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Microbiology > Lab exam 3 > Flashcards

Lab exam 3 Flashcards

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1
Q

What reagent is used in an oxidase test?

A

oxidase

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2
Q

What is the purpose of the oxidase test?

A

to determine if an organism possesses Cytochrome C Oxidase

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3
Q

How is the oxidase test performed?

A

The bacteria is first spread onto sterile filter paper. Then oxidase reagent is dropped onto the filter paper. Tetramethyl-p-phenylenediamine is used as the color indicator. If the indicator is oxidized, the test will change color and it is positive for cytochrome c oxidase.

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4
Q

Why should you read the Oxidase test results within 30 seconds?

A

the reagents of this test are unstable, so they may oxidize independently after becoming moist

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5
Q

What color is a positive oxidase test?

A

dark blue/purple

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6
Q

What color is a negative oxidase test

A

it remains white

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7
Q

what reagent is used in a catalase test?

A

hydrogen peroxide

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8
Q

Why is the catalase test used?

A

It determines the presence or absence of the enzyme catalase. It is typically used to differentiate between gram-positive cocci organisms.

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9
Q

How is the catalase test performed?

A

The organism is spread onto a microscope slide and hydrogen peroxide is dropped on top of it. the more of the organism there is, the better the result. If the result is positive it will have bubbles, and if it is negative it will do nothing.

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10
Q

What does a positive catalase test look like?

A

Any sign of bubbles indicates a positive test

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11
Q

What does a negative catalase test look like?

A

the bacterial smear will show and there will be no bubbles

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12
Q

Why is MacConkey used?

A

It is used to selectively isolate gram-negative and enteric bacteria and differentiate them based on lactose fermentation

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13
Q

What does the bacteria look like on the MacConkey media?

A
  • Lactose Fermenters turn the agar red or pink
  • Non-fermenters do not change the color of the agar
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14
Q

Is the MacConkey test selective or differential?

A

It is BOTH Selective and Differential.

It is Selective to gram-negative enteric bacteria and it differentiates between lactose fermenters and non-fermenters

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15
Q

What does a positive Malonate test look like?

A

Blue

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16
Q

What does a negative Malonate test look like?

A

Green

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17
Q

Why is the Malonate test used?

A

It is used to determine if a microorganism has the enzymes needed to use malonate as a sole carbon source

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18
Q

What is the chemical reaction that occurs during the Malonate test?

A

In one portion of the Krebs cycle, which requires the enzyme succinate dehydrogenase, the coenzyme FAD is reduced to FADH2. Malonate, when added to the media, is similar enough to succinate to replace it as the substrate in the reaction. When combined with subsequent build up of succinate in the cell, shuts down the Krebs cycle and will kill the organism unless it can ferment malonate as its sole remaining carbon source.

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19
Q

What causes the color change in the Malonate test?

A

the color indicator, Bromthymol blue dye, will change color when there is a change in pH.

If the organism uses malonate, it will alkalize the medium and change the color from green to blue

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20
Q

What does a positive Simmon’s citrate test look like?

A

blue

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21
Q

What does a negative Simmon’s citrate test look like?

A

green

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22
Q

Why is the Citrate Test Used?

A

The citrate test determines the ability of an organism to use citrate as its sole source of carbon.

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23
Q

Determine the chemical reaction that takes place during the Citrate Test

A

In a medium containing citrate as the only available carbon source, bacteria that possess citrate-permease can transport molecules into the cell and enzymatically convert it to pyruvate. Pyruvate then can be converted to a variety of products, depending on pH of the environment. The color change occurs because the ammonium phosphate is converted to ammonia and ammonium hydroxide that make the agar overall alkaline.

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24
Q

What cause the color change in the Citrate Test?

A

Bromthymol blue dye, whihc is green at the pH of 6.9 and blue at pH 7.6, is added as an indicator.

Bacteria that survive in the medium and utilize the citrate also convert NH3 and ammonium hydroxide, both of which tend to alkanize the agar. As the pH goes up, the medium changes from green to blue.

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25
Q

What does a positive BEA (Bile Esculin Agar) test look like?

A

dark brown

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26
Q

What does a negative BEA (Bile Esculin Agar) test look like?

A

light brown

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27
Q

What is the purpose of the bile esculin agar?

A

To presumptively identify Group D Streptococci and certain Enterococcus species

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28
Q

Is the Bile Esculin Agar selective or differential? How so?

A

Both; it is selective because only Group D streptococci can grow on the medium. It is differential bc it is differentiating whether or not an organism can hydrolyze esculin in the presence of bile.

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29
Q

Describe the reaction that takes place in a positive bile esculin agar test.

A

The esculin found in the medium is hydrolyzed by esculinase to make B-D-glucose and esculetin as products. The esculetin product will then react with the ferric citrate indicator to create a black precipitate indicative of a positive reaction.

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30
Q

What is the indicator in BEA (Bile Esculin Agar)?

A

ferric citrate

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31
Q

What test uses Sulfanilic Acid and alpha-napthylamine as reagents?

A

Nitrate reduction test

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32
Q

What does a positive nitrogen reductase test look like?

A
  • turning Red immediately after adding reagents A and B
  • no color change of a clear result after adding Zinc
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33
Q

What does a positive methyl red (MR) test look like?

A

Red

34
Q

What does a negative methyl red (MR) test look like?

A

Orange

35
Q

How do you test for Methyl Red (MR) and what reagents are used?

A

3 drops of methyl red reagent are added to the inoculated and incubated tubes and mixed before seeing results. If the test turns red, it is positive for mixed acid fermentation. If no color change happens, it is negative.

36
Q

Why is the methyl red test used?

A

To identify the bacteria capable of fermenting glucose

37
Q

How do you test for Voges-Proskauer and what reagents are used?

A

Add 15 drops of Barritt’s A and 5 drops of Barritt’s B to the inoculated and incubated tube and mix.The color change can take from 10 minutes to an hour to occur. If it is positive for 2,3-butanediol fermentation it is red, and if it is negative it doesn’t change color.

38
Q

What does a Positive VP test look like?

A

Red

39
Q

What does a negative VP test look like?

A

No color change (orange)

40
Q

MR and VP have ________ results

A

Inverse

41
Q

What does a positive urea test look like?

A

Pink

42
Q

What does a negative urea test look like?

A

Orange

43
Q

Why is the Urease Hydrolysis test used?

A

To identify bacteria capable of hydrolyzing urea using urease

44
Q

What does a positive MSA (mannitol salt agar) test look like?

A

Yellow slant, red butt

45
Q

What does a negative MSA test look like?

A

Red slant, red butt

46
Q

Why is the MOI test done in a semisolid medium?

A

Because it allows motile bacteria to move through it

47
Q

What three tests does the medium in MOI test for?

A

Motility, Ornithine, and Indole

48
Q

What does a positive test for motility look like?

A

uniform cloudiness where no stab lines are visible

49
Q

What does a negative motility test look like?

A

it is clear and the stab line can be clearly seen

50
Q

What does an Ornithine Positive test look like?

A

Purple

51
Q

What does a negative Ornithine Negative test look like?

A

Yellow/brown

52
Q

What does a positive indole production test look like?

A

a red ring at the top of the medium

53
Q

What does a negative indole production test look like?

A

no ring formed

54
Q

What reagent is used to test for indole production?

A

Kovac’s reagent

55
Q

In what order should you read the MOI tests?

A
  1. Motility
  2. Ornithine Decarboxylation
  3. Indole
56
Q

In Lysine Iron Agar (LIA), what does purple slant and purple butt mean?

A

Positive for Lysine decarboxylation

57
Q

In LIA, what does purple slant and yellow butt mean?

A

negative for Lysine decarboxylation

58
Q

in LIA, what does red slant and yellow butt mean?

A

negative for Lysine decarboxylation and positive for Lysine deamination

59
Q

Why is the Lysine iron agar test used?

A
  • It is often used to differentiate enteric bacteria
  • the slant tests for anaerobic and aerobic bacteria
  • the ability of the microorganism to produce gas
  • to test for the presence of two different enzymes: Lysine decarboxylase and Lysine deaminase
60
Q

If an LIA slant is burgundy, how is that helpful diagnostically?

A

if the slant is burgundy/red, the test is automatically positive for Lysine Deaminase, and therefore is aerobic since the enzyme works in the presence of oxygen.

61
Q

What causes a burgundy slant and what three genera is this indicative of?

A

Deaminase is the enzyme that works in the presence of oxygen and causes the burgundy slant.

Providencia, Proteus, Morganella.

62
Q

What do the carbohydrate fermentation tests test for?

A

It tests for whichever carbohydrate is being used

ex: Glucose tests for the presence of glucose

63
Q

What is the medium used for a carbohydrate fermentation test?

A

phenol red broth

64
Q

What does a positive carbohydrate fermentation test look like?

A

yellow

65
Q

What does a negative carbohydrate fermentation test look like?

A

red/pink

66
Q

What is the Triple Sugar Iron Agar (TSIA) test used for?

A
  • to differentiate different gram negative enteric (gut) bacteria
  • tests the ability of the microorganism to ferment glucose, sucrose, and/or lactose
  • test for gas production
  • test for H2S production
67
Q

Describe a TSIA tube that is A/A/+/-

A

yellow slant, yellow butt, positive for gas formation, negative for H2S formation

68
Q

Describe a TSIA tube that is A/A/+/+

A

yellow and black, positive for gas production, positive for H2S production

69
Q

Describe a TSIA tube that is K/A/-/-

A

red slant, yellow butt, no gas formation, no H2S

70
Q

Describe a TSIA tube that is K/K/-/-

A

red slant, red butt, no gas formation, and no H2S

71
Q

True or False: you stab AND streak a TSIA tube

A

true

72
Q

in TSIA, what causes the color change in the Slant?

A

aerobic reactions

73
Q

In TSIA, what causes the change in color?

A

Depending on what kind of fermentation, the amount of acid produced affects the pH level of the medium thus affecting the color. (Color turns yellow from fermentation)

74
Q

What butt reaction will you see for all enterics?

A

All enterics will ferment glucose within the first few hours resulting in a color change to yellow.

75
Q

What are the three sugars and what is the purpose of their concentrations? (TSIA)

A

The three sugars are:
-Glucose
-Lactose
-Sucrose

Glucose (.1%) is fermented first followed by Lactose and Sucrose (1%). This helps differentiate whether or not a organism is capable of fermenting either Lactose or Sucrose or both after fermenting Glucose, which is in a shorter supply.

76
Q

What are the indicators in TSIA?

A

ferric iron and phenol red

77
Q

What is differential staining?

A

Differential staining is a staining process which uses more than one chemical stain. Using multiple stains can better differentiate between different microorganisms or structures/cellular components of a single organism.

78
Q

What cellular structures differ between gram positive and gram negative bacteria?

A

Gram positive bacteria have a thick peptidoglycan cell wall with a single internal plasma membrane. They do not contain LPS but they do contain teichoic and lipoteichoic acid.
Gram negative bacteria have a thinner peptidoglycan cell wall with an outer membrane and an inner plasma membrane (dual membrane). They contain LPS.

79
Q

What does a positive gram stain look like?

A

purple

80
Q

What does a negative gram stain look like?

A

pink

81
Q

What is the order of a gram staining procedure?

A
  1. DI water droplet in the middle of the slide
  2. inoculate your loop and smear the bacteria into the water droplet
  3. let it completely dry on the benchtop
  4. after completely dry, attach a clothespin to one end and heat fix with the incinerator
  5. cover with crystal violet and let sit for one minute
  6. rinse with DI water
  7. decolorize with ethanol
  8. immediately rinse with DI water
  9. cover with safranin and let sit for one minute
  10. rinse with DI water
    11.dab with paper towel to dry off and observe with immersion oil under the microscope
82
Q
A

Microbiology (11 decks)

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