Book on Biological Laboratory fifth penalty
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Policy & Procedure Manual
Policy # MI\TECH\11\v01 Page 5 of 6
Section: Technical Manual Subject Title: Cryptococcal Antigen
Interpretation of results
Negative: Negative result in in itial screening tests against ACGR.
Positive: The titre is reporte d as the highest dilution showing a 2+ or greater reaction
with ACGR and negative with NGR.
Nonspecific Interference: The titre with ACGR is at least 4-fold higher than the titre
with NGR.
Uninterpretable test: The titre with ACGR is less than 4-fold greater than the titre with
NGR.
III. Reporting
Telephone all positive reports.
Negative Report: "Cryptococcal antigen not detected by latex agglutination."
Positive Report: "Cryptococcal antigen detected at a titre of by latex
agglutination."
Non-specific or Uninterpretable Report:
"Cryptococcal antigen uninterpretable by latex agglutination."
IV. Precautions
The ring slide must be thoroughly cleaned after each use as follows:
(a) Soak in hypochlorite overnight.
(b) Scrub using detergent.
(c) Rinse well with tap water.
(d) Rinse 3 times with distilled water.
(e) Dry thoroughly using paper towels.
(f) Wipe clean with lint-free tissue.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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Policy & Procedure Manual
Policy # MI\TECH\11\v01 Page 6 of 6
Section: Technical Manual Subject Title: Cryptococcal Antigen
V. Quality Control
The pattern of reactions for the controls must be as follows.
NGR - - +
ACGR + -
CAC NC AGC
Failure to obtain this pattern indicates that the test must be repeated and the patient test
results cannot be reported.
VI. References
Product Insert, 1986. Meridian Diagnostics Inc., 3471 Rive r Hills Dr., Cinc innati, Ohio
45244. (513)-271-3700.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\12\v01 Page 1 of 2
Section: Technical Manual Subject Title: Crystal MRSA ID System
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
CRYSTAL MRSA IDENTIFICATION SYSTEM
Principle
Used as a screening test for the detec tion of intrinsic methicillin-resistant Staphylococcus aureus
from isolated colonies.
Materials
BBL Crystal panel (lid and base)
MRSA Id both-3.2ml
transfer pipette
(all provided in kit)
Method
1. Suspend test S. aureus in 2 ml of Vitek saline a nd adjust to a McFarland 0.5.
2. Vortex and transfer 0.5mL to the tube of MRSA Id broth and Vortex.
3. Remove lid from panel base without touching lid prongs and discard desiccant.
4. Place a drop of sterile saline in the first well (positive control).
5. Using the same pipette, place 3 drops of the ID broth suspension into the same well.
6. Place 4 drops of the broth suspension into th e next 2 wells of the panel (oxacillin and
negative control). Leave the fourth well empty. Remove any bubbles.
7. Cover the panel base with the lid. Gently pre ss lid onto panel base w ith the lid onto panel
base with a snap. Lid should no longer be removed. Do not invert panel.
8. Incubate at 35
o
C for at least 4 but not more than 5 hours.
9. Expose panel to UV light and record which wells are fluorescing.
Interpretation
Bacteria Well#1 Well#2 Well#3
Methicillin Sensitive: + _ _
Methicillin Resistant: + + -
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Technical Manual
Quality Control
Results are uninterpretable if positive control well is negative or the negative control well is
positive.
Reference
1. BBL crystal MRSA ID System package insert August 1993.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\13\v01 Page 1 of 2
Section: Technical Manual Subject Title: Denka MRSA Screen
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
DENKA MRSA SCREEN
Principle
To be used as a screen ing test for the detectio n of Methicillin Resistant S. aureus (MRSA) from
isolated colonies.
Reagents
MRSA screening kit
Microcentrifuge tubes
Boiling water bath
Wooden sticks
Loops
Micropipettes
Method
1. Add 4 drops of extraction reagent #1 into a microcentrifuge tube.
2. Take one heavy loopful of the Staphylococcus aureus colonies from a blood plate and
suspend the cells in the microcentrifuge tube.
3. Place in a boiling water bath for 3 minutes.
4. Remove microcentrifuge tube and let cool to room temperature.
5. Add one drop of extraction reagent #2 to the tube and mix well.
6. Centrifuge at high speed for 5 minutes.
7. For each specimen to be tested , allot and label one circle of the test card for testing with
sensitized latex and one with control latex.
8. Place 50 microliters of the specimen onto 2 of the test ci rcles and add one drop of the
sensitized cells to one circle and one drop of the latex control to the other.
9. Mix the sample and latex together.
10. Rotate the card by hand for 3 minut es and observe fo r agglutination.
PROCEDURE MANUAL
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Technical Manual
11. If negative rotate for another 3 minutes.
Interpretation
Sensitized latex Control latex Result
+ - MRSA
- - Not MRSA
+ or - + Indeterminant
Quality Control
Positive and negative controls must be set up once per week.
Positive: S. aureus (ATCC 43330)
Negative: S. aureus (ATCC 29213)
Reference
1. Denka Seiken Co., Ltd., Tokyo, Japan, Denka MRSA Screen package insert June 1998.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\14\v01 Page 1 of 1
Section: Technical Manual Subject Title: DNAse Test
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
DNAse TEST
Principle
This test determines the ability of an organism to produce deoxyribonuclease (DNAse). This test
used, in conjunction with othe rs, for the identification of S. aureus , M. catarrhalis and Serratia
species.
Reagents
DNAse test agar with methyl green
Other Materials
Culture loop or wooden applicator stick
Procedure
1. Spot inoculate an isolate using gr owth from an 18-24 hr pure culture.
2. Incubate in O
2 at 35
o
C X 18-24 hr.
Interpretation
The plate should be observed against a white background.
Positive: Distinct clear zone surrounding spot inoculum
Negative: No clear zone
Quality Control
Test each new batch of media and when in use with positi ve and negative controls.
Positive: S. aureus (ATCC 25923)
Negative: S. epidermidis (ATCC 12228)
Reference
1. MacFaddin JF, Biochemical Tests for Identification of Medi cal Bacteria, 2nd ed., Williams
and Wilkins, Baltimore MD., 1980, p94-113.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\15\v01 Page 1 of 3
Section: Technical Manual Subject Title: E. coli O157 Latex Test
(Oxoid)
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
E. coli O157 LATEX TEST (OXOID)
Principle
The Latex test will demonstrate by slide agglutination, E. coli strains possessing the O157
antigen. Sorbitol MacConkey Agar (SMAC) should be used as the primary screen. Non-sorbital
fermenting colonies (NSF) are test ed with the latex reag ents, to determine if the isolate belongs
to the O157 serogroup, and is therefore a poten tial vero-cytotoxin (VT) producing strain.
Reagents
DR621 Test Latex - consists of latex partic les sensitized with specific rabbit antibody
reactive with the O157 antigen.
DR622 Control Latex - consists of latex particles sensitized with pre-immune rabbit
globulins.
Storage
Do not freeze. Store at 2
0
C - 8
0
C. Do not use kit beyond the expiry date.
Procedure
NSF colonies may be taken from SMAC plates or alternatively NSF isolates may be inoculated
onto non-selective agar media for testing.
It is necessary to test up to 10 separate NSF co lonies to ensure a high probability of detection
from mixed cultures.
1) Bring the latex reagents to room temperature. Make sure the latex suspensions are mixed
by vigorous shaking. Expel any latex from the dropper pipette for complete mixing.
2) Dispense 1 drop of the Test latex onto a circle of the black slide. Place it close to the
edge of the circle.
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TML/MSH Microbiology Department
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Technical Manual
3) Add some loopfuls or a pasteur pipette drop of sa line to the circle. Ensure that the latex
and saline do not mix at this stage.
4) Using a loop, pick off a portion of the colony to be tested and carefully emulsify in the
saline drop.
5) Mix the Test latex and suspension together and spread to cover most of the reaction area
using the loop. Flame the loop.
6) Rock the slide in a circular motion, observing for agglutination. Do not rock the card for
more than 1 minute and do not use a magnifying glass.
7) If no agglutination occurs, then proceed to te st other NSF colonies if these are present.
8) If agglutination with the test reagent does o ccur, then it is necessary to test a further
portion of the colony with the control reagent to ensure that the isolate is not an
autoagglutinating strain.
9) When finished, dispose of the reaction slide into disinfectant.
Interpretation
a) Positive result - Agglutination of th e Test latex occurs within 1 minute.
No agglutination of the Control latex. *Perform
biochemical tests to confirm that the organism is an E. coli
strain.
b) Negative result - no agglutination of the Test latex.
c) Non-interpretable result - clumping of the Control latex.
References
1. Borczyk A., Lior H., Crebin B. 1987. Int. J. Food. Microbiol. 4, 347-349.
2. Konowalchuk J., Speirs J. and Stavric S. 1977. Infect. Immune. 18, 775-779.
3. Scotland S., Day N. and Rowe B. 19 80. FEMS Microbiol. Lett. 7, 15-17.
4. Centers for Disease Control. 1982. Morbid Mortal Wkly. 31, 580-585.
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Technical Manual
5. Karmali M., Steel B., Petric M. and Lim C. 1983. Lancet 8, 619-620.
6. Johnson W., Lior H. and Bezanson. 1983. Lancet 8, 76.
7. March S. and Ratnam. 1986. J. Clin. Microbiol. 23, 869-872.
8. Krishnan C., Fitzgerald V., Dakin S. and Behme R. 1987. J. Clin. Microbiol. 25, 1043-1047.
PROCEDURE MANUAL
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Policy # MI\TECH\16\v01 Page1 of 2
Section: Technical Manual Subject Title: Germ Tube Test
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
GERM TUBE TEST
Principle
This is a rapid test for the presumptive identification of C. albicans.
Reagents
Bovine serum
A small volume to be used as a working solution may be stored
at 2 to 8
o
C. Stock soluti on can be dispensed into small
tubes and stored at -20
o
C.
Other Materials
Clean glass microscope slides
Glass coverslips
Vitek tubes (13 x 100 mm)
Pasteur pipettes
Procedure
1. Put 3 drops of serum into a small Vitek tube.
2. Using a Pasteur pipette, touch a colony of yeas t and gently emulsify it in the serum. The
pipette can be left in the tube.
3. Incubate at 37
o
C for 2-4 hours but no longer.
4. Transfer a drop of the serum to a slide for examination.
5. Coverslip and examine microscopically using x 40 objective.
Interpretation
Germ tubes are appendages half the width and 3 to 4 times the length of the ye ast cell from which
they arise. There is no co nstriction between the yeast cell and the germination tube.
Positive test: presence of short lateral filament s (germ tubes)
Negative test: yeast cells only
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Precaution
C. tropicalis may form pseudohyphae which may be falsely interpreted as germ tubes.
Quality Control
Set up known controls each time a test is run.
Positive: C. albicans (ATCC 10231)
Negative: C. tropicalis (ATCC 13803)
Reference
1. Murray PA, et al. Manual of Clinical Microbiology, 7
th
ed., 1999; pp. 1189-1191.
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