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From Flashcardlet > Micro Lab Practical 2 (also study quiz 4) > Flashcards

Micro Lab Practical 2 (also study quiz 4) Flashcards

1
Q

How is a phenol red broth differential?

A

It is used to determine if an organism can ferment a particular sugar

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2
Q

What are the products of fermentation?

A

Acids, alcohol, CO2

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3
Q

Is fermentation an/aerobic?

A

Anaerobic respiration

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4
Q

What are the key ingredients of the phenol red broth?

A 
Peptone (protein digest)
Single carb (glucose, sucrose, or lactose)
Durham tube (collects gases)
Phenol red (pH indicator)
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5
Q

What happens to the color of phenol red if the solution is acidic?

A

It turns yellow

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6
Q

What happens to the color of phenol red if the solution is neutral?

A

No change - red

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7
Q

What happens to the color of phenol red if the solution is basic?

A

Hot pink

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8
Q

How would you know if the organism is positive for fermentation?

A

It the phenol red turns yellow

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9
Q

How would you know if the organism can deaminate proteins?

A

The phenol red turns hot pink

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10
Q

What is deamination?

A

Taking the amino group off of a protein

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11
Q

What will the organism use in the broth first as energy? Are the two sources dependent?

A

Carbs, then proteins - independent of one another

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12
Q

Why is the phenol broth necessary to check at 24 hours?

A

Because you need to see if the broth turns yellow first before it turns pink. If it turns pink, you could have missed the yellow

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13
Q

What if the broth is pink but the Durham tube is yellow?

A

The organism is positive for fermentation and deamination

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14
Q

What is the final TEA in nitrate reduction?

A

Nitrate

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15
Q

Is nitrate reduction an/aerobic respiration?

A

Anaerobic

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16
Q

What is nitrate reduction used for?

A

As a way to obtain nitrogen to build compounds

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17
Q

What is the order of the reduction?

A

NO3 - NO2 - (NH4) NO - N2O - N2

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18
Q

What do our reagents detect?

A

NO2

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19
Q

If after 48 hours there are bubbles in the Durham tube, what does that mean?

A

It could be NO2, but it could also be CO2 from fermentation

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20
Q

If there are bubbles in the Durham tube, how could you be sure it was NO2?

A

If you knew it was a nonfermentor and therefore didn’t produce CO2

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21
Q

What are the reagents you add after 48 hours?

A

8 drops of sulfanilic acid and 8 drops of naphthylamine

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22
Q

What happens if the solution is red after adding the first two reagents?

A

Red means it is positive for NO2 production

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23
Q

What happens if the solution does not change after adding the first two reagents?

A

It is negative for NO2 production, OR it reduces NO2 further

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24
Q

If there is no change in the solution, what do you add?

A

Zinc powder

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25
Q

What happens if the solution is red after adding the zinc powder?

A

It is negative for nitrate reduction - NO2 is present but the zinc reduced it, not the bacteria

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26
Q

What happens if the solution does not change after adding the zinc powder?

A

It is positive for nitrate reduction - NO2 was reduced beyond (pink after zinc is negative)

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27
Q

What is the catalase test used for?

A

See if the organism produces the detoxifier enzyme, catalase

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28
Q

What molecule does catalase detoxify and where does it come from?

A

Hydrogen peroxide is a natural by product of aerobic respiration and is toxic, therefore it must be broken down

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29
Q

What is the reaction for the detoxification?

A

H2O2 —(catalase)—-> 2 H2O + O2

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30
Q

How do you test positive for catalase?

A

If the culture bubbles, it uses catalase - the bubbles are the oxygen being released from the reaction

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31
Q

What is KIA (two names)?

A

Kligler Iron Agar or Triple Sugar Iron Agar

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32
Q

What is KIA differential for?

A

Fermentation of glucose and lactose/sucrose

Sulfur reduction

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33
Q

What is the pH indicator of KIA?

A

Phenol red

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34
Q

What is the hydrogen sulfide indicator of KIA?

A

Iron (from FeS)

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35
Q

How do you inoculate KIA?

A

A stab with a fishtail

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36
Q

What does the slant look like if the organism ferments glucose and lactose?

A

The whole slant is yellow

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37
Q

What if the KIA butt is yellow and the slant is red?

A

Ferments glucose, not lactose

Deaminates protein

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38
Q

What is the KIA has a red butt and slant?

A

No fermentation occurred

Deaminates protein

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39
Q

What if the KIA is lifted off the bottom of the tube?

A

A gas is produced from fermentation and appears as fissures that lifts the agar off the tube

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40
Q

What are the key ingredient of KIA?

A

Glucose and lactose (substrates)
Phenol red (pH indicator)
Thiosulfate (sulfate TEA - reduced molecule)
Ferrous iron (FeS - reacts to form sulfate)

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41
Q

What is H2S produced from?

A

The reduction of thiosulfate by the breakdown of cysteine from peptone

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42
Q

Why does H2S appear as a black precipitate?

A

Ferrous sulfate reacts with H2S to form the precipitate

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43
Q

What does it mean if you see a black butt and a red slant?

A

Glucose only fermentation and H2S production

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44
Q

What does it mean if you see a black butt and a yellow slant?

A

Glucose and lactose fermentation and H2S production

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45
Q

What is the reaction for the reduction of thiosulfate?

A

SO4 –> H2S

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46
Q

What does H2S look like?

A

A colorless gas

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47
Q

What is the reaction for the formation of a black precipitate?

A

H2S + Fe –> FeS (black precipitate)

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48
Q

What are we testing for overall using KIA (2)?

A

Can the organism ferment glucose and/or lactose?

Can the organism use SO4 as a TEA (does it produce H2S)?

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49
Q

How do we know if the organism can ferment glucose/lactose?

A

If the organism can ferment, it releases acids and increases the pH of the medium, indicating the color change to yellow. A yellow butt means glucose fermentation, and a yellow slant means lactose fermentation

50
Q

How do we know if the organism can use SO4 as a TEA?

A

FeS will appear as a black precipitate because it was formed by the reaction of Fe and H2S. H2S was produced when SO4 was reduced while being used as a TEA

51
Q

How is interpreting the starch hydrolysis different than other lab exercises?

A

Instead of looking for the presence of a product after a reaction, we look for the depletion of the substrate (starch)

52
Q

What is the key ingredient in the starch hydrolysis?

A

Starch

53
Q

What is the purpose of the starch hydrolysis test?

A

Does the organism produce amylase?

54
Q

What does amylase do?

A

It hydrolyzes starch

55
Q

What is the reaction for starch hydrolysis?

A

Starch —(amylase-hydrolysis)–> glucose

56
Q

What is the starch homolog in humans?

A

Glycogen

57
Q

What organisms produce starch?

A

Plants photosynthesize to make sugars and polymerize extra glucose into starch

58
Q

What sugars is starch made of?

A

Only glucose

59
Q

Why does starch’s size matter?

A

Starch is really big so it can’t be transported into the cell

60
Q

How does a bacteria bring starch into the cell?

A

It releases amylase (extracellular enzyme) into the ECF to break down the starch outside of the cell into glucose so the cell can then allow transport of glucose into the cell

61
Q

How do you interpret the starch hydrolysis agar?

A

You pour iodine over the plate after 5 days - if starch is present, the iodine will react with it and you will see a purple-brown color. If the organism produces amylase it will have utilized the starch around it and the plate will be colorless there

62
Q

What does it mean if a plate is purple brown all around the growth?

A

It means that starch is present and the organism did not utilize the starch, and therefore does not make amylase

63
Q

What does it mean if there is a clear halo around the growth

A

The organism utilized the starch around it and therefore it produces amylase

64
Q

What is the purpose of the urease test?

A

To see if the organism produces urease

65
Q

What is urease?

A

The organism that catalyzes the hydrolysis of urea

66
Q

What is the reaction for the hydrolysis of urea?

A

Urea –(urease-hydrolysis)—> CO2 + NH3

67
Q

Why do we care about urease?

A
  1. Helps distinguish microbes

2. Some pathogenic microbes are urease +

68
Q

What are two examples of microbes that is urease positive?

A

Helicobacter pylori, P. vulgaris

69
Q

What does H pylori cause in humans?

A

Ulcers

70
Q

How does H pylori survive its acidic environment?

A

It releases the basic NH3 to neutralize its environment

71
Q

What does P vulgaris cause in humans?

A

UTI

72
Q

How does P vulgaris use urease?

A

It uses NH3 as a nitrogen source to grow

73
Q

Which urease microbe survives its environment? Takes advantage of its environment?

A

Survives - H. pylori

Takes advantage - P. vulgaris

74
Q

What are the key ingredients of the urease test?

A 
Phenol red (pH indicator)
Urea (substrate)
75
Q

What does it mean if the slant turns hot pink?

A

The pH of the slant increased and is more basic, which means it is urease positive

76
Q

What term do we use to describe fecal coliform molecules?

A

Indicator microbes

77
Q

Why are fecal coliforms indicator microbes?

A

Their presence and density suggests that the sample has been contaminated with feces of a warm blooded animal (risk for potential transmission)

78
Q

What is the influent?

A

Poop water

79
Q

Where does the influent go in the first step?

A

It passes through primary and secondary filtration

80
Q

Where does the solution go after the first and second filtration?

A

The aeration basin

81
Q

What is added to the aeration basin? Why?

A

Oxygen is added to the tank in order to stimulate the growth of aerobic microbes so that they degrade the organic material in the waste water

82
Q

What is the sediment called out of the aeration basin?

A

The sediment is called activated sludge

83
Q

What happens to the activated sludge?

A

It gets used over again, added to the next aeration basin as an inoculum

84
Q

Where does the solution go after the aeration basin?

A

The settling basin (clarifier)

85
Q

What happens to the solution after the settling basin?

A

The water is poured off and is filtered through tertiary filters

86
Q

What happens to the triple filtered solution?

A

It is treated with Uv radiation and sodium hyperchlorite

87
Q

What does UV radiation do to the solution?

A

It causes damage to microbial DNA

88
Q

What does the sodium hyperchlorite do to the solution?

A

It causes microbial protein denaturation

89
Q

What happens to the solution that has been treated with sodium hyperchlorite and UV radiation?

A

It is now output water that is reused but is not potable

90
Q

What does it mean to be not potable?

A

Not drinkable

91
Q

What is the name of the media that we use for fecal coliform?

A

mFC media

92
Q

How does mFC select for FC (4)?

A

Roseolic acid
Bile salts (both inhibit the growth of gram +/- bacteria)
44 degree incubation
Anaerobic incubation (under water)

93
Q

How does the mFC media differentiate for FC?

A

Anniline blue dye

94
Q

Why does anniline blue dye change color?

A

When lactose in the media is fermented, acid is produced which causes a pH drop. When the pH of the media drops, anniline blue dye forms blue precipitates

95
Q

What process of filtration do we use for fecal coliforms?

A

Membrane filtration

96
Q

What is it impractical to test for in water quality?

A

The presence of every single disease causing organism

97
Q

What do we test for which is practical?

A

We test for indicator microbes, microbes that indicate or suggest that a water sample has been contaminated by some, any, or all of these disease causing organisms

98
Q

What is another name for the Kirby Bauer test?

A

The antimicrobial susceptibility test

99
Q

What is the purpose of the Kirby Bauer test?

A

To test if a specific strain is susceptible or resistant to a given antibiotic

100
Q

What are the ingredients of the Kirby Bauer test?

A
  1. Mueller-Hinton plates

2. Antibiotic discs

101
Q

What are Mueller-Hinton plates?

A

They have a standard thickness, composition, and pH that allows consistency in results for any strain/antibiotic combination

102
Q

Is a Mueller-Hinton plate un/defined media?

A

Defined media

103
Q

What is an antibiotic disc?

A

A dry paper disc saturated with a known concentration of one antibiotic

104
Q

How do you interpret the results of the Kirby Bauer test?

A

Measure the zone of inhibition with a ruler an compare it with the chart

105
Q

What does a wide zone of inhibition say about the antibiotic?

A

The antibiotic is very effective against the susceptible strain

106
Q

What is the purpose of the Beta Lactamase test?

A

Does an organism make beta lactamase? Is the organism resistant/susceptible to Beta lactam antibiotics?

107
Q

Where does an organism usually acquire the ability to make beta lactamase?

A

Horizontal gene transfer

108
Q

Which organisms are resistant to beta lactam drugs?

A

All penicillins and all cephalosporins

109
Q

How are all beta lactam antibiotics structurally similar?

A

Beta lactam rings that are essential to their function

110
Q

What do beta lactam drugs inhibit?

A

Cell wall synthesis - they can’t cross link peptidoglycan strands so the cell wall is no longer stable and can lyse
They also inhibit the enzyme penicillin binding protein

111
Q

How do bacteria develop resistance to beta lactam drugs?

A

They produce beta lactamase enzymes

112
Q

What does the beta lactamase enzyme do?

A

It attacks the beta lactam ring and degrades it, therefore the drug loses its structure and function

113
Q

What does the test show if the organism is positive for beta lactamase production?

A

A pink product appears (our antibiotic, nitrocefin, is degraded by beta lactamase and the disc turns pink)

114
Q

What does it mean in terms of susceptibility/resistance if a bacteria produces beta lactamase?

A

It is resistant to beta lactam drugs

115
Q

What is the purpose of the use dilution?

A

To determine the lowest concentration of disinfectant or antiseptic that inhibits a specific microbe

116
Q

What is a disinfectant?

A

It inhibits the microbes on nonliving surfaces

117
Q

What is an antiseptic?

A

It inhibits microbes on living surfaces

118
Q

What is an example of a disinfectant?

A

Bleach

119
Q

What is an example of an antiseptic?

A

Iodine

120
Q

What is a general overview of the use dilution exercise?

A

Place four sterile beads in the culture, then dry them on a pad in a glass petrie dish
Transfer each bead into high, medium, and low germicide solutions, and into sterile water
Then transfer the beads each into a sterile TSB tube and examine for growth (put one sterile bead in sterile TSB)

121
Q

What is the negative control for the use dilution?

A

The sterile bead in sterile TSB - there should be no growth

122
Q

What is the positive control for the use dilution?

A

The inoculated bead in the sterile water - there should be growth

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