BIOCHEMICAL TESTS Flashcards
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.
hydrogen peroxide (H2 O2 ) and superoxide radical (O2 )
catalase
water and oxygen
oxygen bubbles
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 [?].
loop or sterile wooden stick
30% hydrogen peroxide (H2 O2 )
oxygen bubbles
Catalase Test Expected Results
Positive:
Negative:
Copious bubbles are produced
No or few bubbles are produced
Catalase Test Quality Control
Positive:
Negative:
Staphylococcus aureus (ATCC25923)
Streptococcus pyogenes (ATCC19615)
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 [?].
citrate-permease
citrate to pyruvate
Pyruvate
citrate
ammonium phosphate ; ammonia and ammonium hydroxide
alkaline
green to blue
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.
Simmons citrate
18 to 24 hours
35°C to 37°C ; 7 days
blue color
Citrate Utilization Expected Results
Positive:
Negative:
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
Citrate Utilization Quality Control
Positive:
Negative:
Enterobacter aerogenes (ATCC13048)—growth; blue color
Escherichia coli (ATCC25922)—little to no growth; no color change
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.
S. aureus
Bound coagulase ; clump
free coagulase ; clot
plasma coagulase-reacting factor (CRF)
fibrinogen
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
- Incubate tube at [?] in ambient air for [?].
- Check for [?].
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
Coagulase Test Expected Results
Positive:
Negative:
Clot of any size
No clot
Coagulase Test Quality Control
Positive:
Negative:
Staphylococcus aureus (ATCC25923)
Staphylococcus epidermidis (ATCC12228)
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.
tryptophan
indole
Tryptophan
tryptophanase
pyruvate, ammonia, and indole
Kovac’s reagent (dimethylamine-benzaldehyde and hydrochloride) ; indole ; red color
absolute ethyl alcohol
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.
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
Indole Production Expected Results
Positive:
Negative:
Pink- to wine-colored ring after addition of appropriate reagent
No color change after addition of the appropriate reagent
Indole Production Quality Control
A. Kovac’s Method
Positive:
Negative:
B. Ehrlich’s Method
Positive:
Negative:
C. Ehrlich’s Method (Anaerobic)
Positive:
Negative:
Escherichia coli (ATCC25922)
Klebsiella pneumoniae (ATCC13883)
Haemophilus influenzae (ATCC49766)
Haemophilus parainfluenza (ATCC76901)
Porphyromonas asaccharolytica (ATCC25260)
Bacteroides fragilis (ATCC25285)
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.
stabbed into the center
out from the line
entire
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 [?].
straight needle ; 18- to 24-hour
Stab ; 1/3 to 1/4 inch
7 days
Motility Testing Expected Results
Positive:
Negative:
Motile organisms will spread out into the medium from the site of inoculation
Nonmotile organisms remain at the site of inoculation
Motility Testing Quality Control
Positive:
Negative:
Escherichia coli (ATCC25922)
Staphylococcus aureus (ATCC25923)
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.
ferric ions or lead acetate
sodium thiosulfate
peptones, cysteine, and sulfites
Sulfide Testing Method
- Using isolated colonies from an [?] culture or solid media, inoculate the SIM Medium by stabbing the conter of the medium to a depth of [?].
- Incubate the inoculated medium aerobically at 35°C. for [?]
- Observe for [?].
- Once H2S reaction have been read and recorded, apply[?] drops of [?] to the surface of ine medium.
- Observe for the development of a [?] color.
18-24 hour
1/2 inch
18-24 hours
H2S production
three ; Kovacs Reagent
pink to red
Sulfide Testing Expected Results
Positive:
Negative:
Blackening of the medium along the line of inoculation
absence of blackening.
Sulfide Testing Quality Control
Positive:
Negative:
Erysipelothrix
Arcanobacterium
Gardnerella vaginalis
Lactobacillus
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.
mixed acid fermentation
pH 4.4 ; pH 6.2
clear red ; yellow ; various shades of orange
Methyl Red Test Method
- Inoculate MRVP broth with one drop from a [?] broth culture.
- 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.
- Split broth into aliquots for MR test and VP test.
- Add [?] of methyl red reagent per [?] of broth.
- Read reaction immediately.
24-hour brainheart infusion
48 hours ; 4 to 5 days
five or six drops ; 5 mL
Methyl Red Test Expected Results
Weakly positive:
Negative:
Red-orange color.
Yellow color
MR-VP Test Quality Control
MR positive/VP negative:
MR negative/VP positive:
Escherichia coli (ATCC25922)
Enterobacter aerogenes (ATCC13048)
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.
acetoin and 2,3-butanediol
alpha-naphthol
potassium hydroxide (KOH)
red
Voges-Proskauer Test Method
- Inoculate MRVP broth with one drop from a [?] broth culture.
- 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.
- Split broth into aliquots for MR test and VP test.
- Add [?] of solution A [?] and [?]of solution B [?] to [?] of MRVP broth.
- [?] after addition of each reagent.
- Observe for [?].
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
Voges-Proskauer Test Expected Results
Positive:
Negative:
Red color, indicative of acetoin production
Yellow color
Voges-Proskauer Test Quality Control
MR positive/VP negative:
MR negative/VP positive:
Escherichia coli (ATCC25922)
Enterobacter aerogenes (ATCC13048)
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 [?].
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
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 [?].
twice stabbing
center ; bottom
streaking
18 to 24 hours
Lysine Iron Agar (LIA) Expected Results
—lysine decarboxylation and no fermentation of glucose
—glucose fermentation
—lysine deamination and glucose fermentation
Alkaline slant/alkaline butt (K/K)
Alkaline slant/acid butt (K/A)
Red slant/acid butt (R/A)
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:
Citrobacter freundii (ATCC8090)
Escherichia coli (ATCC25922)
Salmonella typhimurium (ATCC14028)
Proteus mirabilis (ATCC12453)
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.
tetramethyl-p-phenylenediamine dihydrochloride
indophenol
dark purple
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 ?.
1% tetramethyl-pphenylenediamine dihydrochloride
24 hours old
filter paper or commercial disk
deep blue or purple ; 10 seconds
Oxidase Test (Kovac’s Method) Expected Results
Positive:
Negative:
Development of a dark purple color within 10 seconds
Absence of color
Oxidase Test (Kovac’s Method) Quality Control
Positive:
Negative:
Pseudomonas aeruginosa (ATCC27853)
Escherichia coli (ATCC25922)
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.
phenylalanine deaminase
amine (NH2 )
ammonia (NH3 ) and phenylpyruvic acid
phenylpyruvic acid ; 10% ferric chloride
green-colored complex
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.
one drop
18 to 24 hours
four to five drops ; 10% aqueous ferric chloride
Phenylalanine Deaminase Agar (PDA) Expected Results
Positive:
Negative:
Green color develops on slant after ferric chloride is added
Slant remains original color after the addition of ferric chloride
Phenylalanine Deaminase Agar (PDA) Quality Control
Positive:
Negative:
Proteus mirabilis (ATCC12453)
Escherichia coli (ATCC25922)
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.
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
Triple Sugar Iron (TSI) Agar Method
- With a straight inoculation needle, touch the [?] of a wellisolated colony.
- Inoculate TSI by first [?] through the center of the medium to the bottom of the tube and then [?] the surface of the agar slant.
- Leave the cap on loosely and incubate the tube at 35°C to 37°C in ambient air for [?].
top
stabbing ; streaking
18 to 24 hours
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
Alkaline slant/no change in the butt (K/NC)
Alkaline slant/acid butt (K/A)
Acid slant/acid butt (A/A)
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.
ferrous sulfide and H2 S gas (H2 S )
CO2 or H2
glucose and sucrose; glucose and lactose; or glucose, sucrose, and lactose
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:
Escherichia coli (ATCC25922)
Salmonella typhimurium (ATCC14028)
Pseudomonas aeruginosa (ATCC27853)
Proteus mirabilis (ATCC12453)
Shigella flexneri (ATCC12022)
g
G
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.
Urea
ammonia and CO2
alkalinizes
6.8 ; 8.1
pink ; 24 hours
yellow
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 [?].
one to two
48 hours to 7 days
Urease Test (Christensen’s Method) Expected Results
Positive:
Negative:
Change in color of slant from light orange to magenta
No color change (agar slant and butt remain light orange)
Urease Test (Christensen’s Method) Quality Control
Positive:
Weak positive:
Negative:
Proteus vulgaris (ATCC13315)
Klebsiella pneumoniae (ATCC13883)
Escherichia coli (ATCC25922)
