BIOCHEMICAL TESTS

Question 1

Catalase Test Principle

Aerobic and facultative anaerobic organisms produce two toxins that detoxify the products of normal metabolism [?]. One of these enzymes, [?], is capable of converting hydrogen peroxide to [?]. The presence of the catalase in a bacterial isolate is evidenced when a small inoculum introduced into hydrogen peroxide (30% for the slide test) causes rapid elaboration of [?]. The lack of catalase is evident by a lack of or weak bubble production.

Answer 1

hydrogen peroxide (H2 O2 ) and superoxide radical (O2 )

catalase

water and oxygen

oxygen bubbles

Question 2

Catalase Test Method
1. Use a [?]to transfer a small amount of colony growth to the surface of a clean, dry glass slide.
2. Place a drop of [?]onto the medium. (3% can also be used for most organisms.)
3. Observe for the evolution of [?].

Answer 2

loop or sterile wooden stick

30% hydrogen peroxide (H2 O2 )

oxygen bubbles

Question 3

Catalase Test Expected Results
Positive:
Negative:

Answer 3

Copious bubbles are produced

No or few bubbles are produced

Question 4

Catalase Test Quality Control
Positive:
Negative:

Answer 4

Staphylococcus aureus (ATCC25923)

Streptococcus pyogenes (ATCC19615)

Question 5

Citrate Utilization Principle

Bacteria that can grow on this medium produce an enzyme, [?], capable of converting [?]. [?] can then enter the organism’s metabolic cycle for the production of energy. Bacteria capable of growth in this medium use the [?] and convert [?] to [?], creating an [?] pH. The pH change turns the bromothymol blue indicator from [?].

Answer 5

citrate-permease

citrate to pyruvate

Pyruvate

citrate

ammonium phosphate ; ammonia and ammonium hydroxide

alkaline

green to blue

Question 6

Citrate Utilization Method
1. Inoculate [?] agar lightly on the slant by touching the tip of a needle to a colony that is [?] old. Do not inoculate from a broth culture, because the inoculum will be too heavy.
2. Incubate at [?] for up to [?].
3. Observe for growth and the development of [?], denoting alkalinization.

Answer 6

Simmons citrate

18 to 24 hours

35°C to 37°C ; 7 days

blue color

Question 7

Citrate Utilization Expected Results
Positive:
Negative:

Answer 7

Growth on the medium, with or without a change in the color of the indicator. Growth typically results in the bromothymol blue indicator turning from green to blue

Absence of growth

Question 8

Citrate Utilization Quality Control
Positive:
Negative:

Answer 8

Enterobacter aerogenes (ATCC13048)—growth; blue color

Escherichia coli (ATCC25922)—little to no growth; no color change

Question 9

Coagulase Test Principle

[?] produces two forms of coagulase, bound and free. [?], or “clumping factor,” is bound to the bacterial cell wall and reacts directly with fibrinogen. This results in precipitation of fibrinogen on the staphylococcal cell, causing the cells to [?] when a bacterial suspension is mixed with plasma. The presence of bound coagulase correlates with [?], an extracellular protein enzyme that causes the formation of a [?] when S. aureus colonies are incubated with plasma. The clotting mechanism involves activation of a [?], which is a modified or derived thrombin molecule, to form a coagulase-CRF complex. This complex in turn reacts with [?] to produce the fibrin clot.

Answer 9

S. aureus

Bound coagulase ; clump

free coagulase ; clot

plasma coagulase-reacting factor (CRF)

fibrinogen

Question 10

Coagulase Test
1. Emulsify several colonies of the unknown clinical isolate in [?] to give a milky suspension.
2. Place a drop of (?) next to the drop of
plasma in an adjacent reaction well as a negative control.
3. Place a drop of (?) in a third adjacent
reaction well as a positive control.
4. With a loop, emulsify a portion of the isolated colony being tested in the (?). Try to create a smooth suspension.
5. (?) with a wooden applicator stick.
6. With a loop, emulsify a known Staphylococcus aureus in the (?).
7. Mix all samples well with a new wooden applicator stick for each sample

2. Incubate tube at [?] in ambient air for [?].
3. Check for [?].

Answer 10

0.5 mL of rabbit plasma (with EDTA)

distilled water or saline

coagulase plasma reagent

rabbit plasma reagent

Mix well

positive and negative control wells

35°C to 37°C ; 1 to 4 hours

clot formation

Question 11

Coagulase Test Expected Results
Positive:
Negative:

Answer 11

Clot of any size

No clot

Question 12

Coagulase Test Quality Control
Positive:
Negative:

Answer 12

Staphylococcus aureus (ATCC25923)

Staphylococcus epidermidis (ATCC12228)

Question 13

Indole Production Principle

The test is used to determine an organism’s ability to hydrolyze [?] to form the compound [?]. [?] is present in casein and animal protein. Bacteria with [?] are capable of hydrolyzing tryptophan to [?]. [?], when added to the broth culture, reacts with the [?], producing a [?]. An alternative method uses Ehrlich’s reagent. Ehrlich’s reagent has the same chemicals as the Kovac preparation, but it also contains [?], making it flammable. Ehrlich’s reagent is more sensitive for detecting small amounts of indole.

Answer 13

tryptophan

indole

Tryptophan

tryptophanase

pyruvate, ammonia, and indole

Kovac’s reagent (dimethylamine-benzaldehyde and hydrochloride) ; indole ; red color

absolute ethyl alcohol

Question 14

Indole Production

A. Enterobacteriaceae
1. Inoculate tryptophane broth with one drop from a [?].
2. Incubate at 35°C to 37°C in ambient air for [?].
3. Add [?] to the broth culture.

B. Other Gram-Negative Bacilli
1. Inoculate tryptophane broth with one drop of a [?].
2. Incubate at 35°C to 37°C in ambient air for [?].
3. Add [?] to the culture.
4. Shake the mixture vigorously to extract the [?] and allow it to stand until the xylene forms a layer on top of the aqueous phase.
5. Add [?] down the side of the tube.

Answer 14

24-hour brain-heart infusion broth culture
48 hours
0.5 mL of Kovac’s reagent

24-hour broth culture
48 hours
1 mL of xylene
indole
0.5 mL of Ehrlich’s reagent

Question 15

Indole Production Expected Results
Positive:
Negative:

Answer 15

Pink- to wine-colored ring after addition of appropriate reagent

No color change after addition of the appropriate reagent

Question 16

Indole Production Quality Control
A. Kovac’s Method
Positive:
Negative:
B. Ehrlich’s Method
Positive:
Negative:
C. Ehrlich’s Method (Anaerobic)
Positive:
Negative:

Answer 16

Escherichia coli (ATCC25922)
Klebsiella pneumoniae (ATCC13883)

Haemophilus influenzae (ATCC49766)
Haemophilus parainfluenza (ATCC76901)

Porphyromonas asaccharolytica (ATCC25260)
Bacteroides fragilis (ATCC25285)

Question 17

Motility Testing Principle

The inoculum is [?] of a semisolid agar deep. Bacterial motility is evident by a diffuse zone of growth extending [?] of inoculation. Some organisms grow throughout the [?] medium, whereas others show small areas or nodules that grow out from the line of inoculation.

Answer 17

stabbed into the center

out from the line

entire

Question 18

Motility Testing Method
1. Touch a [?] to a colony of a young (?) culture growing on agar medium.
2. [?] once to a depth of only [?] in the middle of the tube.
3. Incubate at 35°C to 37°C and examine daily for up to [?].

Answer 18

straight needle ; 18- to 24-hour

Stab ; 1/3 to 1/4 inch

7 days

Question 19

Motility Testing Expected Results
Positive:
Negative:

Answer 19

Motile organisms will spread out into the medium from the site of inoculation

Nonmotile organisms remain at the site of inoculation

Question 20

Motility Testing Quality Control
Positive:
Negative:

Answer 20

Escherichia coli (ATCC25922)

Staphylococcus aureus (ATCC25923)

Question 21

Sulfide Testing

Principle

Microbial production of H2S is detected by allowing it to react with [?] to produce black-colored ferrous sulfide or lead sulfide respectively. In the H2S test, [?]is mainly used as a sulfur source. Alternatively, some media also contains [?]as sulfur sources. As an indicator to detect the production of H2S gas, ferrous sulfate, ferric ammonium citrate, ferric citrate, peptonized iron, and lead acetate is used in H2S detecting medium.

Answer 21

ferric ions or lead acetate

sodium thiosulfate

peptones, cysteine, and sulfites

Question 22

Sulfide Testing Method

1. Using isolated colonies from an [?] culture or solid media, inoculate the SIM Medium by stabbing the conter of the medium to a depth of [?].
2. Incubate the inoculated medium aerobically at 35°C. for [?]
3. Observe for [?].
4. Once H2S reaction have been read and recorded, apply[?] drops of [?] to the surface of ine medium.
5. Observe for the development of a [?] color.

Answer 22

18-24 hour

1/2 inch

18-24 hours

H2S production

three ; Kovacs Reagent

pink to red

Question 23

Sulfide Testing Expected Results

Positive:
Negative:

Answer 23

Blackening of the medium along the line of inoculation

absence of blackening.

Question 24

Sulfide Testing Quality Control

Positive:
Negative:

Answer 24

Erysipelothrix

Arcanobacterium
Gardnerella vaginalis
Lactobacillus

Question 25

Methyl Red Test Principle

The methyl red detects [?] that lowers the pH of the broth. The MR indicator is added after incubation. MR is red at [?] and yellow at[?]. A [?] is a positive result; [?] is a negative result; and [?] are negative or inconclusive.

Answer 25

mixed acid fermentation

pH 4.4 ; pH 6.2

clear red ; yellow ; various shades of orange

Question 26

Methyl Red Test Method

1. Inoculate MRVP broth with one drop from a [?] broth culture.
2. Incubate at 35°C to 37°C for a minimum of [?] in ambient air. Tests should not be made with cultures incubated less than 48 hours, because the end products build up to detectable levels over time. If results are equivocal at 48 hours, repeat the tests with cultures incubated at 35°C to 37°C for [?] in ambient air; in such instances, duplicate tests should be incubated at 25°C.
3. Split broth into aliquots for MR test and VP test.
4. Add [?] of methyl red reagent per [?] of broth.
5. Read reaction immediately.

Answer 26

24-hour brainheart infusion

48 hours ; 4 to 5 days

five or six drops ; 5 mL

Question 27

Methyl Red Test Expected Results

Weakly positive:
Negative:

Answer 27

Red-orange color.

Yellow color

Question 28

MR-VP Test Quality Control

MR positive/VP negative:
MR negative/VP positive:

Answer 28

Escherichia coli (ATCC25922)

Enterobacter aerogenes (ATCC13048)

Question 29

Voges-Proskauer Test Principle

The VP detects the organism’s ability to convert the acid products to [?]. Organisms capable of using the VP pathway produce a smaller amount of acid during glucose fermentation and therefore do not produce a color change when the MR indicator is added. A secondary reagent is added, [?], followed by [?]; a positive test result is indicated by a [?] color complex.

Answer 29

acetoin and 2,3-butanediol

alpha-naphthol

potassium hydroxide (KOH)

red

Question 30

Voges-Proskauer Test Method

1. Inoculate MRVP broth with one drop from a [?] broth culture.
2. Incubate at 35°C to 37°C for a minimum of [?] in ambient air. Tests should not be made with cultures incubated less than 48 hours, because the end products build up to detectable levels over time. If results are equivocal at 48 hours, repeat the tests with cultures incubated at 35°C to 37°C for [?] in ambient air; in such instances, duplicate tests should be incubated at 25°C.
3. Split broth into aliquots for MR test and VP test.
4. Add [?] of solution A [?] and [?]of solution B [?] to [?] of MRVP broth.
5. [?] after addition of each reagent.
6. Observe for [?].

Answer 30

24-hour brainheart infusion

48 hours ; 4 to 5 days

five or six drops ; 5 mL

0.6 mL (6 drops) ; (alpha-naphthol)

0.2 mL (2 drops) ; (KOH)

1 mL

Shake well

5 minutes

Question 31

Voges-Proskauer Test Expected Results

Positive:
Negative:

Answer 31

Red color, indicative of acetoin production

Yellow color

Question 32

Voges-Proskauer Test Quality Control

MR positive/VP negative:
MR negative/VP positive:

Answer 32

Escherichia coli (ATCC25922)

Enterobacter aerogenes (ATCC13048)

Question 33

Lysine Iron Agar (LIA) Principle

Lysine iron agar contains lysine, peptones, a small amount of[?]. The medium has an [?] slant and an [?] butt. When glucose is fermented, the butt of the medium becomes [?]. If the organism produces [?], cadaverine is formed. [?] neutralizes the organic acids formed by glucose fermentation, and the butt of the medium reverts to the [?]. If the decarboxylase is not produced, the butt remains [?]. If oxidative deamination of lysine occurs, a compound is formed that, in the presence of [?] and a coenzyme, [?], forms a [?] color on the slant. If deamination does not occur, the LIA slant remains [?]. Bromocresol purple, the pH indicator, is yellow at or below pH [?] and purple at or above pH [?].

Answer 33

glucose, ferric ammonium citrate, and sodium thiosulfate

aerobic ; anaerobic

acidic (yellow)

lysine decarboxylase

Cadaverine

alkaline state (purple)

acidic (yellow)

ferric ammonium citrate ; flavin mononucleotide ; burgundy

purple

5.2 ; 6.8

Question 34

Lysine Iron Agar (LIA) Method
1. With a straight inoculating needle, inoculate LIA by [?] through the [?] of the medium to the [?] of the tube and then [?] the slant.
2. Cap the tube tightly and incubate at 35°C to 37°C in ambient air for [?].

Answer 34

twice stabbing

center ; bottom

streaking

18 to 24 hours

Question 35

Lysine Iron Agar (LIA) Expected Results
—lysine decarboxylation and no fermentation of glucose
—glucose fermentation
—lysine deamination and glucose fermentation

Answer 35

Alkaline slant/alkaline butt (K/K)

Alkaline slant/acid butt (K/A)

Red slant/acid butt (R/A)

Question 36

Lysine Iron Agar (LIA) Quality Control

Alkaline slant and butt: H2 S positive:
Alkaline slant and butt:
Alkaline slant and butt: H2 S positive:
Red slant, acid butt:

Answer 36

Citrobacter freundii (ATCC8090)

Escherichia coli (ATCC25922)

Salmonella typhimurium (ATCC14028)

Proteus mirabilis (ATCC12453)

Question 37

Oxidase Test (Kovac’s Method) Principle

To determine the presence of bacterial cytochrome oxidase using the oxidation of the substrate [?] to [?], a dark purple–colored end product. A positive test (presence of oxidase) is indicated by the development of a [?] color. No color development indicates a negative test and the absence of the enzyme.

Answer 37

tetramethyl-p-phenylenediamine dihydrochloride

indophenol

dark purple

Question 38

Oxidase Test (Kovac’s Method) Method
1. Moisten filter paper with the substrate (?) or select a commercially available paper disk that has been impregnated with the substrate.
2. Use a platinum wire or wooden stick to remove a small portion of a bacterial colony (preferably not more than[?]) from the agar surface and rub the sample on the [?].
3. Observe the inoculated area of paper or disk for a color change to[?] within ?.

Answer 38

1% tetramethyl-pphenylenediamine dihydrochloride

24 hours old

filter paper or commercial disk

deep blue or purple ; 10 seconds

Question 39

Oxidase Test (Kovac’s Method) Expected Results
Positive:
Negative:

Answer 39

Development of a dark purple color within 10 seconds

Absence of color

Question 40

Oxidase Test (Kovac’s Method) Quality Control
Positive:
Negative:

Answer 40

Pseudomonas aeruginosa (ATCC27853)

Escherichia coli (ATCC25922)

Question 41

Phenylalanine Deaminase Agar (PDA) Principle

Microorganisms that produce [?] remove the [?] from phenylalanine. The reaction results in the production of [?]. The [?] is detected by adding a few drops of [?]; a [?] is formed between these two compounds.

Answer 41

phenylalanine deaminase

amine (NH2 )

ammonia (NH3 ) and phenylpyruvic acid

phenylpyruvic acid ; 10% ferric chloride

green-colored complex

Question 42

Phenylalanine Deaminase Agar (PDA) Method
1. Inoculate phenylalanine slant with [?] of a 24-hour brain-heart infusion broth.
2. Incubate [?] (or until good growth is apparent) at 35°C to 37°C in ambient air with the cap loose.
3. After incubation, add [?] of [?] to the slant.

Answer 42

one drop

18 to 24 hours

four to five drops ; 10% aqueous ferric chloride

Question 43

Phenylalanine Deaminase Agar (PDA) Expected Results
Positive:
Negative:

Answer 43

Green color develops on slant after ferric chloride is added

Slant remains original color after the addition of ferric chloride

Question 44

Phenylalanine Deaminase Agar (PDA) Quality Control
Positive:
Negative:

Answer 44

Proteus mirabilis (ATCC12453)

Escherichia coli (ATCC25922)

Question 45

Triple Sugar Iron (TSI) Agar Principle

The composition of TSI is 10 parts lactose:10 parts sucrose:1 part glucose and peptone. [?]serve as indicators of acidification and H2 S formation, respectively. A glucose-fermenting organism turns the entire medium [?] in [?]. The butt remains acidic after the recommended [?] incubation period because of the presence of organic acids resulting from the fermentation of [?] under [?] conditions in the butt of the tube. The slant, however, reverts to the [?] state because of oxidation of the fermentation products under [?] conditions on the slant. This change is a result of the formation of CO2 and H2O and the oxidation of [?] in the medium to alkaline amines. When, in addition to [?] are fermented, the large amount of fermentation products formed on the slant neutralizes the alkaline amines and renders the slant [?], provided the reaction is read in 18 to 24 hours. Reactions in TSI should not be read beyond 24 hours of incubation, because aerobic oxidation of the fermentation products from lactose [?] proceeds, and the slant eventually reverts to the [?] state. The formation of CO2 and hydrogen gas (H2 ) is indicated by the presence of [?] in the agar or by separation of the agar from the sides or bottom of the tube. The production of H2 S (sodium thiosulfate reduced to H2 S) requires an [?] environment, and reaction with the [?] produces a blackening of the agar butt in the tube.

Answer 45

Phenol red and ferrous sulfate

acidic (yellow) anaerobic ; 8 to 12 hours

18- to 24-hour

glucose

alkaline (red) ; aerobic

peptones

glucose, lactose and/or sucrose

acidic (yellow)

and/or sucrose ; alkaline

bubbles or cracks

acidic ; ferric ammonium citrate

Question 46

Triple Sugar Iron (TSI) Agar Method

1. With a straight inoculation needle, touch the [?] of a wellisolated colony.
2. Inoculate TSI by first [?] through the center of the medium to the bottom of the tube and then [?] the surface of the agar slant.
3. Leave the cap on loosely and incubate the tube at 35°C to 37°C in ambient air for [?].

Answer 46

top

stabbing ; streaking

18 to 24 hours

Question 47

Triple Sugar Iron (TSI) Agar Expected Results

: glucose, lactose, and sucrose nonutilizer; this may also be recorded as K/K (alkaline slant/alkaline butt)
: glucose fermentation only.
: glucose, sucrose, and/or lactose fermenter

Answer 47

Alkaline slant/no change in the butt (K/NC)

Alkaline slant/acid butt (K/A)

Acid slant/acid butt (A/A)

Question 48

TSIA Note: A black precipitate in the butt indicates production of [?]. Bubbles or cracks in the tube indicate the production of [?] . Drawing a circle around the A for the acid butt; that is,Ⓐ, usually indicates this means the organism ferments [?], with the production of gas.

Answer 48

ferrous sulfide and H2 S gas (H2 S )

CO2 or H2

glucose and sucrose; glucose and lactose; or glucose, sucrose, and lactose

Question 49

Triple Sugar Iron (TSI) Agar Quality Control
Ⓐ, gas production:
K/A, / gas production, H2 S :
K/K:
K/A, H2 S :
K/A:

Note: gas production may also be indicated with a lower case (g) or upper case (G) as indicated:
Small amount of gas:
Large amoung of gas:

Answer 49

Escherichia coli (ATCC25922)

Salmonella typhimurium (ATCC14028)

Pseudomonas aeruginosa (ATCC27853)

Proteus mirabilis (ATCC12453)

Shigella flexneri (ATCC12022)

g

G

Question 50

Urease Test (Christensen’s Method) Principle

[?] is the product of decarboxylation of amino acids. Hydrolysis of urea produces [?] . The formation of ammonia [?] the medium, and the pH shift is detected by the color change of phenol red from light orange at pH [?] to magenta (pink) at pH [?]. Rapid urease-positive organisms turn the entire medium [?] within [?]. Weakly positive organisms may take several days, and negative organisms produce no color change or [?] as a result of acid production.

Answer 50

Urea

ammonia and CO2

alkalinizes

6.8 ; 8.1

pink ; 24 hours

yellow

Question 51

Urease Test (Christensen’s Method) Method
1. Streak the surface of a urea agar slant with a portion of a well-isolated colony or inoculate slant with [?]drops from an overnight brain-heart infusion broth culture.
2. Leave the cap on loosely and incubate the tube at 35°C to 37°C in ambient air for [?].

Answer 51

one to two

48 hours to 7 days

Question 52

Urease Test (Christensen’s Method) Expected Results
Positive:
Negative:

Answer 52

Change in color of slant from light orange to magenta

No color change (agar slant and butt remain light orange)

Question 53

Urease Test (Christensen’s Method) Quality Control
Positive:
Weak positive:
Negative:

Answer 53

Proteus vulgaris (ATCC13315)

Klebsiella pneumoniae (ATCC13883)

Escherichia coli (ATCC25922)