CLINICAL BACTERIOLOGY- LABORATORY

POST MIDTERM- IDENTIFICATION OF UNKNOWN

Mr. Alvin John L. Erese

IDENTIFICATION OF UNKNOWN BACTERIA (+) : deamination of acetamide resulting a blue

- Identification is the process of determining color
to which established taxon a new isolate or (-) : No color change
strain belongs.
- Samples of blood , tissue, water, food and
cosmetics are examined for the presence
of pathologic microorganisms, and cross
contamination.
- Pharma industries and research institutes
are constantly screening soil , water and
marine samples to isolate new antibiotic, ,
enzymes and vitamin producing
microorganism.
IMPORTANCE IDENTIFICATION
METHODS
Medical Phenotypic/ Morphology QUALITY CONTROL
Diagnostics
POSITIVE P. auruginosa
Food and brewage Immunological
CONTROL
industries
Negative Stenotrophomonas maltophila
Research setting Molecular/ Genetic
control

Staining Techniques
ACETATE UTILIZATION
Simple staining -Use of -For visualization
PRINCIPLE

🔘This test is to determine if an organism can use

single a of morphological
stain. shape and
arrangement
acetate as the sole source of carbon.

🔘Positive reaction: green to blue color.

Differential -Use of two -For
staining contrasting Identification:
stains 1. Gram stain Procedure:
separated by 2. Acid fast stain 1.Using an inoculating loop; inoculate acetate
a -For Visualization slant lightly from an 18 – 24 hour medium.
decolorizing of structure: 2.Incubate at 35 degree centigrade for up to 7
agent. 1. Spore stain days.
2. Capsule stain
EXPECTED RESULT
Acetamide Utilization Test
POSITIVE BLUE COLOR MEDIUM
Principle: (alkalinized
🔘Used to determine the ability of the NEGATIVE No growth/ no color
change
microorganism to use acetamide as the sole
source of carbon.

🔘Bacteria that can grow on this medium deaminate

acetamide to release ammonia.

🔘Positive result: color change of the medium from

green to blue.
Procedure:
1. Inoculate acetamide slant lightly with
inoculating needle from an 18 – 24 hours culture
2. Incubate at 35 degree Celsius for 7 days
Expected result:

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Montalban Kimberly N.
MED21A
CLINICAL BACTERIOLOGY- LABORATORY
POST MIDTERM- IDENTIFICATION OF UNKNOWN
Mr. Alvin John L. Erese

EXPECTED RESULT
QUALITY CONTROL POSITIVE Colony disintegrates
Positive control E. coli NEGATIVE Intact colonies
Negative control -
QUALITY CONTROL
Positive control S. pneumoniae
Negative control E. faecalis

CAMP TEST
• Certain organism (including group B
streptococci) produce a diffusible extracellular
protein (CAMP factor) that acts synergistically
with the beta-lysin of S. aureus to cause an
enhance RBC lysis.
Procedure:
• Streak a beta lysin producing strain of S. aureus
at the center of a sheep’s BAP
• Streak test organism across the plate
perpendicular to S. aureus streak ( multiple
organisms can be tested on a single plate if they
are 3-4 mm apart)
• Incubate overnight at 35C.

EXPECTED RESULT
POSITIVE ARROW HEAD zone of
beta hemolysis
NEGATIVE No Hemolysis

QUALITY CONTROL

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MED21A
CLINICAL BACTERIOLOGY- LABORATORY
POST MIDTERM- IDENTIFICATION OF UNKNOWN
Mr. Alvin John L. Erese

Positive control S. agalactiae 1. Inoculate tubes with 1 drop of an 18- to 24-hour

Negative control S. pyogenes brain-heart infusion broth culture.
2. Add a 4-mm layer of sterile mineral oil to each
CETRIMIDE tube.
• Used to determine the ability of an organism to 3. Incubate the cultures for 4 days at 35° C in
grow in the presence of cetrimide – a toxic ambient air.
substance that inhibits the growth of bacteria.
Procedure:
• Inoculate a cetrimide agar slant with 1 drop of an
18-24 hr culture in brain-heart infusion broth.
• Incubate at 35 C for 7 days.
• Examine the growth on slant.

QUALITY CONTROL
Positive control P. aeruginosa
Negative control E. coli

EXPECTED RESULT
POSITIVE Alkaline (Purple) color
change
NEGATIVE No color change or acid
(yellow) color in test and
control tube.
DNA HYDROLYSIS
• Used to determine the ability of an organism to
hydrolyse DNA
• If the organism growing on the medium
DECARBOXYLASE TEST hydrolyses DNA – green color fades and the colony
• Measures the enzymatic ability of an organism to is surrounded by colorless zone.
decarboxylate (hydrolyse) an amino acid to form Procedure:
amine. • Inoculate the DNAse agar with the organism to
• Hydrolysis of the amino acid results in an be tested and streak for isolation
alkaline pH change. • Incubate aerobically at 35 C for 13 to 24 hours
Procedure:
A.Glucose nonfermenting organisms
1. Prepare a very heavy suspension
(McFarland No. 5 turbidity standard) in
brain-heart infusion broth from young
bacteria (18 to 24 hours old) growing on 5%
sheep blood agar.
2. Inoculate each of the three decar-boxylase QUALITY CONTROL
broths (arginine, lysine, and ornithine) and Positive control S. aureus
the control broth (no amino acid) with 4 Negative control S. epidermidis
drops of broth. FERMENTATION MEDIA
3. Add a 4-mm layer of sterile mineral oil to • Used to determine the ability of an organism to
each tube. ferment a specific carbohydrate that is
4. Incubate the cultures at 35° C in ambient incorporated in a basal medium , thereby
air for up to 7 days. producing acid with or without visible gas.
B. Glucose-fermenting organisms Procedure:

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CLINICAL BACTERIOLOGY- LABORATORY
POST MIDTERM- IDENTIFICATION OF UNKNOWN
Mr. Alvin John L. Erese

 Peptone medium with Andrade’s indicator EXPECTED RESULT

1. Inoculate each tube with 1 drop of an 18 – 24 hr POSITIVE partial or total
brain-heart infusion broth culture liquefaction of the
2. Incubate at 35 C for 7 days inoculated tube
3. Examine the tubes for acid (indicated by pink NEGATIVE complete solidification at
color) and gas production 4C
Note: No growth in 24 hours – add 1- 2 drops of
sterile rabbit serum per 5 ml of fermentation broth QUALITY CONTROL
to each tube Positive control P. vulgaris
Negative control E. aerogenes
 Blood heart infusion broth with bromcresol
HIPPURATE TEST
purple indicator (for streptococci and
• The test medium must contain only hippurate.
enterococci)
• The end product of hydrolysis of hippuric acid by
1. Inoculate tube with 2 drops of an 18-24 hr brain-
hippuricase include glycine and benzoic acid.
heart infusion broth
• The end product of ninhydrin oxidation reacts to
2. Incubate at 35 C for 4 days
form a purple color.
3. Observe daily change of the bromcresol
Procedure:
indicator from purple to yellow (acid)
• Add 0.1 ml sterile water to a test tube.
• Make a heavy suspension of the organism tested
• Place a hippurate disk in a mixture
• Cap and incubate at 35 C for 2 hours; waterbath
is preffered
• Add ninhydrin reagent and re-incubate for 15-30
min
• Observe a deep purple color.

GELATIN HYDROLYSIS
• Used to determine the ability of an organism to
produce proteolytic enzymes that liquefy the
gelatin.
Procedure:
• Inoculate the gelatin deep with 4-5 drops of a 24
hr culture
• Incubate at 35 C for 14 days
• Remove the gelatin tube daily from the incubator
and place at 4C to check liquefaction ( do not
QUALITY CONTROL
invert)
Positive control S. agalactiae
• Refrigerate an uninoculated control , along with
Negative control S. pyogenes
the Inoculated tube
Note: Liquefaction Is determined only after the
control hardened LAP TEST
• Used to identify catalase negative gram positive
cocci
• Rapid test for the detection of enzyme leucine
aminopeptidase.
Procedure:
1.Before incubation, slightly dampen the LAP disk
with reagent grade water. Do not supersaturate
the disk.
2.Using a wooden applicator stick, rub a small
amount of several colonies of an 18- to 24-hour
pure culture onto a small area of the LAP disk.

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CLINICAL BACTERIOLOGY- LABORATORY
POST MIDTERM- IDENTIFICATION OF UNKNOWN
Mr. Alvin John L. Erese

3.Incubate at room temperature for 5 minutes. • Used to determine gram negative rod
4.After this incubation period, add 1 drop of decarboxylates or deaminase lysine and forms
cinnamaldehyde reagent. H2S.
• When glucose is fermented: the butt medium
EXPECTED RESULT becomes acidic (yellow)
POSITIVE red color upon addition of • If the organism produces lysine, Cadaverine is
cinnamaldehyde reagent formed – neutralizes organic acid; reverts to
NEGATIVE - alkaline state – purple
Procedure:
QUALITY CONTROL • Inoculate LIA by twice stabbing through the
Positive control E. faecalis center medium to the bottom of the tube then
Negative control Leuconostoc sp. streaking the slant.
• Cap the tube and incubate at 35 C for 18-24
hours.
EXPECTED RESULT
K/K No fermentation of
glucose
K/A Glucose fermentation
Black Precipitate H2S+
R/A Lysine deamination and
glucose fermentation

LITMUS MILK
• Used to determine microorganisms ability to
metabolize milk.
• Fermentation of lactose is evidenced by the
litmus turning pink as a result of acid production.
Procedure:
• Inoculate with 4 drops of a 24 hr broth culture
• Incubate at 35-37C
• Observe daily for 7 days for alk. Reaction and
acid reaction, indicator reduction, acid clot and
peptonization
• Multiple changes can occur over the observation
period
• Record all chages
• Alkaline: Alcaligenes faecalis
• Acid: Enterococcus faecium
• Peptonization: Burkholderia cepacia
LYSINE IRON AGAR
OPTOCHIN TEST
• Used to determine the effect of optochin
• Optochin lyses pneumococci (positive) but alpha
streptococci are resistant (negative).
Procedure:
1. Using an inoculating loop, streak two or
three suspect colonies of a pure culture
onto half of a 5% sheep blood agar plate.
2. Using heated forceps, place an optochin
disk in the upper third of the streaked area.
Gently tap the disk to ensure adequate
contact with the agar surface.
3. Incubate plate for 18 to 24 hours at
35° C in 5% CO,.
NOTE: Cultures do not grow as well in
ambient air, and larger zones of inhibition
occur.
4. Measure zone of inhibition in millimeters,
including diameter of disk

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Montalban Kimberly N.
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CLINICAL BACTERIOLOGY- LABORATORY
POST MIDTERM- IDENTIFICATION OF UNKNOWN
Mr. Alvin John L. Erese

EXPECTED RESULT EXPECTED RESULT

POSITIVE Zone of inhibition POSITIVE BRIGHT RED COLOR
NEGATIVE No zone of inhibition NEGATIVE No color change or an
orange color
QUALITY CONTROL
Positive control S. pneumoniae QUALITY CONTROL
Negative control S. mitis Positive control E. faecalis
Negative control S. mitis
PHENYLALANINE DEAMINASE
• Used to determine the ability of the organism to PYRUVATE BROTH
oxidatively deaminate phenylalanine to • Aids in the differentiation of E. faecalis (positive)
phenylpyruvic acid and E. faecium (negative)
• Phenylpyruvic acid is detected by adding few • Used to determine the ability of the organism to
drops of 10% FeCl ; resulting a green – colored utilize pyruvate.
complex. Procedure:
Procedure: 1. Lightly inoculate the pyruvate broth with an
1.Inoculate phenylalanine slant with 1 drop of a 24- 18- to 24-hour culture of the organism from
hour brain-heart infusion broth. 5% sheep blood
2.Incubate 18 to 24 hours (or until good growth is agar.
apparent) at 35°C in ambient air with cap loose. 2. Incubate at 35°C in ambient air for
3. After incubation, add 4 to 5 drops of 10% 24 to 48 hours.
aqueous ferric chloride to the slant. EXPECTED RESULT
EXPECTED RESULT POSITIVE Indicator changes from
POSITIVE Green color GREEN TO YELLOW.
NEGATIVE No color change NEGATIVE No color change

QUALITY CONTROL QUALITY CONTROL

Positive control P. vulgaris Positive control E. faecalis
Negative control E. coli Negative control S. faecium

X AND Y FACTOR TEST

PYR TEST
• Used for the identification of gram positive cocci
• Detected in the presence of
N,Nmethylaminocinnamaldehyde reagent which
gives a positive result = bright red color.
Procedure:
1. Before inoculation, moisten disk slightly
with reagent grade water. Do not flood
disk.
2. Using a wooden applicator stick, rub a
small amount of several colonies of an 18-
to 24-hour pure culture onto a small area of
the PYR disk.
3. Incubate at room temperature for 2
minutes.
4. Add a drop of detector reagent, N, N-
dimethylaminocinnamaldehyde and observe
for a red color within 1 minute.
• Members of genus Haemophilus require.
• Some Haemophilus spp. Require X factor (hemin),
V factor (NAD), or both.
Procedure:
1. Make a very light suspension (McFarland
0.5) of the organism in sterile saline.
NOTE: It is important not to carry over any
X-factor contained in the medium that the
organism is taken from. Therefore, a loop,
not a swab, should be used to make the
suspension.
2. Dip a sterile swab into the organism
suspension. Roll the swab over the entire
surface of a trypticase soy-agar plate.
3. Place the X, V, and XV factor disks on the
agar surface. If using separate disks, place
them at least 4 to 5 cm apart.
4. Incubate overnight at 35° C in ambient air.

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Montalban Kimberly N.
MED21A
CLINICAL BACTERIOLOGY- LABORATORY
POST MIDTERM- IDENTIFICATION OF UNKNOWN
Mr. Alvin John L. Erese

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Montalban Kimberly N.
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