Book on Biological Laboratory penalty 12
Page 87
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\32\v01 Page 1 of 2
Section: Technical Manual Subject Title: Pastorex Staph Plus Test
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
PASTOREX STAPH PLUS TEST
Principle
A rapid slide latex agglutination test for the detection of clumping factor, capsular
polysaccharide and protein A produced by most strains of S. aureus .
Reagents and Materials
Pastorex test latex suspension (store refrigerated)
Disposable reaction cards
Plastic stick
Procedure
1. Confirm the identification of a suspect Sta phylococcus by Gram stain and catalase test.
2. Allow the latex reagent to warm to room temperature before use.
3. Shake the reagent so that all of the particles are resuspended.
4. Dispense one drop of latex test reagent in one of the circles on the reaction card.
5. Dispense one drop of negative control r eagent in another ci rcle on the card.
6. Emulsify 1 to 3 colonies into the test latex with a loop for 10 seconds.
7. Repeat step 6 for the negative control reagent.
8. Gently rock the card for 30 seconds and look for clumping.
9. Discard the card into a discard container.
Interpretation
Positive test: Clumping within 20 seconds with the test latex particles only.
Negative test: No clumping in either latex.
Uninterpretable test: Clumping in the negative control.
Precautions
1. False positive results may occur after 40 seconds.
2. False positive agglutination can occur with organisms other than staphylococci.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 88
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\32\v01 Page 2 of 2
Technical Manual
Quality Control
Test known positive and negative controls daily.
Positive: S. aureus (ATCC 29213)
Negative: S. epidermidis (ATCC 12228)
References
1. Pastorex Staph Plus package insert Feb. 1999.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 89
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\33\v01 Page 1 of 2
Section: Technical Manual Subject Title: Plate Streaking Methods
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
PLATE STREAKING METHODS
Blood Agar and MacConkey Agar for Urine Cultures
1 uL disposable loop
Inoculate in one continuous streak down the middle of the plate. With the sa me loop, streak out the
entire plate at 90
o
to the initial inocul um. Streak a minimum of 15 lines.
1o
inoculum
Martin-Lewis Agar
Inoculate plate with specimen swab in a "Z" pattern across the plate (with continuous rotation of the
swab while inoculating). Streak out the entire plate with a sterile loop at 90
o
to the initial inoculum.
Streak a minimum of 15 lines.
1o
inoculum
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 90
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\33\v01 Page 2 of 2
Technical Manual
Isoplater Streaking
Manual Streaking
Inoculate specimen with swab or loop onto the entire first quadrant of the agar plate. Use a sterile
loop and streak out the se cond, third and fourth quadrants as per diagram:
1 2
Use a sterile loop
3 4
Growth Quantitation:
+ / -
1 +
2 +
3 +
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 91
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\34\v01 Page 1 of 2
Section: Technical Manual Subject Title: Pro-Amp Glu-Amp Tests
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
PRO-AMP GLU-AMP TESTS
Principle
Rapid chromogenic tests for the identi fication of pathogenic Neisseria.
Reagents
Pro-Amp tablets
Glu-Amp tablets
Fast Blue BB solution
Sterile Saline
Other Materials
Sterile Tubes (13 x 100mm)
Procedure
1. Suspend the growth from Choc media in 2 tubes of 0.25 ml sa line to achieve the turbidity >
#2 McFarland standard.
2. Add 1 tablet to the respective tube.
3. Incubate at 36
o
C x 4 hours.
4. After incubation add 3 drops of Fast Blue BB solution to each tube and read results after 10
minutes.
Interpretation
Positive: Orange/salmon colour
Negative: Yellow colour
Organism Glu-Amp Pro-Amp
N. gonorrhoeae - +
N. meningitidis + v
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 92
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\34\v01 Page 2 of 2
Technical Manual
Quality Control
Test with control organisms when test is run:
N. gonorrhoeae (ATCC 43069)
N. menigitidis (ATCC 13090)
Reference
1. Pro lab package insert, February 1985.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 93
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\35\v01 Page 1 of 2
Section: Technical Manual Subject Title: PYR Test
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
PYR TEST
Principle
PYR (L-pyrrolidonyl- β-naphthylamide) impregnated disks serve as a substrate for the detection
of pyrrolidonyl peptidase. Following the hydrolysis of the substrate by the enzyme the resulting
β -naphthylamine produces a red co lour upon the addition of cinnamald ehyde reagent. This test
is used, in conjunction with others, for the identi fication of catalase negative, gram positive cocci
including Enterococci and Group A Streptococci.
Reagents
PYR discs
Cinnamaldehyde reagent (0.01% p-dimethylamino-cinnamaldehyde)
(disks and reagents are both in PYR kit)
Glass slide
Innoculating loop
Forceps
Sterile distilled water
Procedure
1. Place a PYR disk onto a glass slide and moisten it with one drop of st erile distilled water.
2. Rub a loopful of the culture onto the moistened disk holding it in place with sterile
forceps.
3. Leave at room temperature for 2 minutes.
4. After 2 minutes, add 1 drop of cinnamaldehyde reagent.
Interpretation
Positive: Pink or cherry red colour within one minute
Negative: No colour change or slight yellow colour
Quality Control
Test knows positive and negative controls each time an unknown is run.
Positive: Group A streptococcus (ATCC 19615)
Negative: Group B strept ococcus (ATCC 13813)
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 94
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\35\v01 Page 2 of 2
Technical Manual
Reference
1. Carr-Scarborough Microbiologicals package insert 1990.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 95
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\36\v01 Page 1 of 1
Section: Technical Manual Subject Title: Quantitation of Organisms &
Cells on Smears & Culture
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
QUANTITATION OF ORGANISMS & CELLS ON SMEARS & CULTURE
Microscopic:
Report as:
± --- <1 per oil immersion field ±
+ --- 1 - 5 per oil immersion field +
++ --- 5 - 10 per oil immersion field ++
+++ --- >10 per oil immersion field +++
Culture:
Report as:
± --- few colonies in primary inoculum scant growth
+ --- confluent growth in prim ary inoculum light growth
++ --- growth up to 2nd quadrant moderate growth
+++ --- growth in or >3rd quadrant heavy growth
Note: Quantitation precedes identification.
Size of colonies
lg - large
med - medium
sm - small
tiny - tiny
ppt - pinpoint
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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