green is a cation that binds to DNA due to its negative charge
• organisms are spot-inoculated (single streak) onto the agar plate. if an organism produces DNase, it is secreted outside the bacterial cell and degrades the DNA into smaller pieces. detection of depolymerized DNA is accomplished by flooding the agar surface with 1N HCl and observing the medium for the development of a clear zone surrounding the bacterial growth.
• clear zone is a positive test, no clearing is negative.
• test is important for distinguishing between gram + cocci that are catalase positive