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TML/MSH Microbiology Department
Policy & Procedure Manual
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Precautions
1. E. coli O157:H7 and non-motile stra ins which produce verotoxi n are MUG test negative.
Quality Control
The following controls are tested weekly:
MUG INDOLE
E. coli (ATCC 25922) + +
P. mirabilis (ATCC 12453) - -
Reference
1. Prolab package insert
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Policy & Procedure Manual
Policy #MI\TECH\26\v01 Page 1 of 2
Section: Technical Manual Subject Title: Neisseria Identification
Sugars
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
NEISSERIA IDENTIFICATION SUGARS
Principle
The test determines the ability of bacteria to produ ce acid products from carb ohydrates. Used as a
method to identify Neisseria species and other fastidious organisms.
Materials
Cystine Proteose Peptone Agar (CPPA) media: - glucose, maltose, lactose, sucrose, control (no
sugar).
Inoculating loop.
Procedure
1. For each tube, scrape a full 3 mm loopful of growth from the surface of a 24 hour chocolate
agar subculture plate.
2. Deposit this inoculum a few millimetres below the surface of the medium.
3. Incubate at 35
o
C.
4. Examine tubes after 1, 4 and 24 hours incubation.
Interpretation
Positive: Yellow colour at top of tube
Negative: Red (alkaline) to orange (neutral) colour.
Organism Glu Mal Lac Suc Cont
N. gonorrhoeae + - - - -
N. meningitides + + - - -
M. catarrhalis - - - - -
Precautions
1. Inoculum must be heavy.
2. False positive results may occur if tubes are incubated in CO
2
.
3. Tubes that appear bright yellow should be gram stained to check for contamination.
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Quality Control
The following controls are run each time the test is performed:
N. gonorrhoeae (ATCC 43069)
N. meningitidis (ATCC 13090)
M. catarrhalis (ATCC 8176)
Reference
1. Murray PA, et al. Manual of Clinical Microbiology, 7th ed ., 1999; pp. 592-598.
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Policy & Procedure Manual
Policy #MI\TECH\27\v01 Page 1 of 2
Section: Technical Manual Subject Title: ONPG Test
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
ONPG TEST
Principle
This test is used to demonstrat e the presence or absence of the enzyme B-galactosidase using the
substrate ortho-nitrophenyl-D-galactopyranoside in order to differ entiate lactose-fermenting from
non lactose-fermenting organism s and in the identification of B. cepacia.
Reagents
ONPG disks (Store refrigerated)
Sterile saline
Other materials
Sterile tube (13 x 100 mm)
Bacteriology loop
Sterile graduated Pasteur pipette
Procedure
1. Place an ONPG disk into a sterile tube and add 0.2 mL saline.
2. Heavily inoculate the tube with a loopful of the test isolate.
3. Incubate at 35
o
C for up to 4 hours.
Interpretation
Positive: yellow colour within 4 hours
Negative: colourless at 4 hours
Precautions
1. A heavy inoculum is necessary to obtain a high concentration of enzyme.
Quality Control
Test with known positive and negative controls each time the test is performed.
Positive: E. coli (ATCC 25922)
Negative: P. vulgaris (ATCC 13315)
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Policy & Procedure Manual
Policy # MI\TECH\27\v01 Page 2 of 2
Technical Manual
References
1. MacFaddin JF, Biochemical Tests for Identification of Medi cal Bacteria, 2nd ed., Williams
and Wilkins, Baltimore MD., 1980, p120-128.
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Policy & Procedure Manual
Policy #MI\TECH\28\v01 Page 1 of 2
Section: Technical Manual Subject Title: ONPG-Phenylalanine-
Motility Medium (ONPG-PAM)
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
ONPG-PHENYLALANINE-MOTILITY MEDIUM (ONPG-PAM)
Principle
This test is used to determine an organism's motility, its ability to ferm ent lactose and produce
phenylalanine deaminase. The medium is primarily used as a screening procedure for the detection
of enteric pathogens.
Reagents
ONPG-PAM tube
10% Ferric chloride
Other materials
Culture wire
Procedure
1. Inoculate the ONPG-PAM tube by stabbing the centre of it to the bottom of the tube.
2. Incubate the tube in O
2
at 35
o
C X 18 hours.
3. Read the tube for ONPG, motility and indole.
4. Add 2 drops of 10% ferric chloride so lution and read the ph enylalanine result.
Interpretation
ONPG: positive: yellow
negative: no colour change
Motility: positive: diffuse growth from line of inoculum
negative: growth does not spread from line of inoculum
Phenylalanine (PPA): positive: green
negative: yellow/brown
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Policy & Procedure Manual
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Quality Control
Test with control organisms each time a new batch of meda is prepared.
ONPG Motility PPA K. pneumoniae (ATCC 13883) + - -
P. vulgaris (ATCC 13315) - + +
References
1. Murray, PA, et al. 1999. Manual of Clinical Microbiology, 7
th
ed., American Society for
Microbiology, Washington, D.C.
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Policy & Procedure Manual
Policy #MI\TECH\29\v01 Page 1 of 2
Section: Technical Manual Subject Title: Optochin Sensitivity Test
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
OPTOCHIN SENSITIVITY TEST
Principle
This test is used to determine an organism's susceptibilit y to the chemic al optochin
(ethylhydrocupreine hydrochloride) fo r the presumptive identification of S. pneumoniae .
Reagents
Bacto Optochin Disks (5 µg disk) Store refrigerated
Mueller Hinton Sheep Blood Agar (MHSBA)
Other Materials
Culture loop
Forceps
Cotton swabs
Procedure
1. Inoculate the suspected alpha haemolytic colony onto a MHSBA to ob tain confluent growth.
2. Using aseptic technique place an optochin disk onto the surface of the inoculated agar.
Press down with forceps.
3. Incubate at 35
o
C in CO2
for 18-24 hours.
Interpretation
Susceptibile: Zone of inhi bition of at least 14 mm
Resistant: Zone of inhi bition less than 14 mm
Quality Control
Test with known susceptible and resistant control strains weekly:
Susceptible: S. pneumoniae (ATCC 6303)
Resistant: Viridans Strep. (LPTP 8610)
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References
1. MacFaddin JF, Biochemical Tests for Identification of Medi cal Bacteria, 2nd ed., Williams
and Wilkins, Baltimore MD., 1980, p245-249.
2. Difco package insert, July 1983.
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Policy & Procedure Manual
Policy #MI\TECH\30\v01 Page 1 of 2
Section: Technical Manual Subject Title: Oxidase (API Strip)
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
OXIDASE (API STRIP)
Principle
This test determines whether an isolate produces oxidase enzymes. This test is mainly used, in
conjunction with other tests, for the identification of gram negative organisms and Bacillus species.
Reagents
API Oxidase Reagent
1. 0.2% Aqueous ascorbic acid: Reconstitute ascorbic acid with 25 ml ster ile distilled water.
This solution may be refrigerated for up to 28 days. The expiry date mu st be written on the
bottle.
2. N,N,N,-Tetramethyl-p-phenylenediamine-dihydrochloride:
Reconstitute with 5 ml of the 0.2% aqueous ascorbic acid. It is recommended that this be
re-constituted 4-5 hours before use. This solu tion may be refrigerated fo r up to 7 days at 2 -
8
o
C. The expiry date must be written on the bottle.
Other Materials
API filter paper
API oxidase tray
Wooden applicator stick
Procedure
1. Place a filter paper in the oxidase tray and moisten entire pape r with oxidase reagent. Allow
to air dry. May be used for up to 1 week.
2. Transfer a portion of the colony to the filter paper using a wooden applicator stick.
3. Observe for 30 seconds.
Interpretation
Positive: Development of a purple colour within 30 seconds
Negative: No colour change
Precautions
Nichrome wire may cause false positive reactions.
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Policy & Procedure Manual
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Quality Control
Test daily with known positive and negative controls.
Positive: P. aeruginosa (ATCC 27853)
Negative: K. pneumoniae (ATCC 13883)
References
1. API Oxidase package insert 3/80.
2. MacFaddin JF, Biochemical Tests for Identification of Medi cal Bacteria, 2nd ed., Williams
and Wilkins, Baltimore MD, 1980, p249-260.
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Policy # MI\TECH\31\v01 Page 1 of 1
Section: Technical Manual Subject Title: Oxidase
(Spot Test Dropper)
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
OXIDASE (SPOT TEST DROPPER)
Principle
This test determines whether an isolate produces oxidase enzymes and is used for the identification
of Neisseria species isolated from primary plates.
Reagents
Spot Test dropper. Store at room temperature.
Procedure
1. Hold the dropper upright and squeeze gently to crush the glass ampule inside the dispenser.
2. Add 2 - 3 drops directly to the colonies to be tested and observe for 30 seconds.
Interpretation
Positive: Development of a purple colour within 30 seconds
Negative: No colour change
Note: Colonies which are positive must be subcultured immediately since prolonged
exposure will result in death of the organisms.
Quality Control
Test daily with known positive and negative controls.
P. aeruginosa (ATCC 27853) : positive
E. coli (ATCC 25922) : negative
References
1. Difco Spot Text Oxidase reagent package insert 1985.
2. MacFaddin JF, Biochemical Tests for Identification of Medi cal Bacteria, 2nd ed., Williams
and Wilkins, Baltimore MD, 1980, p249-260.
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