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Lab 15 Flashcards

Biochemical testing

1
Q

Glucose/Lactose/Sucrose fermentation test

A

Medium is glucose broth/lactose broth/sucrose broth and non fermentable source of nitrogen
Regent/Indicator is PH indicator phenol red, inverted Durham tube that collects gas
Positive result: acidity with pH <6–>yellow
Neutral result: no change->red
Old test: alkaline with pH >7–>magenta
Positive–> gas, negative–> no gas

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2
Q

Are Enterobacteriaceae pathogenic or non pathogenic

A

The family consists of both

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3
Q

Where do Enterobacteriaceae species inhabit? What infections do they cause?

A
  1. Usually in the intestines of humans and other animals, but can also be found in all natural habitats.
  2. They can cause meningitis, bacilliary dysentery, typhoid, and food poisoning
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4
Q

What is Enterobacteriaceae morphology under a microscope

A

Large gram negative rods

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5
Q

What similar characteristics do bacteria in the Enterobacteriaceae family share ?
What is the difference between non-pathogenic and pathogenic?

A
  1. They are oxidase negative, glucose fermenters, and nitrate reducers
  2. Non-pathogenic species usually metabolize lactose while pathogenic ones do not
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6
Q

How can it be determined what type of enteric species is responsible for an infection

A

Biochemical tests

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7
Q

IMViC is usually used to test for

A

Differentiation of enteric bacteria

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8
Q

Methyl Red portion of MRVP test

A
  • Fermentation test, MRVP broth 2.5 mL
  • Differentiate between intestinal bacteria:coliforms
  • Contains dextrose as carbohydrate source
  • Some coliforms ferment dextrose–>pH drop <5
  • pH indicator: 5 drops of methyl red dye added after inoculation and incubation of 48 hrs at 37°C
  • Positive (pH <4.4): red–>mixed acid fermentation, Negative: yellow–> less acidic byproduct–>ethanol or butanediol
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9
Q

Voges-Prokauer part of MRVP test

A
  1. Differentiate between coliforms that convert dextrose to mixed acid fermentation vs. acetoin a precursor to butanediol
  2. Second part, after MR test performed, from same medium of MRVP broth take 1mL
  3. Reagents: VP-A add 15 drops, VP-B add 5 drops
  4. Shake to expose to atmospheric oxygen, wait 20 minutes, or up to 1 hour and no more
  5. Positive: t/o or on surface pink-red, VP negative no color change
  6. Positive–> reaction for production of acetoin
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10
Q

Exoenzyme

A

Enzymes that are excretes, used to degrade large polymers into smaller compounds

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11
Q

Examples of exoenzymes

A

Amylase, Gelatinase, Caseinase, Lipase

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12
Q

Amylase

A

Breaks starch into smaller sugar residues that can enter the cell and be processed by respiration or fermentation

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13
Q

Gelatinase

A

Cause liquefaction of media solidified by gelatin

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14
Q

Caseinase

A

Hydrolyzes casein, major protein in milk–> proteolysis–> milk loses white appearance–>transparent

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15
Q

Lipase

A

Breaks fats into components glycerol and fatty acids–> agar loses its opacity surrounding bacteria producing lipase

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16
Q

Endoenzymes

A

Enzymes produced in cell and catalyze intracellular reactions.

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17
Q

What kind of intracellular reactions are catalyzed by endoenzymes

A
  1. Breakdown of toxic waste such as hydrogen peroxide and urea
  2. The reduction of nitrate or oxygen
  3. The degradation of specific amino acids
  4. The utilization of non carbohydrate carbon sources for growth
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18
Q

Catalase

A

An enzyme that splits hydrogen peroxide into water and oxygen.

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19
Q

Catalase production test

A

A glob of bacteria is placed on a clean slide.
A few drops of 3% hydrogen peroxide are added.
If bubbles are produced–> positive for catalase

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20
Q

What organisms test positive/ negative for catalase

A

Positive–>E coli

Negative–>Streptococcus mutans

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21
Q

MRVP positive/negative organisms

A

Positive MR–> E.coli
Negative MR–>E.aerogenes
Positive VP–> E.aerogenes
Negative VP–> E.coli

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22
Q

Positive fermentation of lactose/glucose/sucrose

A

E.coli

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23
Q

Negative fermentation of lactose/glucose/sucrose

A

Pseudomonas

24
Q

Procedure for fermentation reactions

A
  1. 3 tubes, 1 lactose broth, 1 glucose broth, 1 sucrose broth
  2. Inoculate each with loopful of organism, incubate for 24 to 48 hours at 37° C
  3. Examine for growth(+), gas(G), acid (A)
25
Oxidase production procedure
1. Use toothpick to transfer a heavy inoculum of bacteria on to oxidase paper 2. Observe for color change within 30 seconds for a strong positive 3. Positive reaction appears black or purple
26
Morgans positive/negative for oxidase
Positive--> Pseudomonas fluorescence | Negative--> E.coli
27
Oxidase
Enzyme that catalyze the reduction of oxygen during respiration. Cytochrome oxidase performs final step in electron transport, reducing oxygen to water
28
Oxidase test
1. Used to detect oxidase production 2. Oxidase paper 3. Colorless reagent 4. If negative, possible Enterobacteriaceae species 5. Positive-->black or purple, Negative--> no color change
29
Citrate Utilization in organisms
Some bacteria may be able to use organic compounds other than sugars as their sole source of carbon. Such compounds include citrate.
30
Citrate utilization test
1. Selective allowing bacterial growth of organisms that utilize citrate as sole source of carbon 2. Medium: Simmons Citrate agar containing citrate and ammonium salts (sole nitrogen source) 3. Reagent: Bromo thymol blue indicator dye 4. Green--> neutral pH/ negative, deep blue-->pH above 7.6--> alkaline-->positive 5. MOA: Organisms that metabolize citrate utilize the ammonium salts releasing ammonia and increasing the pH of the medium.
31
Citrate utilization procedure
1. Using a sterile inoculating needle, streak one organism over the surface of the agar slant then stab the butt. 2. Incubate the tubes at 37ﾟC for at least 48 hours 3. Examine for growth
32
Positive/Negative citrate utilization organisms
Positive--> alkaline-->Enterobacter aerogenes | Negative-->neutral--> E.coli
33
Urease
An enzyme that breaks the carbon-nitrogen bond of amides to form carbon dioxide, ammonia, and water
34
Urease test
1. Selective, to identify bacteria capable of hydrolyzing urea with urease enzyme 2. Medium contains amide, urea 3. Reagent: phenol red is pH indicator, turns pink in basic environment 4. Positive --> pink, Negative--> yellow 5. MOA: when urea is broken down, ammonia is released and the pH of the medium increases (more basic)
35
Urease test procedure
1. Inoculate bacteria into urea slants and incubate at least for 48 hours
36
Negative/Positive urease organisms
Positive--> members of Proteus | Negative--> E. Coli
37
The urease test can help distinguish between which genus members
Proteus and other enteric bacteria
38
SIM TEST
1. Sulfur: Production of hydrogen sulfide from sulfur containing amino acids Indole: Indole production Motility: do organisms have flagella 2. SIM 0.3 % agar semi-solid deep 3. Differentiate mainly among gram negative enteric bacteria 4. Reagent: Ferrous sulfate, addition of Indole (Kovac's reagent) 5. Part if IMViC test
39
SIM MOA
1. The liberation of hydrogen sulfide, Sulfur binds to iron--> blackening of medium 2. Indole--> the ability to degrade amino acids to identifiable end products--> red 3. Motility is observed by checking for migration of the inoculum from stab line
40
SIM procedure
1. Inoculate SIM tube with organisms 2. Incubate at 37°C for 48 hours 3. Add 3 drops of Kivacs reagent 4. Red color in the alcohol layer is positive
41
SIM Positives
Positive for hydrogen sulfide-->black Positive for indole production-->red on surface Positive for motility-->turbidity away from stab line
42
Organisms positive for indole
E.coli
43
Organisms positive for motility
E.coli, E.aerogenes, Citrobacter, Pseudomonas
44
Organisms positive for hydrogen sulfide
Citrobacter
45
TSI procedure
1. Inoculate TSI agar slant by streaking surface and stabbing agar 2. Insert lead acetate strip between plug and inner wall of tube, above level of medium. Place cap, but do not tighten 3. Incubate for 24 to 48 hours
46
TSI test
1. For identification of gram negative bacteria, particularly Enterobacteriaceae 2. Triple sugar iron agar: sucrose, dextrose, lactose, and peptone 3. Reagent: phenol red indicator of pH, and sodium thiosulfate and ferrous sulfate as indicators of hydrogen sulfide production 4. Positive for fermentation/acid(A)--> yellow Positive for gas-->lifting of agar/ ripping of agar Positive for hydrogen sulfide-->black Negative fermentation/alkaline(K)--> red No change(NC)
47
What type of environment are bacteria exposed to in TSI agar
1.The bacteria are exposed to both anaerobic (butt) and aerobic (slant) environments
48
TSI MOA of Non fermenters
Non-fermenters can grow in slant by aerobic degradation of protein components in medium to alkaline products--> slant and butt red
49
TSI MOA of glucose only fermenters
Only glucose fermenters--> acid--> butt and slant yellow. Glucose used up in 12 hours, surface bacteria grow and degrade proteins. 18-24 hours, alkaline in slant--> revert to red, 24 hours--> butt yellow, slant red
50
TSI MOA of fermenters of lactose and/or sucrose and glucose
Fermentation of lactose and/or sucrose and glucose--> slant and butt yellow. Even after long incubation and degradation of protein, slant remains acidic due to high concentrations of lactose.
51
If Salmonella spp that ferments dextrose, but not lactose, is incubated on TSI agar, what would be the result? Why?
It will yield an acidic butt with a black precipitate and an alkaline slant. It fermented glucose, not lactose and produced hydrogen sulfide. The acidic slant reverted to red after degradation of proteins on slant made medium alkaline.
52
How much sucrose and lactose are in TSI compared to glucose
10x amount of glucose
53
The degradation of proteins makes medium more
Basic or alkaline
54
Nitrate
Nitrate is used as the terminal electron acceptor to carry out nitrate respiration. The enzyme nitrate reductase catalyzes the transfer of electrons from cytochrome b to nitrate reducing it to nitrate.
55
Is nitrate reductase an inducible enzyme
It is an inducible enzyme that is repressed when oxygen is present
56
Nitrate Reduction test
1. Differential, Used to identify Enterobacteriaceae that are usually nitrate reducers 2. Nitrate broth contains beef extract and potassium nitrate, inverted Durham tube 3. Reagents: Nitrate A: sulfanilic acid, Nitrate B: dimethyl-alpha-naphthylamine, Nitrate C (Zinc powder) 4. Positive--> gas in Durham tube Positive -->red-->nitrate reduction (after reagents A and B) Negative--> clear 5. If negative, need to verify by adding Zinc Results: Positive for reduction--> Clear Negative for reduction--> red
57
Nitrate reduction procedure
1. Inoculate small amount of culture in nitrate broth for 48 hours 2. Observe for gas in Durham tube 3. If no gas, add reagents 5 drops of Nitrate A and then 5 drops of Nitrate B, shake gently to mix, wait 1-2 minutes. Observe for red color 4. If no change, add dash of Zinc powder with toothpick and wait 5 -10 minutes. 5. Red confirms negative and clear indicates nitrate reduction

Microbiology LAB (10 decks)

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