Lab 3

Front 1

What is meant by a plaque-forming unit?

Back 1

A plaque-forming unit occurs where in the plated preparation one infective virus (virion) wasadjacent to, and infected, a bacterial cell.

hole in lawn of bacteria

Front 2

What are coliphages?

Back 2

They are viruses that infect Escherichia coli.

Front 3

Describe the similarities between the plaque technique in this exercise and the standard platecount for bacteria.

Back 3

In both cases you are counting the desired microorganisms on a background medium. In the bacterialplate count, you count the bacterial colonies on an agar surface. For a viral plaque count, you countareas of viral-infected bacterial lysis on a solid lawn of bacteria growing in an appropriate medium.

Front 4

6. What effect does chloroform have on viruses? On bacteria?

Back 4

It does not kill the viruses but lyses the cell membranes of the bacteria, thus killing them.

Front 5

1. Why are coliforms selected as the indicator of water potability?

Back 5

They indicate the presence of lactose-fermenting gram-negative rods. If they are present, the watermay contain fecal microorganisms (from the intestinal tract of humans or animals) and is not potable

Front 6

Does a positive presumptive test indicate that water is potable?

Back 6

No. It suggests it may not be potable

Front 7

Why is the MPN test qualitative rather than quantitative

Back 7

looks for bubbles produced instead of bacterial count

Front 8

function of lactose broth

Back 8

presumptive indication of coliforms

Front 9

function of Levine’s EMB or LES Endo agar

Back 9

select for gram negatives

Front 10

function of nutrient agar slant

Back 10

grows cells for gram stain

Front 11

function of gram stain

Back 11

shows whether organisms preset are really gram negative rods

Front 12

5. What does a metallic green sheen indicate on an EMB plate?

Back 12

In both cases these results indicate the presence of coliforms in the completed test.

Front 13

What bacterial diseases can be transmitted by polluted water?

Back 13

Bacterial dysentery, shigella, cholera, typhoid fever

Front 14

When doing a throat culture, what specific area of the throat is swabbed, and why?

Back 14

The area around the tonsils is swabbed, because this area is more likely to harbor potential pathogensand a smaller number of transient microorganisms

Front 15

When doing a skin culture, why is the swab first moistened with saline?

Back 15

To more readily absorb microorganisms from the skin onto the swab.

Front 16

What are four reasons for knowing which microorganisms are associated with different parts ofthe body?

Back 16

To learn which microorganisms are common to specific body sites.

b. To be able to associate specific infections with microorganisms from particular body sites.

c. To evaluate the consequences of overgrowth of specific microorganisms in particular body sites.

d. To evaluate how the host immune system reacts to normal flora.

Front 17

Why were the blood agar plates incubated at 35°C, whereas the TSA plates were at roomtemperature?

Back 17

To more accurately reflect the temperatures of the body sites where the microorganisms are normallyfound.

Front 18

Why is EMB used for rectal cultures?

Back 18

It is selective for gram-negative enteric rods and inhibits the growth of most other rectal bacteria.

Front 19

What are fastidious streptococci?

Back 19

need additional nutrients such as iron and enzymes

Front 20

Would you expect to isolate ß-hemolytic streptococci from the skin? Explain your answer

Back 20

No, because these organisms do not grow well on TSA or at room temperature.

Front 21

What precautions should be taken by a nurse who is a “staph” carrier?

Back 21

should not be treating patients until she is cleared of her infection

Front 22

what test can be done to differentiate between staph epi and staph aureus.

Back 22

a coagulase test

Front 23

How would you treat a person who has a carbuncle?

Back 23

go to a doctor that is trained in treating carbuncles

Front 24

Describe the cellular morphology and arrangement of staphylococci.

Back 24

They are gram-positive facultatively anaerobic cocci in clusters.

Front 25

7. Why are staphylococcal infections frequent among hospital patients?

Back 25

a. A more susceptible population in the hospital (at risk).

b. More selection of resistant strains due to the extensive use of antibiotics in hospitals.

c. Contact of health care professionals with staphylococcal-infected patients

Front 26

8. In summary, what is the major purpose of this experiment?

Back 26

To recognize the medical significance of the staphylococci, to isolate staph from the body, and todistinguish the pathogenic species of staph from the non-pathogenic species.

Front 27

1. How does the enzyme coagulase function?

Back 27

It produces fibrin clots around the microorganism in infected tissues.

Front 28

2. How does the enzyme DNase function?

Back 28

It degrades macromolecular DNA into small molecular weight components that are then solubilized.

Front 29

3. False-positive coagulase tests have been reported for some bacteria that can metabolize citrate.

Back 29

Citrate inhibits the nonspecific clotting of fibrin. These microorganisms remove the citrate, and thisallows nonspecific coagulation to take place.

Front 30

Does a coagulase-positive staphylococcus also have to be DNase positive?

Back 30

No. Coagulase and DNase are two distinct enzymes produced from different genes. Therefore, onecould be present and the other absent.

Front 31

5. What is the composition of DNA agar with methyl green?

Back 31

Agar, pancreatic digest of casein, peptic digest of animal tissue, NaCl, DNA, and methyl green.

Front 32

1. How could you determine if a sore throat was caused by S. pyogenes (Group A, ß hemolytic)?

Back 32

By looking for ß-hemolytic gram-positive cocci in chains, bacitracin susceptibility and SXTresistance, and group A Lancefield serotyping

Front 33

7. How would you differentiate between - and ß-hemolysis?

Back 33

alpha- or partial hemolysis results in greenish or grayish zones around the colonies. ß-hemolysis is totalhemolysis of the blood cells resulting in clear or yellowish zones around the colonies.

Front 34

1. Of what use to bacteria is the ability to produce H2S?

Back 34

It allows the bacteria to metabolize sulfur-containing amino acids efficiently and use the products asnutrients.

Front 35

2. How is SIM medium used to detect motility?

Back 35

growth beyond the stab line

Front 36

3. What substrates are acted on in SIM medium in order for H2S to be produced?

Back 36

H2S is produced from cysteine in the peptone and from the reduction of sodium thiosulfate.

Front 37

4. In addition to H2S production and motility, for what other test can SIM medium be used?

Back 37

The production of indole from the degradation of tryptophan in the peptone.

Front 38

5. How does a black precipitate of FeS indicate the production of H2S?

Back 38

When H2S is produced from cysteine or thiosulfate, it combines with ferrous ammonium sulfate toproduce an insoluble, black ferrous sulfide

Front 39

6. What does cysteine desulfurase catalyze? Show the reaction.

Back 39

Cysteine desulfurase catalyzes the removal of sulfur and ammonium ion from the amino acidcysteine. cysteine

H2O + cysteine --------> pyruvic acid + NH3 + H2S. desulfurase

Front 40

7. What does thiosulfate reductase catalyze? Show the reaction.

Back 40

Thiosulfate reductase adds hydrogen to thiosulfate resulting in sulfite and hydrogen sulfide as endproducts. thiosulfate

2S2O32- + 4H + --> 2SO32- + 2H2S. reductase

Front 41

What is the component in the SIM deep tubes that makes this medium suitable to detect theproduction of indole by bacteria?

Back 41

The amino acid tryptophan.

Front 42

2. What organic molecule is necessary to detect mixed acid fermentation by bacteria?

Back 42

The pH indicator methyl red.

Front 43

3. Why did you shake the MR-VP culture?

Back 43

To aerate the culture and facilitate the color change.

Front 44

Can a bacterium that ferments using the 2, 3-butanediol pathway also use the mixed acid route?

Back 44

Yes, but fewer acidic end products will be produced, and the pH will not be significantly lowered inthe medium.

Front 45

Why is a chemically defined medium necessary for the detection of citrate utilization bybacteria?

Back 45

To ensure that no alternative carbon sources are in the medium, which might preferentially be utilizedin place of the citrate.

Front 46

1. What metabolic property characterizes bacteria that possess oxidase activity?

Back 46

Bacteria that possess oxidase activity have cytochrome c and its associated oxydase. Electrons aretransferred to cyt c and then to cyt c oxydase to molecular oxygen.

Front 47

2. What is the importance of cytochrome oxidase to bacteria that possess it?

Back 47

It allows the bacteria to use oxygen (O2) as an electron acceptor and form water utilizing reducedcytochrome c as the electron (hydrogen) donor.

Front 48

3. Do anaerobic bacteria require oxidase? Explain your answer.

Back 48

No, because they will not grow or survive in the presence of oxygen; therefore, they lack the ability toreduce oxygen.

Front 49

4. What is the function of the test reagent in the oxidase test?

Back 49

The reduced indicator reagents (oxidase reagents) serve as artificial electron donors for cytochromeoxidase. When they are oxidized (donate their electrons to cytochrome oxidase) they change from alight pink to a dark purple.

Front 50

5. The oxidase test is used to differentiate among which groups of bacteria?

Back 50

The oxidase test differentiates among gram-negative pathogenic bacteria that are oxidase positive andthe Enterobacteriaceae which are oxidase negative.

Front 51

Why should nichrome or other iron-containing inoculating devices not be used in the oxidasetest?

Back 51

They would serve as an electron donor and might cause false-positive results.

Front 52

1. From your results, which bacteria are negative for nitrate reduction? Which are positive?

Back 52

Negative = Staphylococcus epidermidis

Positive = Escherichia coli, Pseudomonas fluorescens

Front 53

2. How do you explain the results from the soil sample?

Back 53

The answer will vary depending upon the student’s results.

Front 54

3. Why is the development of a red color a negative test when zinc is added?

Back 54

The red color in the presence of zinc depends upon the presence of substantial amounts of NO-3within the medium; thus it is not reduced by the bacteria

Front 55

What are the end products that may result from the action of bacteria with nitrate-reducingenzymes?

Back 55

Nitrite (NO2), Ammonia (NH+3), and molecular nitrogen (N2).

Front 56

5. What is the purpose of a control tube in this exercise?

Back 56

To test for microbial contamination.b. To see if NO-3 is spontaneously degenerated within the medium.

Front 57

6. How would you perform a complete test for the presence of nitrate reduction?

Back 57

After growth of the pure culture for 24 to 48 hours in nitrate broth, add sulfanilic acid and N,Ndimethyl-l-naphthylamine and look for a red color (positive result). If no red color appears, then add apinch of zinc powder and observe whether the solution remains colorless (a positive result) or if itturns red (a negative result).

Front 58

1. Explain the biochemistry of the urease reaction.

Back 58

Urea is enzymatically degraded to its components: ammonia, carbon dioxide, and water.

Front 59

2. What is the purpose of the phenol red in the urea broth medium?

Back 59

To indicate a change in pH (if urea is degraded, ammonia will be produced and the pH will increaseresulting in a reddish-pink color).

Front 60

3. When would you use the urease test?

Back 60

To distinguish Proteus from other nonlactose-fermenting enteric bacteria.

Front 61

4. In a positive urease test, why does the urea disk change color?

Back 61

Because the pH becomes alkaline as ammonia is produced.

Front 62

6. What is in urea broth? In a urea slant?

Back 62

In the urea broth, various nutrients (see next sentence) including a substantial amount of urea and anappropriate acid-base indicator dye such as phenol red. In the urea slant, agar, NaCl, Na2HPO4,peptone, glucose, KH2PO4, phenol red, urea solution.

Front 63

7. What color is cerise?

Back 63

A deep pink-red color.

Front 64

1. Explain what occurs during decarboxylation.

Back 64

The carboxyl group is removed from an amino acid resulting in CO2 and an amine.

Front 65

2. Why does the LDC broth or lysine iron agar turn purple when lysine is decarboxylated?

Back 65

The production of alkaline amines raises the pH of the medium.

Front 66

Why does the LDC medium always turn yellow regardless of the ability of the bacteria toproduce lysine decarboxylase?

Back 66

Because the sugar present in the medium (usually glucose) is fermented first, lowering the pH andthus activating the decarboxylases.

Front 67

4. Why is the lysine decarboxylase test negative if both LDC and DC broths turn purple?

Back 67

The microorganism is producing alkaline metabolites but did not ferment the sugar present due toaerobic conditions.

Front 68

5. Why is sterile mineral oil added to LDC test media?

Back 68

To bring about oxygen deficiency within the medium.

Front 69

7. How does the pH indicator bromcresol purple indicate a change in pH?

Back 69

At acidic pH it is gray or yellow, and at alkaline pH it changes to purple.

Front 70

1. What are two ways that phenylalanine can be used by P. vulgaris?

Back 70

a. It can deaminate it to form phenylpyruvic acid.b. It can put it into protein as is.

Front 71

2. What is the purpose of the ferric chloride in the phenylalanine deamination test?

Back 71

It reacts with phenylpyruvic acid to form a green complex.

Front 72

3. When would you use the phenylalanine deamination test?

Back 72

When attempting to distinguish between enteric bacteria such as Escherichia coli and Proteusvulgaris.

Front 73

4. Name some bacteria that can deaminate phenylalanine.

Back 73

a. Proteus species

b. Morganella species

c. Providencia species

Front 74

5. Describe the process of deamination.

Back 74

This is the enzymatic removal of an amino group from an organic compound such as an amino acid.

Front 75

6. Why must the phenylalanine test be determined within 5 minutes?

Back 75

Because the color fades very quickly.

Front 76

7. Describe the color of an uninoculated tube of phenylalanine agar.

Back 76

A yellow-brown color.