A book about the biological laboratory fourth penalty
References
1. MacFaddin JF, Bioche mical Tests for Identification of Medical Bacter ia, 2nd ed., Williams
and Wilkins, Baltimore MD., 1980, p51-58.
2. Murray PA, et al. Manual of Clinical Microbiology, 7th ed ., 1999; pp 426-427.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 41
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\10\v01 Page 1 of 1
Section: Technical Manual Subject Title: Cetrimide Pseudomonas
Selective Agar
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
CETRIMIDE PSEUDOMONAS SELECTIVE AGAR
Principle
Cetrimide Selective Agar is used for the identification of Pseudomonas aeruginosa. Cetrimide is a
compound that has germ icidal activity against most organisms except Pseudomonas aeruginosa.
Also pigment production is enhanced on this media.
Procedure
1. Divide the plate into approximately 8 pie shaped divisions.
2. Streak the test organism (pure culture) onto one of the pie shaped divisions.
3. Incubate at 35
0
C for 18 - 24 hours.
Interpretation
Pseudomonas aeruginosa will grow on this media and will be pigmen ted a pale green to dark blue-green colour. All other organisms will not grow or will be non-pigmented.
Quality Control
Test with positive and nega tive controls ea ch time the test is set up.
Positive: Pseudomonas aeruginosa (ATCC 27853)
Negative: Escherichia coli (ATCC 25922)
Reference
1. PML Technical Manual, 1990.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 42
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\11\v01 Page 1 of 6
Section: Technical Manual Subject Title: Cryptococcal Antigen
Issued by: LABORATORY MANAGER Original Date: March 20, 2000
Approved by: Laboratory Director
Revision Date: July 26, 2000
CRYPTOCOCCAL ANTIGEN
Latex particles coated with anti-cryptococcal globulin (ACGR) reacts with cryptococcal
polysaccharide antigen (in CSF or serum) causing a visible agglutination.
I. Specimen Collection and Processing
5 mL of blood is collected in a serum separator tube and separated by centrifugation. The
serum is removed to a vial and refrigerated until te sting. Specimens are stored a
-70o
C after testing.
Spinal fluid is collected in clean, sterile, cent rifuge tubes. Specimens are stored refrigerated
after testing.
Note: Fungus culture sh ould also be set up.
II. Procedure
Reagents
Meridian CALAS (Cryptococcal Antigen Latex Agglutination System)
1. GBDA - Glycine buffe red diluent with albumin.
2. ACGR - Anti-cryptococcal globulin reagent.
3. NGR - Normal globulin reagent.
4. AGC - Antiglobulin contro l. Rehydrate with 1.5 mL dH
2
O.
5. NC - Negative control. Rehydrate with 2.5 mL dH
2
O and inactivate the negative
at 56
o
C for 30 minutes .
6. CAC - Cryptococcal antigen control (Positive control).
7. Pronase - Rehydrate with 2.5 mL dH
2
O.
Note: Ensure that all reconstituted vials are thoroughly dissolved before use
All reagents are stored refri gerated. Do not interchange reagents with a kit having a
different lot number. Allow reag ents to warm to room temperature before use. Mix gently
before use.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 43
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\11\v01 Page 2 of 6
Section: Technical Manual Subject Title: Cryptococcal Antigen
Other Materials
Boiling water bath
56o
C heating block
1.0 x 0.1 mL pipettes
Rotator
Small serologic test tubes
Test tube rack
Marking pen
Applicator sticks
The following are provided by Meridian:
Capillary pipettes
Rubber bulb
Ring slide
Method
Specimen preparation:
1. Store refrigerated if testing is not done immediately.
(a) Inactivate serum by mixing 500 µL of serum and 500 µL of pronase solution
in a 12 x 75 mm tube and incubate at 56
o
C for 15 minutes. Further
inactivate in a boiling water bath for 5 minutes. This constitutes a 1:2
dilution.
(b) Centrifuge CSF at 3500 rpm for 15 mins. Inactivate the supernatant in a
boiling water bath for 5 minutes.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 44
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\11\v01 Page 3 of 6
Section: Technical Manual Subject Title: Cryptococcal Antigen
Performing the tests:
Note: Controls must be run each time a patient specimen is tested.
1. Set up and label the slide as follows:
2. Gently resuspend the la tex particles in the ACGR an d NGR reagents. Rock each
reagent just prior to use.
Place one drop of ACGR or NGR into the designated rings.
3. Place 25 µL (one drop) of the cryptococcal antigen control (CAC) into the
designated rings. Repeat with the negativ e control (NC) and anti-globulin control
(AGC)
4. Place 25 µL of specimen in the designated rings.
5. Using a separate applicator stick, mi x the contents of each ring thoroughly,
spreading the contents over the entire surface area.
N
G
R
A
C
G
R
CAC NC AGC TEST
(POS (NEG (Anti-
Control) Control) globulin
Control)
Not
Used
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 45
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\11\v01 Page 4 of 6
Section: Technical Manual Subject Title: Cryptococcal Antigen
6. Place the slide on the rotator and rotate at 125 rpm for 5 minutes.
7. Read the reactions immediately.
8. Rate the agglutination as follows:
Positive = any evidence of agglutination (granula tion or clumping)
Negative = a homogenous suspension of particles with no visible clumping.
9. Patient specimens showi ng any agglutination in ACGR should be titrated against
both ACGR and NGR reagents.
(a) Prepare two-fold serial dilutions of the specimen using 200 µL of GBDA in
each of 8 test tubes labelled as follows:
Tube 1 2 3 4 5 6 7 8
Serum 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512
CSF 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256
(b) Transfer one drop of each dilution into 2 rings.
(c) Add one drop of ACGR to one ring of each dilution.
(d) Add one drop of NGR to each of the other rings.
(e) Mix using separate applicator sticks.
(f) Place the slide on the rotator and rotate at 125 rpm for 5 minutes.
(g) Read the results as follows:
1+ = fine granulation against a milky background
2+ = small but definite clumps against a slightly cloudy background
3+ = large and small clumps against a clear background
4+ = large clumps against a clear background
(h) If tube #8 gives an agglutination of 2+ or
greater, the specimen must be further serially
diluted until a titre may be obtained.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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