A book about the sixth penalty Biological Laboratory
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\17\v01 Page 1 of 2
Section: Technical Manual Subject Title: Gonogen
(GC Coagglutination) Test
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
GONOGEN (GC COAGGLUTINATION) TEST
Principle
The Gonogen II test is a coagglutination test for the confirmato ry identification of N. gonorrhoeae.
Reagents
I: Buffer
II: Gonogen reagent (antibodies)
Positive control reagent
Negative control reagent
Other Materials
Test tray: consists of wells with special
matrix and absorbent material
Glass tubes (12 x 75mm) (not provided)
Glass dropper rod assembly
Plastic transfer pipets
Procedure
1. Preparation of sample
a) In a 12x75 mm tube dispense 500 µL of reagent I (buffer) using the glass dropper
rod assembly provided (demarcation line).
b) Using a swab, make a suspension of approximately 30 colonies to match a
McFarland 1 turbidity standard.
c) Press swab against side of tube to express as much liquid as possible.
d) Vortex reagent II an d add 1 drop to the tube.
e) Mix and set sit fo r at least 5 minutes.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\17\v01 Page 2 of 2
Technical Manual
2. Test
a) With a plastic transfer pi pet, add 2 drops of each test suspension into a well of the
test tray using a separa te well for each test.
b) using a clean plastic transfer pipet, add 1 drop of reagent I (buffer) to each
completed test well.
Interpretation
Positive: Pink to red do t in well of test tray.
Negative: White to pale pink dot in well of test tray.
Note: 1. A colour reaction more intense th an the negative control should be
interpreted as positive.
2. If color reaction is questionable, reincubate tube at RT for 3 minutes
and repeat test.
3. If specimen suspension is made too turbid a faint background colour
will occur. This should NOT be interpreted as a positive result.
Quality Control
The positive and negative controls must be tested whenever a test is run. The test is performed in
the same manner except 1 drop of the control reagent is added to 500 µL of buffer rather than a
suspension of the test organism.
References
1. Gonogen II package insert, October 1993.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 60
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\18\v01 Page 1 of 2
Section: Technical Manual Subject Title: High Level
Aminoglycoside Testing
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
HIGH LEVEL AMINOGLYCOSIDE TESTING
Principle
Enterococcal species where an identification and sensitivity has been performed must be tested
for resistance to vancomycin and high level gentamicin and streptomycin (HLAR).
Materials
Entero HLAR Bi-plates
Brain Heart Infusion Agar (BHI) - Control Plates
Procedure
1. Using the VITEK colorimeter, prepare a 0.5 McFarland suspension in sterile saline
(inoculum from VITEK can be used).
2. Using a sterile swab, spot inoculate the suspension onto each half of the plates. Three
organisms can be tested on each plate.
3. After the inocula has dried, incubate the plate at 35
o
C for up to 48 hours.
Interpretation
Check the control plate for adequate growth. Then check the drug pl ates for absence or presence of
growth; any growth is considered sign ificant. Read plates at 24 hours and record results. If there is
no growth on the streptomycin plate, reincubate plate for an add itional 24 hours.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\18\v01 Page 2 of 2
Technical Manual
Quality Control
Control strains are tested daily.
Expected results
C G S
E. faecalis (ATCC 19966) + + -
E. gallinarum (ATCC 35038) + - -
E. casseliflavus (ATCC 12755) + - +
C = Growth Control; G = Gent amicin; S = Streptomycin; + = growth; - = no growth
Reporting Results
Blood cultures and sterile fluids -- - Report with canned comment (Ref er to Susceptibility Testing
Manual).
Urines and other sites --- Do not report HLAR.
Reference
1. PML Technical Manual data sheet No. 323, Nov. 1993.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 62
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\19\v01 Page 1 of 2
Section: Technical Manual Subject Title: Hippurate Test
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
HIPPURATE TEST
Principle
This test determines the ability of bacteria to hydrolyse sodium hippurate. One of the end products,
glycine is detected by the addition of ninhydrin reagent.
Reagents
Hippurate disks (store refrigerated)
Ninhydrin reagent
Sterile distilled water
Other Materials
Sterile tube (13 x 100mm)
Bacteriology loop
Sterile graduated pasteur pipette
Procedure
1. Place a Hippurate disk into a sterile tube and add 0.4 mL sterile water.
2. Heavily inoculate the tube with a loopful of the test organism.
3. Incubate at 35
0
C for 2 hours.
4. Following incubation add 5 drops of ninhydrin reagent to the tube and shake gently.
5. Reincubate tube for 10 minutes and read reaction.
Interpretation
Positive: Deep purple-blue colour
Negative: No colour change or light purple
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\19\v01 Page 2 of 2
Technical Manual
Precautions
1. A heavy inoculum is necessary to obtain a high concentration of enzyme.
2. Do not incubate longer than 30 minutes after addition of ni nhydrin reagent because a false
positive reaction could result.
Quality Control
Test with known positive and negative controls each time the test is preformed.
Positive: Campylobac ter jejuni (ATCC 29428)
Negative: Campylobacter coli (CPI B7080)
Reference
1. Difco package insert.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 64
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\20\v01 Page 1 of 2
Section: Technical Manual Subject Title: Indole Test
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
INDOLE TEST
Principle
Bacteria that produce the enzyme tryptophanase will deaminate tryptophan to indole, pyruvic
acid and ammonia in the presence of a co-enzyme pyridoxal phosphate.
Indole combines with Ehrlich's / Kovac's reagent to form a red-coloured complex.
Materials
Test A: Filter paper strips impregnated with Ehrlich's reagent.
Test B: Kovac's reagent
2% Tryptone broth (Difco, Oxoid)
Method
A: Filter paper strips are suspended over tubes of ONPG / PAM media, and incubated at
35
0
C overnight.
Interpretation
Positive test - development of red colour on the strip.
Negative test - white-yellow colour.
B: 1. Inoculate the tryptone broth, and incubate at 35
0
C overnight.
2. Add a few drops of Kovac's reagent to the broth.
Interpretation
Positive test - red colour in the upper layer.
Negative test - light-yellow colour in the upper layer.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\20\v01 Page 2 of 2
Technical Manual
Quality Control
Test the following positive and negative controls weekly:
Positive: Proteus vulgaris (ATCC 13315)
Negative: Klebsiella pneumoniae (ATCC 13883)
Reference
1. Murray, PA, et al. Manual of Clinical Microbiology 7
th
ed. 1999.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 66
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\21\v01 Page 1 of 1
Section: Technical Manual Subject Title: Koehler Illumination
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
KOEHLER ILLUMINATION
The microscope should be set up using Koehler il lumination for all parasitology examinations.
This ensures that all the light from the lamp is being focused onto the specimen and that the field
to be examined is evenly illuminated.
Procedure
1. Turn the lamp on.
2. Bring the condenser up to the top position, with the top lens swung in.
3. Open the condenser diaphragm.
4. Place a specimen on the stage and focus with the 10x objective.
5. Close the field diaphragm.
6. Lower the condenser until the edge of the field diaphragm is in sharp focus.
7. Center the field diaphragm image with condenser centering screws.
8. Open the field diaphragm until the edge just disappears from view.
9. Remove one eyepiece.
10. Looking down the eyepiece tube, close the c ondenser diaphragm until the illumination is
approximately 2/3 full.
11. Replace the eyepiece.
12. Repeat for each objective lens when changed.
Reference
1. Baron E., Finegold S.M., Bailey & Scott's Diagnostic Microbiology, 8
th
ed., The C.V.
Mosby Company, p64.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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