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Microbio > Lab Test 3 > Flashcards

Lab Test 3 Flashcards

1
Q

What are exoenzymes

A

Enzymes secreted out of the cell to act upon a large substrates (macromolecules) in the environment
ex. hemolysin

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2
Q

Endoenzymes

A

Enzymes whose substrate is located within the cell to perform a reaction. such as = ATP production, creation or repair of cell parts, creating movement, DNA transcription, replication, and translation.

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3
Q

How do bacteria feed

A

By diffusion, secreting exoenzymes into the watery medium surrounding them, and then absorbing the dissolved end products through the cell wall and membrane.
-dependence on water is a weakness - usually why dried foods can resist bacterial spoilage without the need for preservatives or refrigeration.

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4
Q

Exoenzyme reactions are classified as _______ reactions because they use water to split the large molecules

A

Hydrolytic

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5
Q

The substrates of exoenzymes are typically _____

A

Large, polymeric macromolecules such as carbohydrates, lipids, and proteins of various sorts.

  • These molecules are usually poorly soluble in water, existing as suspensions which give the watery medium a turbid, opaque appearance.
  • End products of exoenzymes are small enough to diffuse through the membrane and are usually soluble making solutions containing them clear.
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6
Q

The substrates of exoenzymes give a ___ appearance to a medium

A

Cloudy

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7
Q

The end products of exoenzyme activity give a _____ appearance to a medium

A

Clear

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8
Q

Which organism would produce more exoenzymes: one that lives in the intestine or one that lives in dirt

A

Dirt - organisms living in intestine don’t need to use many exoenzymes because usually food is already broken down for them via hosts enzymes

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9
Q

What is the enzyme that digests the substrate starch?

A

Amylase splits the starch into disaccharides (maltose), then maltase splits maltose into individual glucose molecules

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10
Q

What is the indication of a positive test for starch hydrolysis & what is the reagent used

A

Learning of medium; Iodine

  • Bacteria is grown on a plate with starch incorporated into the agar during preparation. The substrate is opaque, but is the exoenzymes amylase and maltase are produced and released into the agar, an area around the streak of growth will show a clearing
  • Iodine turns purple in presence of starch but nothing happens with just maltose or glucose
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11
Q

What bacteria is the starch hydrolysis test useful for?

A

All different types of bacteria

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12
Q

What substrate does amylase work on? Maltase?

A 
Amylase = starch
Maltase = Maltose
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13
Q

What substrate does Lipase work on?

A

Triglycerides

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14
Q

What is/are the end products of lipid hydrolysis

A

Three fatty acids and a glycerol

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15
Q

Indication of a positive lipid hydrolysis test, reagents? useful for?

A

clearing of medium (spirit blue agar plate used), no reagent used

  • triglycerides = insoluble, but glycerol and FA = soluble
  • a lot of bacteria
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16
Q

What is the substrate that caseinase works on

A

Casein

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17
Q

What are the end products of casein hydrolysis

A

Amino acids

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18
Q

Indication of a positive test: casein hydrolysis, reagent used, useful for

A

Clearing of medium (milk plate)

  • The insoluble polymer, casein, is cloudy in suspension, but the amino acids produced by its breakdown by the enzyme casinos are soluble, and therefore clear in solution
  • no reagent used
  • useful for a lot of bacteria
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19
Q

What substrate does the enzyme gelatinase work on?

A

Gelatin

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20
Q

What is/are the end product (s) of gelatin hydrolysis

A

Amino acids

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21
Q

Indication of a positive test - gelatin hydrolysis, reagent, useful for

A

Liquid appearance: Inoculating bacteria into a deep tube using gelatin as the solidifying agent. After incubation if the bacteria have digested the gelatin, the medium will be liquefied

  • no reagent
  • useful for a lot of bacteria
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22
Q

Fermentation

A

Is an anaerobic energy producing process utilized by anaerobes, facultative organisms, and some microaerophiles to produce ATP.

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23
Q

Why is testing for fermentation of a single sugar not useful?

A

For most aerobes, fermentation isn’t an important process therefore performing fermentation tests would be useless. Strict anaerobes ferment all sugars so they are not easily differentiated by which sugars they ferment giving positive results for all.

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24
Q

Any media used to detect carbohydrate fermentations must have the following features:

A

Only one carbohydrate, a pH indicator, a durham tube (to catch any CO2 gas produced during the fermentation process)

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25
Q

Carbohydrate fermentation reaction

A

Carbohydrates (glucose, sucrose, lactose) —fermentation enzymes —> organic acids, alc, CO2, H2

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26
Q

Indication of a positive test for carbohydrate fermentation, reagent, and organisms it is most useful for

A

Color change from purple to yellow, from acids produced (pH indicator), and bubbles in durham tube**

  • most useful for facultative organisms
  • *no bubbles is a NEGATIVE test, even if there is a color change
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27
Q

Two important pathogenic genera of gram positive cocci

A

Staphylococcus and streptococcus

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28
Q

What is the substrate for the enzyme catalase?

A

H2O2

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29
Q

Catalase Test

A
  • done to separate the genera of gram positive cocci
  • strep (catalase -)
  • staph and micrococcus (catalase +)
  • a positive test is not definitive for staph
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30
Q

What are the end products of catalase

A

H20 and 02

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31
Q

Indication of a positive catalase test and what organisms is it useful for

A

bubbles

  • confirm or rule out strep (-)
  • suggest staph or micrococcus (+)
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32
Q

Mannitol reaction

A

Mannitol —digestive enzymes–> acids

-color change from red to yellow, suggests staph a.

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33
Q

What is the most definitive test for determining staph

A

Coagulase, only pathogenic staph produce this enzyme

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34
Q

Coagulase test reaction, indication of a positive test and what is the test useful for

A

Fibrinogen (liquid) —–coagulase–> fibrin (solid)

  • solidification
  • definitive for staph a.
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35
Q

Species of streptococcus are separated from one another by…

A

hemolytic activity and lance field serological groupings

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36
Q

Strep pyogenes

A

Causative agent of “strep throat”

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37
Q

Hemolysis

A

The splitting of red blood cells, an enzymatic reaction carried out by some organisms to feed from the highly nutritive contents of the cells, or to reduce oxygen concentration in the surroundings

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38
Q

Beta hemolysis

A

Complete clearing of blood agar
-Cause not only the rupture of the red blood cell membrane, but also the total digestion of the hemoglobin released from the cell into its iron and amino acid building blocks

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39
Q

Alpha hemolysis

A

Incomplete clearning or greening of blood agar

  • break open red blood cells but do not completely digest the hemoglobin contained inside
  • instead hemoglobin is modified to form hemoverdin, a greenish compound
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40
Q

Gamma hemolysis

A

No clearning

  • non-hemolytic and do not lyse RBC’s.
  • these organisms tend to not be pathogens
41
Q

Beta hemolysin reaction, indication of a positive test, what organism does it indicate

A

Hemoglobin —beta hemolysins–> amino acids + Fe

  • complete clearing
  • strep pyogenes
42
Q

Alpha hemolysin reaction, indication of a positive test, what organism

A

Hemoglobin —-alpha hemolysins—> hemoverdin

  • incomplete clearing/greening
  • strep pneumoniae
43
Q

What are the end products of beta hemolysis

A

Amino acids and iron

44
Q

End products of alpha hemolysis

A

Hemoverdin

45
Q

Lancefield serological groupings

A

Group streps. based on antigens on their cell surface separate from the ability to cause hemolysis

46
Q

Oxidase test

A
  • oxidase is one of the enzymatic components of the electron transport chain
  • all aerobes use the ETC and have oxidase; all anaerobes lack the enzyme, since they use fermentation
47
Q

What organisms is an oxidase test definitive for?

A

Pseudomonas and neisseria

48
Q

Oxidase test, indication of a positive test, reagent, organisms

A

Hydrogen (NADH) & Oxygen (air) —cytochrome oxidase—–> water

  • color change from pink to purple to black within 60 seconds (+)
  • no color change, or a color change to black after 60 seconds is a negative reaction
  • helpful at determining neisseria
49
Q

What do the SIM stand for in SIM medium

A

Sulfur, iodine, motility

-has ingredients to indicate: motility, indole production and H2S production

50
Q

SIM Medium

A
  • contains agar (semi-soft)
  • hard enough that bacteria will be held in place if they don’t have flagella
  • soft enough that bacteria with flagella will be able to move throughout the agar
51
Q

What are the sulfur containing substrates in a sIM medium

A

cysteine, and thiosulfate

-indicator for the reaction = iron

52
Q

Reaction where thiosulfate is the substrate, indication of a positive test, and what organisms useful for

A

Thiosulfate —thiosulfate reductase—> sulfite + hydrogen sulfide gas

  • Black solid (FeS)
  • Gr negative enteric rods
53
Q

Reaction where cysteine is the substrate, indication of a positive test, and what organisms useful for

A

Cysteine —cysteine desulfurase—> pyruvic acid + hydrogen sulfide gas + ammonia

  • Black solid (FeS)
  • Gr negative enteric rods
54
Q

Four possibilities for results in a SIM tube

A
  1. Cloudiness throughout: motile, - for H2S production
  2. Black throughout: motile, + for H2S production
  3. Cloudiness along stab line: non motile and - for H2S production
  4. Black along the stab line: non-motile & positive for H2S production
55
Q

What are the three sugars in the triple sugar iron agar reactions

A

Glucose, lactose, and sucrose

56
Q

What is the TSIA used for

A

To differentiate between facultative organisms, by showing the pattern of which of the three common sugars are fermented to produce ATP when in low oxygen environments. The TSIA medium, since it is a solid agar slant which is stabbed as well as streaked is anaerobic in the lower portion of the stab and aerobic near the surface of the slant

57
Q

What can the TSIA medium determine

A
  • Acid production from glucose fermentation only
  • Acid production from lactose and/or sucrose fermentation
  • gas production from fermentation of sugar
  • and the release of H2S from thiosulfate or cysteine
58
Q

Why is glucose in a smaller amount in the TSIA medium

A

All bacteria capable of fermenting sugars will selectively act upon glucose first, since it is the simplest most widespread sugar. Only after exhausting the supply of glucose will bacteria switch to the fermentation of other sugars. Limiting the glucose concentration forces the bacteria to switch.

59
Q

What will the TSIA tube look like if only glucose was fermented

A

The tube will change yellow initially, but then the neutral/basic end products of respiration of sugars and proteins occurring at the top will turn the slant portion red.

  • neutral from sugars
  • alkaline from proteins as AA’s release ammonia
60
Q

What is the pH indicator in the TSIA tube

A

Phenol red

61
Q

What will a TSIA tube look like if glucose and one of the other two sugars was fermented?

A

First the tube will be all yellow but after 48 hours the slant will be red.
*Fermenters of multiple sugars will yield a false negative result for sucrose/lactose fermentation if recorded after 24 hours.

62
Q

What does a control TSIA tube look like?

A

All orange throughout

63
Q

TSIA tube where no fermentation has occurred

A

Red slant and orange throughout, after 48 hours turns all red

64
Q

What is an end product that always occurs with fermentation

A

Gas! If no fermentation occurs there can be no gas produced beneath the surface

65
Q

If a TSIA tube appears blackened what is going on

A

If the bacteria inoculated produces thiosulfate reductase or cysteine desulfurase, H2S gas is released in the agar, and will combine with iron to form FeS (black precipitate) as it rises to the top

66
Q

A yellow butt is ____ whereas an orange butt is ____

A

acidic; basic

67
Q

What are the tests that make up the iMViC series

A

Indole test, methyl red, voges proskauer test and the citrate test

68
Q

What is the substrate for tryptophanase

A

AA tryptophan

69
Q

Indole test & reaction

A

-E. Coli produces tryptophanase thus yielding a positive reaction
-indole = only unique end produce of the action of the enzyme
Tryptophan —–trypotophanase—> indole + pyruvate + ammonia

70
Q

Indication of a positive indole test, recent and organisms

A

Cherry Red layer at the top, Kovac’s reagent, and to differentiate E. Coli from enterobacter and other lactose fermentors apart

71
Q

Methyl red test and reaction/indication of a positive test/reagent/organisms

A

-Indicates extreme acids produced (like ph 3-4) from fermentation of pyruvate to mixed acids such as: lactic, acetic and formic

glucose—fermentation enzymes–> strong acids
Red, methyl red
-To differentiate E. Coli from enterobacter and other lactose fermenters

72
Q

Voges proskauer test

A

Some glucose fermenters produce less acidic end products (enterobacter) during their fermentation process (ph 6). Therefore the VP test is designed to test specifically for the end product acetylmethylcarbinol
-VPA and VPB are added to the glucose broth after incubation and will react with acetylmethylcarbinol

73
Q

VP test reaction/indication of positive test/reagent/organisms

A

Glucose –fermentation enzymes–> 2,3 butanediol
brick red; VP reagents A &B
-differentiate E. coli from enterobacter and other lactose fermenters apart

74
Q

Citrate test

A

-Enterobacter has citrate but not E.Coli therefor enterobacter can survive in a medium containing only citrate whereas E. Coli cannot

75
Q

Simmons citrate medium

A
  • contains inorganic material rather than organic nutrients

- only carbon chain is therefore citrate and if an organism cannot use citrate it cannot grow

76
Q

Citrate reaction/positive test/reagents/organisms

A

citrate —citrase–> Pyruvate + Co2

  • blue color change due to excess Co2
  • to differentiate E.Coli from enterobacter and other lactose fermenters apart
77
Q

The accepted poor water quality pathogen is __ & why?

A

E.Coli; since E. Coli is found in all human and some animal waste, but never in nature, if E. Coli is in the water, feces contamination has occurred and the water is determined to be undrinkable

78
Q

What does the water analysis do

A

Tests for coliforms = gram - rods such as E. Coli that are common in intestinal tract

79
Q

Quantitative water analysis

A

Involves the filtration of water which collects all bacteria from the water. The filter is disassembled and the filter is placed on a petri dish of EMB liquid a

80
Q

Drawbacks of quantitative water analysis test

A
  • costly and time consuming
  • apparatuses are expensive
  • aseptic techniques require trained personnel
81
Q

What are the three parts of the quantitative water test

A
  • presumptive test; involves innoculating measured amounts of water into 3 lactose fermentation broths. Presence of acid (turning tube from purple to yellow) and gas in any one of the tubes is a positive presumptive test and warrants progression to next test
  • Confirmed test; used to confirm that E coli is present in the water sample. One of the lactose broths is used to streak an EMB plate for isolation. Greenish gold colonies = positive test, no greenish gold just means some other organism
  • Completed test; double check. A greenish gold isolated culture is grown and a gram stain must be done to indicate small, gr- bacilli. Lastly a new tube is innoculated and must show acid and gas production
82
Q

Serology

A

The study of serum, the clear, non-cellular fluid component of the blood

83
Q

Seriological reactions

A

Use antigen-antibody specificity to identify antigen/pathogen involved
-since antibodies selectively bind only to the antigen which solicited ints production the specificity of the binding can be used in clinical settings to either identify the antibody type found in a patients serum or identifying antigens

84
Q

Antigens from bacteria

A
  • proteins of the cell wall
  • cell membrane
  • flagella
  • pilli
  • fimbrae
  • exoenzymes
  • exotoxins
  • capsule
85
Q

Antigens from viruses

A

-proteins of the capsid or peplomer proteins

86
Q

antigens from protozoa

A

-worms/yeasts: cell surface proteins

87
Q

Antigens from our own bodies

A

proteins made during usual or disease conditions not normally present in the body such as HCG found in the urine during pregnancy

88
Q

3 main categories of serological tests

A

precipitation
agglutination
enzyme linked

89
Q

Precipitation

A

The combination of soluble antibody and soluble antigen best seen in a gel immuno-diffusion plate also called as Ouchertolony plate

  • A thick agar plate has wells cut into it and into a central well is placed the unknown component and known potential matching components placed in wells arranged in a circle around the center
  • the soluble components diffuse through the agar and when they meet the binding is visible as a faint milky haze
90
Q

Agglutination

A

Occurs when the antigen is part of the surface of a cell and when antibodies bind the entire cell is bound to the antibody
-ex. blood typing in humans
clumping with the addition of antibodies against antigen B means they have B type blood, clumping with both antibodies means AB, clumping with none means blood type O

91
Q

Titer

A

A rough measurement of how much antibody is present in a patient’s body
-a high titer indicates a more extreme response and thus a more serious infection than a lower titer

92
Q

Enzyme linked immunoabsorbet serological assay (ELISA)

A

Involves a color change to indicate antigen-antibody binding

  • in ELISA tests the known component prepared in the lab is joined to an enzyme capable of a color change
  • common tests = pregnancy and HIV tests
93
Q

Why are homogenization/dilution necessary

A

Homogenization necessary to eliminate possibility of uneven distribution of bacteria on surfaces of food
Dilution necessary to get a countable plate (30-300) when sampling foods if no dilution then colonies will be too close together

94
Q

Cheeses that are mold ripened from the inside out by penicillium roqueforti

A

Blue cheese, roquefort

95
Q

Cheeses red smear ripened from outside in by a mixture of brevibacterium and yeasts

A

Munster, Limberger

96
Q

“eyes” from Co2 bubbles in hardened curd

A

Swiss, havarti, gouda, gruyere

97
Q

Acid curd cheeses (strep lactose and lacto bacillus)

A

Cottage cheese, cream cheese

98
Q

Longest curing time for a hard cheddar cheese

A

Extra sharp cheddar

99
Q

Mold ripened from outside in by penicillium camemberti

A

Camembert (brie if made anywhere other than france)

Microbio (7 decks)

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