BACTERIOLOGY LABORATORY MLS – 110

FINALS (BIOCHEMICAL TESTS- BOOK BASED)

Overview of Bacterial Identification Methods and Strategies Media: NaC2H3O2 (2 g); MgSO4 (0ff1 g); NaCl (5 g);
NH4H2PO4 (1 g); agar (20 g); bromothymol blue indicator (0ff8
Colony morphology g), per 1000 mL, pH 6ff7
- Macroscopic description of the colony
- Quality Control
- First information a microbiologist uses in
o (+) Escherichia coli
identification Process
o (-) Shigella spp.
- Hemolysis, pigment, size, texture (opaque, translucent,
- Results
or transparent), adherence to agar, pitting of agar, and
o (+) blue
pitting of agar
o (-) no color change
After careful observation of Colony…

- Gram stain
o Gram (+)
▪ Catalase test
o Gram (-)
▪ Oxidase test
▪ MAC (G- rods/ coccobacillus)

FUTURE TRENDS OF ORGANISM IDENTIFICATION

1. Acetamide Utilization

- Differentiate on the ability to use acetamide as sole source

of carbon 3. L-Alanine-7-amido-4-methylcourmarin (Gram-Sure)
- Bacteria capable of growth on this medium produce the
- This test is used in conjunction with the Gram stain to
enzyme acylamidase, which deaminates acetamide to
distinguish aerobic gram-positive rods or coccobacilli that
release ammonia. The production of ammonia results in an
may appear gram-negative or gram-variable.
alkaline pH, causing the medium to change color from green
to royal blue. - The compound L-Alanine-7-amido-4-methylcourmarin is
Media: NaCl (5 g), NH4H2PO4 (1 g), K2HPO4 (1 g), agar (15 g), impregnated in a commercially prepared disk (Remel-
Thermo Fisher Scientific, Lenexa, KS) Gram-negative
bromothymol blue indicator (0ff8 g), per 1000 mL, acetamide
organisms produce an aminopeptidase that is capable of
(10 g), pH 6.8
hydrolyzing the reagent in the disk, forming a blue
- Quality Control fluorescent compound that is visible under long-wave UV
o (+) Pseudomonas Aeruginosa light.
o (-) Escherichia coli - Quality Control
- Results o (+) Escherichia coli
o (+) blue o (-) Staphylococcus aureus
o (-) no color change - Results
o (+) Aerobic, gram-negative rods and
coccobacilli will appear fluorescent or blue
o (-) Aerobic, gram-positive rods and
coccobacilli will appear colorless

2. Acetate Utilization
4. Bacitracin Susceptibility

- Differentiate on the ability to use acetate as sole source

of carbon
- Generally used to differentiate Shigella spp. From Escherichia
coli.
- Organisms capable of using sodium acetate grow on the
medium, resulting in an alkaline pH, turning the indicator from
green to blue.
- Presumptive identification and differentiation of beta-
hemolytic group A streptococci (Streptococcus pyogenes–
susceptible) from other beta-hemolytic streptococci It is also
used to distinguish staphylococci species (resistant) from
micrococci (susceptible)ff
- The antibiotic bacitracin inhibits the synthesis of bacterial cell
walls. A disk (TaxoA) impregnated with a small amount of
bacitracin (0.04 units) is placed on an agar plate, allowing the

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antibiotic to diffuse into the medium and inhibit the growth of
susceptible organisms. After incubation, the inoculated plates
are examined for zones of inhibition surrounding the disks
- Quality Control
o (+) Streptococcus pyogenes, Micrococcus luteus
o (-) Streptococcus agalactiae, Staphylococcus aureus
– Results
o (+) Any zone of inhibition greater than 10 mm;
susceptible
o (-) No zone of inhibition; resistant
5. Bile Esculin Test
- Differentiates enterococci and group D streptococci from
non–group D viridans streptococci.
- Identification of enterococci and organisms in the
Streptococcus bovis group.
- Gram-positive bacteria other than some streptococci and
enterococci are inhibited by the bile salts in this medium.
Organisms capable of growth in the presence of 4% bile and
able to hydrolyze esculin to esculetin. Esculetin reacts with
Fe3+ and forms a dark brown to black precipitate.
Media: Beef extract (11 g), enzymatic digest of gelatin (34ff5
g), esculin (1 g), ox bile (2 g), ferric ammonium citrate (0ff5 g),
agar (15 g), per 1000 mL, pH 6ff6
- Quality Control
o (+) Enterococcus faecalis; black ppt
o (-) Escherichia coli; no color change
- Results
o (+) growth; blackening of agar slant
o (-) growth; no blackening
6. Bile Solubility Test
- Differentiates Streptococcus pneumoniae (positive– soluble)
from alpha-hemolytic streptococci (negative– insoluble)
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Bile or a solution of a bile salt (sodium deoxycholate)
rapidly lyses pneumococcal colonies, Lysis depends on the
presence of an intracellular autolytic enzyme, amidase. Bile
salts lower the surface tension between the bacterial cell
membrane and the medium, thus accelerating the
organism’s natural autolytic process
- Quality Control
o (+) Streptococcus pneumoniae - bile soluble
o (-) Enterococcus faecalis – bile insoluble
- Results
o (+) colony disintegrate; imprint of lysed colony
o (-) intact colonies
7. Butyrate Disk (Catarrhalis Test)
- Rapid test to detect the enzyme butyrate esterase, to aid
identification of Moraxella (Branhamella) catarrhalis
- Organisms capable of producing butyrate esterase
hydrolyze bromochlorindolyl butyrate, Hydrolysis of the
substrate in the presence of butyrate esterase releases
indoxyl, which in the presence of oxygen spontaneously forms
indigo, a blue to blue-violet color
- Quality Control
o (+) Moraxella catarrhalis
o (-) Neisseria gonorrhoeae
- Results
o (+) Development of a blue color during the 5-
minute incubation period
o (-) No color change
8. CAMP Test
- The Christie, Atkins, and Munch-Peterson (CAMP) test is used
to differentiate group B streptococci (Streptococcus
agalactiae – positive) from other streptococcal species.
- Listeria monocytogenes also produces a positive CAMP
reaction
- (CAMP factor) - a diffusible extracellular hemolytic protein
acts synergistically with the beta-lysin of Staphylococcus
aureus to cause enhanced lysis of red blood cells
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- The group B streptococci are streaked perpendicular to a 10. Cetrimide Agar
streak of S. aureus on sheep blood agar A positive reaction
appears as an arrowhead zone of hemolysis - primarily used to isolate and purify Pseudomonas aeruginosa
from contaminated specimens.
- Quality Control
o (+) Streptococcus agalactiae/ arrowhead - The test is used to determine the ability of an organism to grow
in the presence of cetrimide, a toxic substance that inhibits the
o (-) Streptococcus pyogenes/ beta hemolysis
growth of many bacteria by causing the release of nitrogen
without enhanced arrowhead’
and phosphorous, which slows or kills the organism. P.
- Result
aeruginosa is resistant to cetrimide
o (+) Enhanced hemolysis is indicated by an
arrowhead-shaped zone of beta hemolysis at the - Quality Control
juncture of the two organisms o (+) Pseudomonas aeruginosa
o (-) No enhancement of hemolysis o (-) Escherichia coli
- Results
o (+) Growth
o (-) no growth

9. Catalase Test
11. Citrate utilization
- This test differentiates catalase-positive micrococcal and
staphylococcal species from catalase-negative streptococcal
- The purpose of this test is to identify organisms capable of
species
using sodium citrate as the sole carbon source and inorganic
- Aerobic and facultative anaerobic organisms produce two ammonium salts as the sole nitrogen source.
toxins during normal metabolism, hydrogen peroxide
The test is part of a series referred to as IMViC (indole, methyl
(H2O2) and superoxide radical (O2−). These bacteria have
red, Voges-Proskauer, and citrate), which is used to
two enzymes that detoxify the products of normal metabolism.
differentiate Enterobacteriaceae from other gram- negative
One of these enzymes, catalase, is capable of converting
rods
hydrogen peroxide to water and oxygen
- Bacteria that can grow on this medium produce an
- False positives may occur if the sample is
enzyme, citrate-permease, capable of converting citrate to
contaminated with blood agar
pyruvate. Pyruvate can then enter the organism’s
- Some organisms (enterococci) produce a peroxidase that metabolic cycle for the production of energy. Bacteria
slowly catalyzes the breakdown of H2O2, and the test may capable of growth in this medium use the citrate and
appear weakly positive. This reaction is not a truly positive test. convert ammonium phosphate to ammonia and
ammonium hydroxide, creating an alkaline pH. The pH
- Quality Control
change turns the bromothymol blue indicator from green
o (+) Staphylococcus aureus
to blue.
o (-) Streptococcus pyogenes
- Results - Quality Control
o (+) Copious bubbles are produce o (+) Enterobacter aerogenes; growth/ blue
o (-) No or few bubbles are produced color
o (-) Escherichia coli; little/ no growth/ no
color change

- Results
o (+) 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
o (-) Absence of growth

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13. Decarboxylase Test (Moeller’s Method)

- Differentiate decarboxylase producing
Enterobacteriaceae from other gram-negative rods

- This test measures the enzymatic ability (decarboxylase) of

an organism to decarboxylate (or hydrolyze) an amino acid
to form an amine. Decarboxylation, or hydrolysis, of the amino
acid results in an alkaline pH and a color change from orange
to purple.

12. Coagulase Test Media: Peptic digest of animal tissue (5 g), beef extract (5 g),
bromocresol purple (0.1 g)), cresol red (0.005), dextrose (0.5 g),
- The test is used to differentiate Staphylococcus aureus pyridoxal (0.0005 g), amino acid (10 g), pH 6.0
(positive) from coagulase-negative staphylococci - Quality Control
(negative)ff o (+) Lysine—Klebsiella pneumoniae
Ornithine—Enterobacter aerogenes
- S. aureus produces two forms of coagulase, bound and free
Arginine—Enterobacter cloacae/ Pseudomonas
Bound coagulase, or “clumping factor,” is bound to the
aeruginosa
bacterial cell wall and reacts directly with fibrinogen. This
o (-) Lysine—Citrobacter freundii
results in precipitation of fibrinogen on the staphylococcal
Ornithine—Proteus vulgaris
cell, causing the cells to clump when a bacterial suspension
Arginine—Escherichia coli
is mixed with plasma. The presence of bound coagulase
correlates with free coagulase, an extracellular protein
enzyme that causes the formation of a clot when S. aureus
colonies are incubated with plasma. The clotting mechanism
involves activation of a plasma coagulase-reacting factor
(CRF), which is a modified or derived thrombin molecule, to
form a coagulase-CRF complex. This complex in turn reacts
with fibrinogen to produce the fibrin clot.

12.1 Slide test – Detection of Bound Coagulase or Free

coagulase

- all negative Slide Test must be confirmed with Tube Test

Results

o (+) Macroscopic clumping in 10 seconds or less

14. DNA Hydrolysis (DNase Test Agar)
o (-) no clumping, positive and negative controls are
appropriate
This test is used to differentiate organisms based on the
12.2 Tube test – Detection of Free coagulase production of deoxyribonuclease. It is used to distinguish
Serratia spp. (positive) from Enterobacter spp., Staphylococcus
1. Test results can be positive at 4 hours and then revert to aureus (positive) from other species, and Moraxella
negative after 24 hours. catarrhalis (positive) from Neisseria spp.
2. If negative at 4 hours, incubate at room temperature - The test is used to determine the ability of an organism to
overnight and check again for clot formation. hydrolyze DNA. The medium is pale green because of the
Results DNA–methyl green complex. If the organism growing on
o (+) Clot (fibrin clot) the medium hydrolyses DNA, the green color fades and the
o (-) No clot colony is surrounded by a colorless zone.
Quality Control
o (+) Staphylococcus aureus Media: Pancreatic digest of casein (10 g), yeast extract (10
o (-) Staphylococcus epidermidis g), deoxyribonucleic acid (2 g), NaCl (5 g), agar (15 g),
methyl green (0.5 g), pH 7.5

- Quality Control
o (+) Staphylococcus aureus
o (-) Escherichia coli
- Results
o (+) When DNA is hydrolyzed, methyl green is
released and combines with highly polymerized
DNA at a pH of 7ff5, turning the medium colorless
around the test organism
o (-) If no degradation of DNA occurs, the
medium remains green

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15. Esculin Hydrolysis

17. Flagella Stain (Wet Mount Technique)- Ryu stain
- This test is used for the presumptive identification and
differentiation of Enterobacteriaceae - Flagella are too thin to be visualized using a bright field
- This test is used to determine whether an organism is able to microscope with ordinary stains, such as the Gram stain, or a
hydrolyze the glycoside esculin. Esculin is hydrolyzed to simple stain. A wet mount technique is used for staining
esculetin, which reacts with Fe3+ and forms a dark brown to bacterial flagella, and it is simple and useful when the number
black precipitate. and arrangement of flagella are critical to the identification of
species of motile bacteria. The staining procedures require the
- Quality Control use of a mordant so that the stain adheres in layers to the
o (+) Enterococcus faecalis flagella, allowing visualization
o (-) Escherichia coli
- Results Expected Results
o (+) Blackening and loss of fluorescence under Observe the slide and note the following:
wood’s lamp
1. Presence or absence of flagella
2. Number of flagella per cell
o (-) No blackening and no loss of fluorescence
under wood’s lamp. 3. Location of flagella per cell
a. Peritrichous
b. Lophotrichous
c. Polar
4. Amplitude of wavelength
a. Short
b. Long
5. Whether or not “tufted”
- Quality Control
o Peritrichous: Escherichia coli
o Polar: Pseudomonas aeruginosa
o Negative: Klebsiella pneumonia
18. Gelatin Hydrolysis
16. Fermentation Media
- The production of gelatinases capable of hydrolyzing
- Fermentation media are used to differentiate organisms
gelatin is used as a presumptive test for the identification
based on their ability to ferment carbohydrates
of various organisms, including Staphylococcus spp.
incorporated into the basal medium. Andrade’s formula is
Enterobacteriaceae, and some gram-positive bacilli
used to differentiate enteric bacteria from coryneform, and
bromocresol purple is used to distinguish enterococci from - This test is used to determine the ability of an organism to
streptococci produce extracellular proteolytic enzymes (gelatinases)
that liquefy gelatin, a component of vertebrate connective
Positive result
tissue.
Andrade’s formula - Indicator change to pink with or without gas Nutrient gelatin medium differs from traditional microbiology
formation in Durham tube media in that the solidifying agent (agar) is replaced with
bromocresol purple - Indicator change to yellow Quality gelatin. When an organism produces gelatinase, the enzyme
liquefies the growth medium.
Control Dextrose
- Quality Control
Positive with gas: Escherichia coli o (+) Bacillus subtilis
o (-) Escherichia coli
- Results
o (+) Partial or total liquefaction of the inoculated
tube (the control tube must be completely

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solidified) at 4°C within 14 days
o (-) Complete solidification of the tube at 4°C

22. Indole Production (tryptophane broth)

The test is used to determine an organism’s ability to

hydrolyze tryptophan to form the compound indole.
Tryptophan is present in casein and animal protein. Bacteria
19. Growth at 42C with tryptophanase are capable of hydrolyzing tryptophan to
- Differentiate a pyocyanogenic pseudomonads from pyruvate, ammonia, and indole. Kovac’s reagent
other Pseudomonas spp. (dimethylamine-benzaldehyde and hydrochloride), when
added to the broth culture, reacts with the indole, producing
- Quality Control a red color. An alternative method uses Ehrlich’s reagent.
o (+) Pseudomonas aeruginosa Ehrlich’s reagent has the same chemicals as the Kovac
o (-) Pseudomonas fluorescens preparation, but it also contains absolute ethyl alcohol,
- Results making it flammable. Ehrlich’s reagent is more sensitive for
o (+) Good growth at both 35°and 42°C detecting small amounts of indole. (The spot indole test)
o (-) No growth at 42°C, but good growth at 35°C 21.1 Enterobacteriaceae Method (Kovac’s)

21.2 Other Gram-Negative Bacilli Method (Ehrlich’s)

- Results
o (+) Pink to wine colored ring
o (-) No color change after addition of reagent
- Quality Control

Kovac’s Method

Positive: Escherichia coli Negative: Klebsiella

pneumoniae
Ehrlich’s Method

Positive: Haemophilus influenzae

Negative: Haemophilus parainfluenzae

Ehrlich’s Method (Anaerobic)

20. Hippurate Hydrolysis Positive: Porphyromonas asaccharolytica
Negative: Bacteroides fragilis
- The end products of hydrolysis of hippuric acid by
hippuricase include glycine and benzoic acid. Glycine is
deaminated by the oxidizing agent ninhydrin, which is
reduced during the process. The end products of the
ninhydrin oxidation react to form a purple-colored product.
The test medium must contain only hippurate, because
ninhydrin might react with any free amino acids present in
growth media or other broths.
-
- Quality Control
o (+) Streptococcus agalactiae
o (-) Streptococcus pyogenes
- Results
o (+) Deep purple color 22. Leucin Aminopeptidase Test (LAP test)
o (-) Colorless or slightly yellow pink - The LAP test is used for the presumptive identification of
catalase-negative gram-positive cocci

- The LAP disk is a rapid test for the detection of the enzyme

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leucine aminopeptidase. Leucine beta- naphthylamide–
impregnated disks serve as a substrate for the detection of
leucine aminopeptidase. After hydrolysis of the substrate by
the enzyme, the resulting beta-naphthylamine produces a
red color upon addition of cinnamaldehyde reagent.
- Quality Control
o (+) Enterococcus faecalis
o (-) Aerococcus viridans
- Results
o (+) Development of a red color within 1 minute
after adding cinnamaldehyde reagent
o (-) No color change or development of a slight
yellow color
23. Litmus Milk Medium
- This test differentiates microorganisms based on various
metabolic reactions in litmus milk, including fermentation,
reduction, clot formation, digestion, and the formation of gas.
Litmus milk is also used to grow lactic acid bacteria.
25. Methyl Red/Voges-Proskauer (Mrvp) Tests
24. Lysine Iron Agar (LIA)
- This test is used to differentiate gram-negative bacilli based
on decarboxylation or deamination of lysine and the
formation of hydrogen sulfide (H2S)
- Lysine iron agar contains lysine, peptones, a small amount
of glucose, ferric ammonium citrate, and sodium
thiosulfate. The medium has an aerobic slant and an
anaerobic but. When glucose is fermented, the butt of the
medium becomes acidic (yellow)ff If the organism
produces lysine decarboxylase, cadaverine is formed.
Cadaverine neutralizes the organic acids formed by
glucose fermentation, and the butt of the medium reverts
to the alkaline state (purple). If the decarboxylase is not
produced, the butt remains acidic (yellow). If oxidative
deamination of lysine occurs, a compound is formed that,
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in the presence of ferric ammonium citrate and a coenzyme,
flavin mononucleotide, forms a burgundy color on the slant.
If deamination does not occur, the LIA slant remains
purple. Bromocresol purple, the pH indicator, is yellow at or
below pH 5.2 and purple at or above pH 6.8
Quality Control
• Alkaline slant and butt: H2S positive: Citrobacter
freundii
• Alkaline slant and butt: Escherichia coli
• Alkaline slant and butt: Salmonella typhimurium
• Red slant, acid butt: Proteus mirabilis
- Combination test methyl red (MR) and Voges- Proskauer
(VP) differentiates members of the Enterobacteriaceae
family.
- The methyl red detects mixed acid fermentation that
lowers the pH of the broth.
- The VP detects the organism’s ability to convert the acid
products to acetoin and 2,3-butanediolff 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 methyl red indicator is
added. A secondary reagent is added, alpha-naphthol,
followed by potassium hydroxide (KOH)
25.1 MR (Methyl Red) Test
Expected Results
Positive: Bright red color, indicative of mixed acid
fermentation
Weakly positive: Red-orange color
Negative: Yellow color
25.2 VP (Voges-Proskauer) Test (Barrit’s Method)
-for Gram-Negative Rods
- 0.6 mL of a-naphthol and 0.2 mL of KOH
25.3 VP (Voges-Proskauer) Test (Coblentz Method)
- for Streptococci
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- 1.2 mL of a-naphthol and 0ff4 mL of 40%KOH with
creatine
Expected Results

Positive: Red color, indicative of acetoin production

Negative: Yellow color
Quality Control

MR positive/VP negative: Escherichia coli

MR negative:/VP positive: Enterobacter aerogenes

28. Lactobacillus MRS Broth

- Determine whether an organism forms gas during glucose

fermentation. Some Lactobacillus spp. and Leuconostoc spp.
produce gas

- The MRS broth contains sources of carbon, nitrogen, and

26. Microdase Test (Modified Oxidase)
- Differentiate gram-positive, catalase-positive cocci
(micrococci from staphylococci
- The microdase test is a rapid method to differentiate
Staphylococcus from Micrococcus spp. by detection of the
enzyme oxidase. In the presence of atmospheric oxygen, the
oxidase enzyme reacts with the oxidase reagent and
cytochrome C to form the colored compound,
indophenol.
- Staphylococci should yield a negative color change,
except for S. sciuri, S. lentus, and S. vitulus.
- Quality Control
o (+) Micrococcus luteus
o (-) Staphylococcus aureus
- Results
o (+) Development of blue to purple color
o (-) No color change
27. Motility Testing
- used to determine whether an enteric organism is motile.
An organism must have flagella to be motile.
- Bacterial motility is evident by a diffuse zone of growth
extending out from the line of inoculation. Some organisms
grow throughout the entire medium, whereas others show
small areas or nodules that grow out from the line of inoculation
vitamins to support the growth of lactobacilli and other
organisms. It is a selective medium that uses sodium acetate
and ammonium citrate to prevent overgrowth by
contaminating organisms. Growth is considered a positive
result. A Durham tube may be added to differentiate
Lactobacillus spp. from Leuconostoc spp.
o Quality Control
o (+) Lactobacillus lactis
o (-) Escherichia coli
o Results
o (+) Leuconostoc spp.—Growth, gas production
indicated by a bubble in the Durham tube
o Lactobacillus spp.—Growth, no gas production
o (-) No growth
29. 4-Methylumbelliferyl-β-D-Glucuronide (MUG) Test
- presumptively identify various genera of
Enterobacteriaceae and verotoxin-producing Escherichia
coli.
- Eff coli and other Enterobacteriaceae produce the
enzyme β-d-glucuronidase, which hydrolyzes β-d-
glucopyranosid-uronic derivatives to aglycons and d-
glucuronic acid
- The substrate 4-methylumbelliferyl-β-d-glucuronide is
impregnated into the disk and is hydrolyzed by the enzyme
to yield the 4-methylumbelliferyl moiety, which fluoresces
blue under long wavelength ultraviolet light.

- However, verotoxin producing strains of Eff coli do not

produce MUG, and a negative test result may indicate the
presence of a clinically important strain
- Only test on oxidase-positive organisms, because some
oxidase-negative organisms naturally fluoresce.
o Quality Control
o (+) Escherichia coli

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o (-) Klebsiella pneumoniae o (-) No zone of inhibition
o Results
34. Oxidase Test (Kovac’s Method)
o (+) Electric blue fluorescence
o (-) No fluorescence
- determines the presence of cytochrome oxidase activity in
30. Nitrate Reduction microorganisms for the identification of oxidase-negative
Enterobacteriaceae, differentiating them from other gram-
- to determine the ability of an organism to reduce nitrate to negative bacilli
nitrite
o Quality Control
- All members of the Enterobacteriaceae family reduce o (+) Pseudomonas aeruginosa
nitrate, but some members further metabolize nitrite to other o (-) Escherichia coli
compounds o Results
Quality Control o (+) Development of a dark purple color within
10 seconds
Positive: NO3+, no gas: Escherichia coli Positive: o (-) Absence of color

NO3+, gas: Pseudomonas aeruginosa Negative:

Acinetobacter baumannii
31. Nitrite Reduction

- determine whether an organism can reduce nitrites to

gaseous nitrogen or to other compounds containing
nitrogen.
- Microorganisms capable of reducing nitrite to nitrogen do
not turn color and do produce gas in the nitrate reduction test.
The test does not require the addition of zinc dust.
35. Oxidation/Fermentation (of) Medium (CDC Method)
- Quality Contro
o (+) Proteus mirabilis - This test is used to differentiate microorganisms based on the
o (-) Acinetobacter baumannii ability to oxidize or ferment specific carbohydrates.
o Results
36. Phenylalanine Deaminase Agar (PDA)
o (+) No color change to red + gas
o (-) Red + no gas
- This test is used to determine the ability of an organism to
32. o-Nitrophenyl-β-D-Galactopyranoside (ONPG) Test oxidatively deaminate phenylalanine to phenylpyruvic acid

- The genera Morganella, Proteus, and Providencia can be

- The test distinguishes late lactose fermenters from non–
differentiated from other members of the
lactose fermenters of Enterobacteriaceaeff
Enterobacteriaceae familyff
- determine the ability of an organism to produce β-
o Quality Control
galactosidase, an enzyme that hydrolyzes the substrate ONPG
o (+) Proteus mirabilis
to form a visible (yellow) product, orthonitrophenol
o (-) Escherichia coli
o Quality Control o Results
o (+) Shigella sonnei o (+)Green color develops on slant after ferric
o (-) Salmonella typhimurium chloride is added
o Results o (-) Slant remains original color after the
o (+) Yellow addition of ferric chloride
o (-) Colorless

33. Optochin (P disk) Susceptibility Test

- This test is used to determine the effect of Optochin (ethyl

hydrocupreine hydrochloride) on an organism. Optochin lyses
pneumococci (positive test), but alpha- streptococci are
resistant (negative test)

-Any zone of inhibition less than 14 mm is questionable for

pneumococci; the strain is identified as a pneumococcus with
confirmation by a positive bile- solubility test

o Quality Control 37. L-Pyrrolidonyl Arylamidase (PYR) Test

o (+) Streptococcus pneumoniae
o (-) Streptococcus pyogenes - For the presumptive identification of group A streptococci
o Results (Streptococcus pyogenes) and enterococci by the presence
o (+) Zone of inhibition ≥ 14 mm in diameter, with of the enzyme L-pyrrolidonyl arylamidase
6-mm disk o Quality Control

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o (+) Enterococcus faecalis/ Streptococcus 41. Triple Sugar iron Agar (TSI)
pyogenes - TSI is used to determine whether a gram-negative rod
o (-) Streptococcus agalactiae ferments glucose and lactose or sucrose and forms hydrogen
o Results sulfide (H2S)
o (+): Bright red color within 5 minutes
o (-) No color change or an orange color - The test is used primarily to differentiate members of the
Enterobacteriaceae family from other gram-negative rods.
38. Pyruvate Broth
Expected Results
- This test is used to determine the ability of an organism
to utilize pyruvate. This aids in the differentiation between Alkaline slant/no change in the butt (K/NC): glucose, lactose,
Enterococcus faecalis (positive) and Enterococcus faecium and sucrose nonutilizer; this may also be recorded as K/K
(negative)ff (alkaline slant/ alkaline but)
- Quality Control Alkaline slant/acid butt (K/A): glucose fermentation only
o (+) Enterococcus faecalis
Acid slant/acid butt (A/A): glucose, sucrose, and/or lactose
o (-) Streptococcus bovis fermenter
- Results
o (+) Indicator changes from green to yellow
o (-) No color change or yellow green
(weak) Salt tolerance
39. Salt tolerance

- to determine the ability of an organism to grow in high

concentrations of saltff It is used to differentiate
enterococci (positive) from nonenterococci (negative)ff
- A heart infusion broth containing 6ff5% NaCl is used as the test
mediumff
- This broth (BHI) also contains a small amount of glucose and
bromcresol purple as the indicator for acid productionff

- Enterococci are resistant to high salt concentration

o Quality Control
o (+) Enterococcus faecalis
o (-) Streptococcus bovis
o Results
o (+) Visible turbidity in the broth, with or without a
color change from purple to yellow
o (-) No turbidity and no color change

40. Spot Indole Test 42. Urease Test (Christensen’s Method

- This test is used to determine the presence of the enzyme - This test is used to determine an organism’s ability to
tryptophanase. It is a rapid method that can be used in lieu produce the enzyme urease, which hydrolyzes urea. Proteus spp.
of the tube test Indole Production (tryptophane broth) may be presumptively identified by the ability to rapidly hydrolyze
urea.
o Quality Control
o (+) Escherichia coli
o (-) Klebsiella pneumoniae
o Results
o (+) Blue color within 20 second
o (-) No color development/ slightly pink

RDME | BSMT – 3C 10
 BACTERIOLOGY LABORATORY MLS – 110
FINALS (BIOCHEMICAL TESTS- BOOK BASED)
43. X and V Factor Test
-The X and V factor test is used to differentiation Haemophilus
species. Members of the genus Haemophilus require accessory
growth factors in vitro. Some Haemophilus spp. require X factor
(hemin) alone, V factor (nicotinamide adenine dinucleotide
[NAD]) alone, or a combination of the two.

Expected Results

Positive: Growth around the XV disk only shows a requirement for

both factors. Growth around the V disk, no growth around the X
disk, and light growth around the XV disk shows a V factor
requirement.
Negative: Growth over the entire surface of the agar indicates
no requirement for either X or V factor (Figure 13-44, C)

Quality Control

Positive: Haemophilus influenza: halo of growth around the XV

disk, no growth on the rest of the agar surface.
o Haemophilus parainfluenzae: halo of growth around the XV
and V disks
o Haemophilus ducreyi: halo of growth around the XV and X
disks

RDME | BSMT – 3C 11