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microbiology lab > Lab Exam 3 Part 1 > Flashcards

Lab Exam 3 Part 1 Flashcards

1
Q

What is a free radical?

A

A molecule with one or more unpaired electron in its outer shell. Unstable molecule that is made during normal cell metabolism

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2
Q

Why do organisms using or living in the presence of oxygen need to eliminate theses chemical supplies (free radicals)?

A

Because some forms of oxygen are toxic. Either strong oxidizing agents or steal electrons and damage cellular components.

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3
Q

In reference to oxygen usage, which organisms must produce enzymes that detoxify free radicals?

A

Aerobes, faculative anaerobes, microaerophiles, and aerotolerant organisms.

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4
Q

In practical terms, name the two bacterial species differentiated by the catalase test.

A

staphylococci vs. streptococci

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5
Q

Both staphylococci and streptococci living in the presence of oxygen and produce the enzyme superoxide dismutase which catalyzes the conversion of superoxide to hydrogen peroxide. Name the enzyme used by staphylcocci and streptococci to detoxify hydrogen peroxide. Which reaction produces oxygen bubbles? Which does not?

A

Enzyme catalase. The reaction catalyzing hydrogen peroxide by Superoxide dismutase (SOD) and the reaction catalyzed by catalase produce oxygen bubbles. The reaction catalyzed by peroxidase does not.

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6
Q

Why can it be difficult to differentiate betweeen staphylcocci and streptococci on the basis of Gram stain results?

A

They are both Gram-positive cocci. Only other difference is staph grows in clumps while strep grows in chains.

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7
Q

Catalase

Define and what are the end products?

A

Enzyme used by orgnaisms that either use oxygen or live in presence of oxygen. It converts toxic hydrogen peroxide into oxygen gas and water.

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8
Q

Hydrogen peroxide (H₂O₂)

A

toxic byproduct produced as oxygen enters respiratory pathways.

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9
Q

What are the three routes that catalyze hydrogen peroxide?

A

1) Superoxide dismutase (SOD) catalyzes peroxide anion O2^-2 into hydrogen peroxide H2O2 then catalase catalyzes it to O2 and water.

2) Catalase catalyzes hydrogen peroxide directly.

3) Peroxidase catalyzes hydrogen peroxide directly.

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10
Q

Catalase test procedure:

A
  1. Need solid media cultures, 3% H2O2, and clean slides.
  2. Prepare smear.
  3. Apply 1-2 drops of 3% H2O2 to the wet smear while holding slide over a dark surface.
  4. Observe for bubbling and record results.
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11
Q

Catalase test results of streptococcus and staphylococcus.

A

Streptococcus= - no bubbles
Staphylococcus= +Bubbles

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12
Q

Support or refute this statement: If an organism is catalase negative it must be anaerobic?

A

Refute, there are other enzymes other then catalase that can be used to detoxify hydrogen peroxide.

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13
Q

Superoxide free radical

Whyis it toxic and how does it occur?

A

Highly reactive form of oxygen. Posses unpaired electron, formed by the incomplete reduction of O2. Detoxified by superoxide dismutase.

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14
Q

Superoxide dismutase

A

Enzyme that detoxifies superoxide radical.

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15
Q

Peroxidase

A

Enzyme that can detoxidy peroxide anion.

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16
Q

The catalase test is performed by adding 3% hydrogen peroxide to a colony of an unknown bacterium on a blood agar plate. Vigorous bubbling is observed. Making a smear from a colony off a tryptic soy agar plate shows no bubbling after adding a couple drops of the hydrogen peroxide. Explain the discrepancy in results. Is the organism really catalyses possessive.

A

No, catalase is abundant in red blood cells. The false positive result was the hydrogen peroxide reacting with the media not the bacteria.

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17
Q

Whys is the coagulase enzyme produced by some staphylococci?

A

Uses coagulase to form a fibrin coat from fibrinogen present in the bloodstream. This helps the bacteria evade detection and phagocytosis by the immune system.

Key feature of pathogenic Staph. The enzyme produces coagulation of blood, allowing the organism to “wall “ its infection off from the host’s protective mechanisms rather effectively.

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18
Q

How is the coagulase test used to differentiate between Staphylococcus aureus and Staphylcoccus epidermidis?

A

Inoculate plasma with bacteria and observe for clots.

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19
Q

Is Staphylococcus aureus or Staphylococcus spidermidis considered to be more pathogenic?

A

Staphylococcus aureus is pathogenic while the other is usually not pathogenic.

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20
Q

Describe the expected reaction of each staphylococcus species on mannitol salt agar.

A

Mannitol fermentation by pathogenic staphylococci, such as Staphylococcus aureus, is indicated by the media changing to yellow, whereas Staphylococcus epidermidis, a non-pathogenic species of staphylococci, does not produce a yellow color.

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21
Q

Coagulase

A

Enzyme. A virulence factor produced by some bacteria that causes blood or plasma to clot.

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22
Q

How can you tell weather Gram positive cocci in clusters are staphylococci and not streptococci wholes normal cell arrangement was disrupted?

A

Preforme a catalase test.

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23
Q

How could you determine whether staphylococci were pathogenic or not? Be specific about the test you would perform and the rusults of those test.

A

Run a coagulase test. The pathogenic strains have the coagulase enzyme as it aids their infiltration of the body. If clots form the strain is pathogenic.

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24
Q

Coagulase test procedure:

A
  1. Gather stock cultures of staphylococcus aureua and Staphylcoccus spidermidis, 2 plastic capped tubes containing 0.5ml ciratad rabbit plasma, and 1 mannitol salt agar plate.
  2. Heavily inoculate each species into a tube of nitrated rabbit plasma. Using the loop thoroughly mix the inoculums in the rabbit plasma.
  3. Incubate up to 4 hours at 37C checking the tube every half hour by tilting them to determine if a clot has formed. Clots=positive and no clots= negative
  4. Divide the mannitol salt agar plate in half with a marker and streak each organism on half the plate. Incubate for 24 hours at 37C.
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25
Q

What do the general results of coagulase test look like?

A

Positive= clots
Negative= no clots.

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26
Q

What does Staphylococcus aureus and Staphylococcus epidermidis look like on mannitol salt agar (MSA)?

A

Both grow as they are salt tolerant. However, Staphylococcus aureus has the ability to ferment mannitol resulting in acid waste products turning the media yellow. Staphylococcus epidermidis does not ferment mannitol so media stays red.

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27
Q

How is cytochrome c oxidase involved in aerobic cellular respiration?

A

It is an electron carrier.

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28
Q

Can cells lacking cytochrome c oxidase still perform aerobic cellular respiration? If so, how?

A

Yes, there are a diverse range of electron carriers used in cellular respiration so other ones get used instead.

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29
Q

cytochrome c oxidase

What is it and where is it found?

A

Electron carrier present in many but not all electron transport chains in archaea, bacteria, and eukaryotes.

Its just and alterantive to NADH and FADH, carries electrons.

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30
Q

Oxidase test procedure:

A
  1. Gather slat cultures, sterile swabs, and oxidase reagent ( N,N-dimethyl-p-phenylenediamine and alpha-naphthol).
  2. Inoculate organisms onto a separate sterile swab.
  3. Add one drop of the oxidase reagent to each swab.
  4. Observe for color change and record results. Blue=positive, no color=negative
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31
Q

What makes up the oxidase reagent?

A

N, N-dimethyl-p-phenylendiamine and alpha-naphthol.

32
Q

How is cytochrome c oxidase involved in aerobic cellular respiration?

A

The role of cytochrome c is to carry electrons from one complex

33
Q

How can some bacteria survive and perform aerobic cellular respiration without cytochrome c oxidase?

A

they produce and use a different enzyme, other than cytochrome c oxidase, to transfer electrons from the electron transport chain to O2.

34
Q

In what specific part of the cell is cytochrome c oxidase found in bacteria that posses it?

A

Mitochondira

35
Q

In what specific part of the cell is cytochrome c oxidase found in humans.

A

Inner mitochondiral membrane

36
Q

Positive vs. negative results of a oxidase test.

A

Blue=positive
No color=negative

37
Q

What is a differential medium and how is it useful?

A

Contains specific ingredients or chemicals that allow the observer to visually distinguish which species possess and which species lack a specific biochemical process

38
Q

At the molecular level, why would one bacterial species possess a particular enzyme while another bacterial species lacks that same enzyme?

A

Because it depends on the substrate the bacterial uses. Enzymes are substrate specific so they need to correctly fit the situation they are in.

39
Q

In general terms, how are molecules other than glucose typically catabolized?

A

Converted to glucose or converted into other intermediates of the aerobic respiration pathway.

40
Q

What is the relevance of pyruvate to aerobic respiration?

A

In aerobic respiration, pyruvate is the bridge between glycolysis and the Kreb’s cycle. It is produced in glycolysis, and then is brought into the mitochondria where it forms acetyl Coenzyme A.

41
Q

What is anaerobic respiration and how does it differ form aerobic respiration?

A

Aerobic respiration needs oxygen to occur, while anaerobic does not. This presence of oxygen determines what products will be created. During aerobic respiration, carbon dioxide, water, and ATP are produced. During anaerobic respiration, lactic acid, ethanol, and ATP are created.

42
Q

What is an indicator molecule and how is it useful?

A

Substances whose solutions change color due to changes in pH. Useful in indicating reactions that cause a pH change like fermentation.

43
Q

What organelle is generally responsible for bacterial motility?

A

Flagellum

44
Q

What does SIM media stands for? and what does it test for?

A

Sulfur, Indole, and Motility Media. Tests for sulfur reduction, indole production, and motility.

45
Q

Sulfure reduction

A

Some bacteria posses the enzyme cysteine desulfurase which reduces the sulfer containing amino acid cysteine to form pyruvate and hydrogen sulfide (H2S). Other bacteria produce thiosulfate as a terminal electron acceptor during anaerobic respiration.

46
Q

How are the results of SIM media interpreted?

A

Black precipitate= H2S positive
No precipitate=H2S negative

Pink/red color at top of tube= Indole postive
No color at top of tube= Indole negative

Growth confined to stab line= lack motility.
Growth extending laterally from the stab line= motility present.

47
Q

Indole production

A

Some bacteria possess the enzyme tryptophanase which converts the amino acid trytpohan into pyruvate, indole, and ammonia.

48
Q

Kovac’s reagent

A

Indole production indicator reagent. Reacts with indole producing a red or pink color at the top of the solution. Color change is caused by a reaction between indole and the para-dimethylaminobenzaldehyde (DMAB) in kovac’s reagent.

49
Q

Motility of SIM

A

Semi solid, meaning low agar content then solid media typically used.

50
Q

Ferric ammonium sulfate can be used as an indicator molecule for hydrogen sulfide production. What indicator molecules have you used in previous labs?

A

Mannatol, Blood agar, and MacConkey agar.

51
Q

What species of the SIM test produced H2S? What is the genetic basis for this phenotype?

Bacillus subtilis
Enterococcus faecalis
Escherichia coli
Pseudomonas aruginosa
Proteus vulgaris
Staphylcoccus epidermidis

A

Proteus vulgaris. The ability to produce the enzyme cysteine desulfurase?

51
Q

What species produced indole? What is the genetic basis for this phenotype?

Bacillus subtilis
Enterococcus faecalis
Escherichia coli
Pseudomonas aruginosa
Proteus vulgaris
Staphylcoccus epidermidis

A

Escherichia coli and Proteus vulgaris. The ability to produce the enzyme tryptophanase.

52
Q

Your SIM tube shows a wide band of growth from one angle and a thin band of growth form another. How would you explain this result? Is this bacterium motile or non-motile?

A

The lack of uniform growth could be due to the stab needle not being pulled straight out. The bacteria should be retested to confirm results.

53
Q

How would you test an H2S producing species for motility?

A

By using a SIM plate.

54
Q

What species produced was motile?

Bacillus subtilis
Enterococcus faecalis
Escherichia coli
Pseudomonas aruginosa
Proteus vulgaris
Staphylcoccus epidermidis

A

Bacillus subtilis
Escherichia coli
Pseudomonas aruginosa
Proteus vulgaris

55
Q

SIM Media Procedure

A
  1. properly label SIM media tubes.
  2. Straighten inoculating needle by rolling it on the benchtop.
  3. Roll the tip of the needle in a pure bacterial culture on solid media.
  4. Carefully stab the media to a depth of about 2/3 of the depth of the tube.
  5. Lossen the slants cap 1/4 to 1/2 turn to allow gas exchange.
  6. Incubate 37C for 48 hours.
  7. Examine tube for growth pattern and color change.
  8. Add 0.5ml of Kovac’s reagent to the top of the tube to test for indole.
  9. Examine for red-pink oclor.
56
Q

Is blood agar synthetic or non-synthetic? Why?

A

Blood agar is a non-synthetic (complex) media. Non-synthetic media contain at least one of the ingredients is not chemically defined. Blood agar is a non-synthetic media because it contains many unspecified nutrients.

57
Q

To which functional category or categories does blood agar belong? Explain.

A

Differential, a visual change occurs depending on the characteristics of the bacteria being gown.

58
Q

Alpha hemolysis and its effect on blood agar

A

Alpha hemolysins partially degrade hemoglobin and results in a greening of the agar around the colonies.

59
Q

Beta hemolysis and its effects on blood agar

A

Beta hemolysins completely break down hemoglobin and results in a clearing of the agar around the colonies.

60
Q

Gamma hemolysis and its effect on bood agar

A

Also called gamma hemolytic. Do not produce hemolysins and results in no change in the appearance of the agar.

61
Q

What is the general term for the enzyme that causes hemolysis?

A

Hemolysins

62
Q

Which bacterial species is responsible for a “strep throat”?

A

Streptococcus pyogenes

63
Q

What type of hemolysis is expected with the pathogen that causes “strep throat”?

A

Alpha and Beta

64
Q

What composes blood agar?

A

5% sheep blood and nutrient agar.

65
Q

Fastidious organism

A

A fastidious organism is defined as any organism which has very complicated nutritional requirements, meaning it will not grow without specific factors present or in specific conditions.

66
Q

Benefits of hemolysins to bacteria?

A

Increases pathogenicity of an orgnaism. Allows them to break down red blood cells and/or hemoglobin. The bacteria can utilize hemolysis to release and utilize nutrients from the host animal cells. Iron e.g., is essential to many pathogenic bacteria, but is only present in very low concentrations outside the cells

67
Q

Blood agar procedure

A
  1. 5% sheep agar plates, Enterobacter aerogenes, Pseudomonas aerginosa, and Stretococcus penumoniae.
  2. With marker divide bottom of blood agar plate into three equal segments and label EA, PA, and SP.
  3. Streak each segment of the plate with the appropriate stock culture. Each streak should be about one cm long in the center of the segment.
  4. Incubate at 37C for 24 hours. Instructor may recommend the use of a candle extinction jar, which helps promote the growth of capnophilic species of streptococci,
68
Q

Procedure for performing a throat culture

A
  1. Sterile swabs, tongue depressor, loop, blood agar plate, slides, and Gram stain supplies.
  2. Have your partner swab the tonsil area of your throat. Be careful to avoid touching the swab to the surfaces of the mouth or tongue. Use a tongue depressor to hold the tongue down. Rotate the swab as you obtain your specimen.
  3. Incubate a small area of your plate as you streak and simultaneously rotate the swab.
  4. Discard the swab in hazardous waste.
  5. Flame an inoculation loop and streak the inoculated sector for isolation.
  6. IIncubate at 37C for 24 hours.

7.Examine the plate for colonies demonstrating a and b hemolysis.

  1. Make smear of each type of hemolytic colony and gram stain.
69
Q

Blood agar results for following bacteria:
Enterobacter aerogenes
Pseudomonas aerginosa
Stretococcus penumoniae

A

Enterobacter aerogenes- Gama hemolytic, no change.

Pseudomonas aerginosa- Beta hemolysis, clearing of agar around colonies.

Stretococcus penumoniae- Alpha hemolysis, greening of the agar around the colonies.

70
Q

One disease caused by Streptococcus pyogenes

A

Strept throat and scarlet fever.

71
Q

One disease caused by Streptococcus pneumoniae

A

sinusitis, otitis media, pneumonia, bacteraemia, osteomyelitis, septic arthritis and meningitis

72
Q

One disease caused by Pseudomonas aeruginosa

A

Pneumonia,endocarditis, meningitis surigical site infections, UTI’s, septicemia.

73
Q

One disease caused by Enterobacter aerogenes

A

urinary tract infections (UTI), respiratory infections, soft tissue infections, osteomyelitis, and endocarditis

74
Q

Why do you incubate blood agar plates at 37C? Use appropriate terms to describe temperature range required for growth of theses organisms.

A

Because the bacteria expected normally grow in the human body at body temperature. Theses bacteria are mesophiles and grow between 20-45C

75
Q

Blood agar results

A

Alpha hemolysins: Greening of the agar around the colonies.

Beta hemolysins: clearing of the agar around colonies.

Gama hemolytic: no change

microbiology lab (7 decks)

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