PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES \ MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 1
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\v07 Page 1 of 128
Section: Technical Manual Subject Title: Table of Contents
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Direct or Revision Date: April 20, 2005
Review Date: April 20, 2005
TECHNICAL MANUAL
TABLE OF CONTENTS
ALA (RAPID PORPHYRIN TEST) ............................................................................................4
ANAEROBIC IDENTIFICATION USI NG SPECIAL POTENCY DISKS
Anaerobic Identification by Means of Special Potency Disks......................................................6
SPS Disk for Differentiation of Anaerobic Cocci.........................................................................9
ANAEROBIC/CAMPYLOBACTER JAR SET UP ..................................................................11
API TEST STRIPS
CORYNEBACTERIUM (API CORYNE) ...........................................................................13
ENTEROBACTERIACEAE (API 20E) ................................................................................17
IDENTIFICATION OF NON-ENTERIC GRAM-NEGATIVE RODS (API 20NE) ..……24
NEISSERIA & HAEMOPHILUS (API NH).......................................................................28
API VOICE RESPONSE.......................................................................................................32
BACITRACIN DISK TEST.......................................................................................................32
BETA-LACTAMASE (CEFINASE) TEST...............................................................................35
BILE ESCULIN TEST...............................................................................................................37
BILE SOLUBILITY TEST .........................................................................................................38
CATALASE TEST .....................................................................................................................39
CETRIMIDE PSEUDOMONAS SELECTIVE AGAR .............................................................41
CRYPTOCOCCAL ANTIGEN...................................................................................................42
CRYSTAL MRSA IDENTIFICATION SYSTEM....................................................................48
DENKA MRSA SCREEN ..........................................................................................................50
DNAse TEST ..............................................................................................................................52
E. coli O157 LATEX TEST (OXOID).......................................................................................53
GERM TUBE TEST...................................................................................................................56
GONOGEN (GC COAGGLUTINATION) TEST .....................................................................58
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 2
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\v07 Page 2 of 128
Section: Technical Manual Subject Title: Table of Contents (Cont'd)
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Direct or Revision Date: April 20, 2005
Review Date: April 20, 2005
HIGH LEVEL AMINOGLYCOSIDE TESTING ......................................................................60
HIPPURATE TEST....................................................................................................................62
INDOLE TEST ...........................................................................................................................64
KOEHLER ILLUMINATION....................................................................................................66
KOH STRING TEST ..................................................................................................................67
LAP TEST ............................................................................................................................... ...69
MOTILITY TEST MEDIUM .....................................................................................................71
MUG TEST (PGUA)..................................................................................................................74
NEISSERIA IDENTIFICATION SUGARS ..............................................................................76
ONPG TEST............................................................................................................................... 78
ONPG-PHENYLALANINE-MOTILITY MEDIUM (ONPG-PAM) ........................................80
OPTOCHIN SENSITIVITY TEST............................................................................................82
OXIDASE (API STRIP) .............................................................................................................84
OXIDASE (SPOT TEST DROPPER)........................................................................................86
PASTOREX STAPH PLUS TEST ............................................................................................87
PLATE STREAKING METHODS............................................................................................89
PRO-AMP GLU-AMP TESTS...................................................................................................92
PYR TEST ............................................................................................................................... ...93
QUANTITATION OF ORGANISMS & CELLS ON SMEARS & CULTURE .......................95
RapID ANA II SYSTEM............................................................................................................96
RapID MGP TEST .....................................................................................................................97
RapID VP TEST .........................................................................................................................99
RapID YEAST PLUS TEST.....................................................................................................100
SIM (SULFIDE-INDOLE-MOTILITY)..................................................................................102
STAINING METHODS:
Acid Fast Stain for Mycobacteria (Kinyoun) .......................................................................104
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 3
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\v07 Page 3 of 128
Section: Technical Manual Subject Title: Table of Contents (Cont'd)
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Direct or Revision Date: April 20, 2005
Review Date: April 20, 2005
Acid fast stain for Nocardia (Modifed Kinyoun).................................................................106
Acridine Orange Stain ..........................................................................................................108
Bacto 3-Step Gram Stain Procedure....................................................................................110
Eosinophil Stain ...................................................................................................................112
Fungi-fluor Stain ..............................................................................................................113
Gram Stain ...........................................................................................................................115
STAPHAUREX TEST..............................................................................................................117
STREPTOCOCCAL GROUPING ...........................................................................................119
TETRAZOLIUM REDUCTION TEST (TTC) ........................................................................121
THERMONUCLEASE TEST..................................................................................................122
TRIBUTYRIN TEST ................................................................................................................124
TSI (TRIPLE SUGAR IRON)..................................................................................................125
TUBE COAGULASE TEST ....................................................................................................128
UREA SLANT..........................................................................................................................130
X AND V DISKS FOR IDENTIFICATION OF HAEMOPHILUS ........................................132
XYLOSE FERMENTATION ...................................................................................................134
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 4
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\01\v01 Page 1 of 2
Section: Technical Manual Subject Title: ALA (Rapid Porphyrin
Test)
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
ALA (RAPID PORPHYRIN TEST)
Principle
This test is used for rapidly detecting porphyrin as a means of speciating Haemophilus species.
Enzymes which convert ALA (delta - aminolevulinic acid) to porp hyrins in the biosynthesis of
hemin (X factor) are produced by Haemophilus parainfluenzae but not by H. influenzae. The
production of porphyrins is detected by examination with an ultra-violet (UV) light.
Reagents
BBL TAXO Differentiati on Disks ALA. (Store refrigerated in the da rk. Allow 10-15 minutes
for the container to reach room temperature before opening).
Sterile distilled water
Other Materials
Petri dish
Inoculating loop
Gauze
Long-wave UV lamp
Forceps
Procedure
1. Place one ALA disk for each organism to be tested on the inside of a Petri dish using
forceps.
2. Moisten each disk with one drop of sterile water.
3. Rub a loopful of the test organism onto the moistened disk holding it in place with sterile
forceps.
4. Saturate gauze with water, squeeze out any excess and place it in the petri dish as far away
from the disks as possible.
5. Incubate at 35
o
C.
6. Examine at hourly intervals for 6 hours by re moving the top of the petri dish and exposing
the disks to UV light in a darkened room. NB: Wear UV safety goggles when using the
UV light.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 5
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\01\v01 Page 2 of 2
Technical Manual
Interpretation
A. Positive: Orange-red fluorescence
B. Negative: No fluorescence observed
Precautions
1. Use for differentiating Haemophilus spp. only.
2. Best results are obtained wh en a heavy inoculum is used.
3. ALA is light sensitive. Disks must be protected from light.
Quality Control
Test the following positive and negative co ntrols each time an unknown is tested:
Positive: H. parainfluenza (ATCC 7901)
Negative: H. influenzae (ATCC 35056)
Reference
BBL TAXO Differentiation Disks ALA package insert, 1999. Becton Dickinson Microbiology
Systems.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 6
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\02\v01 Page 1 of 5
Section: Technical Manual Subject Title: Anaerobic Identification Using
Special-Potency Disks
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
ANAEROBE IDENTIFICATION US ING SPECIAL-POTENCY DISKS
Principle
Special potency antimicrobial disks of vancomycin (5 µg), kanamycin (1,000 µg) and colistin
(10 µg) are used as an aid in determining the Gram reaction of anaerobes as well as in
preliminary categorization of some anaerobic genera and species (Table 1). In general, gram
positive organisms are resistant to colistin and susceptible to vancomycin, while most gram
negative organisms are resistant to vancomycin. Th is difference is especially useful with some
Clostridia that consistently stain gram negative.
Table 1 - Anaerobic Identification by Means of Special Potency Disks
Response
1
to Disk:
Type of Organism
Vancomycin
(5 µ g)
Kanamycin
(1,000 µ g)
Colistin
(10 µ g)
Gram negative
Gram positive
B. fragilis group
B. ureolyticus group
Fusobacterium spp.
Porphyromonas spp.
Veillonella spp.
R
S
R
R
S
R
V
R
S
R
S
V
R
S
R
S
1
R - resistant; S - susceptible; V - variable
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 7
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\02\v01 Page 2 of 5
Technical Manual
Materials
A. Reagents
1. Special potency antibiotic disks:
Vancomycin 5 µg
Kanamycin 1,000 µg
Colistin 10 µg
Store a small supply of disks (one carton each) in a tight container with desiccants
at 4
0
C.
2. Brucella or other anaerobic blood agar plate.
B. Supplies
1. Single disk dispenser or forceps
2. Ruler (divided into millimeters)
Procedure
1. Allow the container with disks to reach room temperature before opening it.
2. Subculture the isolate on a BAP. To ensure an even, heavy lawn of growth, streak the
first quadrant back and forth se veral times. Streak the other quadrants to yield isolated
colonies.
3. Place the three antibiotic disks on the first quadrant will apart fr om each other.
4. If you have several organisms to test, first streak all the plates and then add the disks to
them at the same time.
5. Incubate the plate(s) anaer obically for 48-72 hours at 35-37
0
C.
6. Examine for zones of inhibition of growth around the disks.
Interpretation
A. Susceptible: Zone of inhibition of ≥ 10 mm
B. Resistant: Zone of inhibition of < 10 mm
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 8
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\02\v01 Page 3 of 5
Technical Manual
Quality Control
A. Test special potency antibiotic disks by lot when initially received and weekly thereafter.
B. Test Bacteroides fragilis (ATCC 25285), Clostridium perfringens (ATCC 13124), and
Fusobacterium necrophorum (ATCC 25286) as described below under Procedure. The
results should show the following:
1. B. fragilis: resistant to all three antibiotics
2. F. necrophorum: resistant to vancomycin; susceptible to colistin and kanamycin
3. C. perfringens : susceptible to vancomycin and kanamycin and resistant to colistin
C. Record the results on a QC log.
A book about the first penalty Biological Laboratory
TORONTO MEDICAL LABORATORIES \ MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 1
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\v07 Page 1 of 128
Section: Technical Manual Subject Title: Table of Contents
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Direct or Revision Date: April 20, 2005
Review Date: April 20, 2005
TECHNICAL MANUAL
TABLE OF CONTENTS
ALA (RAPID PORPHYRIN TEST) ............................................................................................4
ANAEROBIC IDENTIFICATION USI NG SPECIAL POTENCY DISKS
Anaerobic Identification by Means of Special Potency Disks......................................................6
SPS Disk for Differentiation of Anaerobic Cocci.........................................................................9
ANAEROBIC/CAMPYLOBACTER JAR SET UP ..................................................................11
API TEST STRIPS
CORYNEBACTERIUM (API CORYNE) ...........................................................................13
ENTEROBACTERIACEAE (API 20E) ................................................................................17
IDENTIFICATION OF NON-ENTERIC GRAM-NEGATIVE RODS (API 20NE) ..……24
NEISSERIA & HAEMOPHILUS (API NH).......................................................................28
API VOICE RESPONSE.......................................................................................................32
BACITRACIN DISK TEST.......................................................................................................32
BETA-LACTAMASE (CEFINASE) TEST...............................................................................35
BILE ESCULIN TEST...............................................................................................................37
BILE SOLUBILITY TEST .........................................................................................................38
CATALASE TEST .....................................................................................................................39
CETRIMIDE PSEUDOMONAS SELECTIVE AGAR .............................................................41
CRYPTOCOCCAL ANTIGEN...................................................................................................42
CRYSTAL MRSA IDENTIFICATION SYSTEM....................................................................48
DENKA MRSA SCREEN ..........................................................................................................50
DNAse TEST ..............................................................................................................................52
E. coli O157 LATEX TEST (OXOID).......................................................................................53
GERM TUBE TEST...................................................................................................................56
GONOGEN (GC COAGGLUTINATION) TEST .....................................................................58
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 2
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\v07 Page 2 of 128
Section: Technical Manual Subject Title: Table of Contents (Cont'd)
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Direct or Revision Date: April 20, 2005
Review Date: April 20, 2005
HIGH LEVEL AMINOGLYCOSIDE TESTING ......................................................................60
HIPPURATE TEST....................................................................................................................62
INDOLE TEST ...........................................................................................................................64
KOEHLER ILLUMINATION....................................................................................................66
KOH STRING TEST ..................................................................................................................67
LAP TEST ............................................................................................................................... ...69
MOTILITY TEST MEDIUM .....................................................................................................71
MUG TEST (PGUA)..................................................................................................................74
NEISSERIA IDENTIFICATION SUGARS ..............................................................................76
ONPG TEST............................................................................................................................... 78
ONPG-PHENYLALANINE-MOTILITY MEDIUM (ONPG-PAM) ........................................80
OPTOCHIN SENSITIVITY TEST............................................................................................82
OXIDASE (API STRIP) .............................................................................................................84
OXIDASE (SPOT TEST DROPPER)........................................................................................86
PASTOREX STAPH PLUS TEST ............................................................................................87
PLATE STREAKING METHODS............................................................................................89
PRO-AMP GLU-AMP TESTS...................................................................................................92
PYR TEST ............................................................................................................................... ...93
QUANTITATION OF ORGANISMS & CELLS ON SMEARS & CULTURE .......................95
RapID ANA II SYSTEM............................................................................................................96
RapID MGP TEST .....................................................................................................................97
RapID VP TEST .........................................................................................................................99
RapID YEAST PLUS TEST.....................................................................................................100
SIM (SULFIDE-INDOLE-MOTILITY)..................................................................................102
STAINING METHODS:
Acid Fast Stain for Mycobacteria (Kinyoun) .......................................................................104
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 3
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\v07 Page 3 of 128
Section: Technical Manual Subject Title: Table of Contents (Cont'd)
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Direct or Revision Date: April 20, 2005
Review Date: April 20, 2005
Acid fast stain for Nocardia (Modifed Kinyoun).................................................................106
Acridine Orange Stain ..........................................................................................................108
Bacto 3-Step Gram Stain Procedure....................................................................................110
Eosinophil Stain ...................................................................................................................112
Fungi-fluor Stain ..............................................................................................................113
Gram Stain ...........................................................................................................................115
STAPHAUREX TEST..............................................................................................................117
STREPTOCOCCAL GROUPING ...........................................................................................119
TETRAZOLIUM REDUCTION TEST (TTC) ........................................................................121
THERMONUCLEASE TEST..................................................................................................122
TRIBUTYRIN TEST ................................................................................................................124
TSI (TRIPLE SUGAR IRON)..................................................................................................125
TUBE COAGULASE TEST ....................................................................................................128
UREA SLANT..........................................................................................................................130
X AND V DISKS FOR IDENTIFICATION OF HAEMOPHILUS ........................................132
XYLOSE FERMENTATION ...................................................................................................134
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 4
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\01\v01 Page 1 of 2
Section: Technical Manual Subject Title: ALA (Rapid Porphyrin
Test)
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
ALA (RAPID PORPHYRIN TEST)
Principle
This test is used for rapidly detecting porphyrin as a means of speciating Haemophilus species.
Enzymes which convert ALA (delta - aminolevulinic acid) to porp hyrins in the biosynthesis of
hemin (X factor) are produced by Haemophilus parainfluenzae but not by H. influenzae. The
production of porphyrins is detected by examination with an ultra-violet (UV) light.
Reagents
BBL TAXO Differentiati on Disks ALA. (Store refrigerated in the da rk. Allow 10-15 minutes
for the container to reach room temperature before opening).
Sterile distilled water
Other Materials
Petri dish
Inoculating loop
Gauze
Long-wave UV lamp
Forceps
Procedure
1. Place one ALA disk for each organism to be tested on the inside of a Petri dish using
forceps.
2. Moisten each disk with one drop of sterile water.
3. Rub a loopful of the test organism onto the moistened disk holding it in place with sterile
forceps.
4. Saturate gauze with water, squeeze out any excess and place it in the petri dish as far away
from the disks as possible.
5. Incubate at 35
o
C.
6. Examine at hourly intervals for 6 hours by re moving the top of the petri dish and exposing
the disks to UV light in a darkened room. NB: Wear UV safety goggles when using the
UV light.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 5
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\01\v01 Page 2 of 2
Technical Manual
Interpretation
A. Positive: Orange-red fluorescence
B. Negative: No fluorescence observed
Precautions
1. Use for differentiating Haemophilus spp. only.
2. Best results are obtained wh en a heavy inoculum is used.
3. ALA is light sensitive. Disks must be protected from light.
Quality Control
Test the following positive and negative co ntrols each time an unknown is tested:
Positive: H. parainfluenza (ATCC 7901)
Negative: H. influenzae (ATCC 35056)
Reference
BBL TAXO Differentiation Disks ALA package insert, 1999. Becton Dickinson Microbiology
Systems.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 6
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\02\v01 Page 1 of 5
Section: Technical Manual Subject Title: Anaerobic Identification Using
Special-Potency Disks
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
ANAEROBE IDENTIFICATION US ING SPECIAL-POTENCY DISKS
Principle
Special potency antimicrobial disks of vancomycin (5 µg), kanamycin (1,000 µg) and colistin
(10 µg) are used as an aid in determining the Gram reaction of anaerobes as well as in
preliminary categorization of some anaerobic genera and species (Table 1). In general, gram
positive organisms are resistant to colistin and susceptible to vancomycin, while most gram
negative organisms are resistant to vancomycin. Th is difference is especially useful with some
Clostridia that consistently stain gram negative.
Table 1 - Anaerobic Identification by Means of Special Potency Disks
Response
1
to Disk:
Type of Organism
Vancomycin
(5 µ g)
Kanamycin
(1,000 µ g)
Colistin
(10 µ g)
Gram negative
Gram positive
B. fragilis group
B. ureolyticus group
Fusobacterium spp.
Porphyromonas spp.
Veillonella spp.
R
S
R
R
S
R
V
R
S
R
S
V
R
S
R
S
1
R - resistant; S - susceptible; V - variable
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 7
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\02\v01 Page 2 of 5
Technical Manual
Materials
A. Reagents
1. Special potency antibiotic disks:
Vancomycin 5 µg
Kanamycin 1,000 µg
Colistin 10 µg
Store a small supply of disks (one carton each) in a tight container with desiccants
at 4
0
C.
2. Brucella or other anaerobic blood agar plate.
B. Supplies
1. Single disk dispenser or forceps
2. Ruler (divided into millimeters)
Procedure
1. Allow the container with disks to reach room temperature before opening it.
2. Subculture the isolate on a BAP. To ensure an even, heavy lawn of growth, streak the
first quadrant back and forth se veral times. Streak the other quadrants to yield isolated
colonies.
3. Place the three antibiotic disks on the first quadrant will apart fr om each other.
4. If you have several organisms to test, first streak all the plates and then add the disks to
them at the same time.
5. Incubate the plate(s) anaer obically for 48-72 hours at 35-37
0
C.
6. Examine for zones of inhibition of growth around the disks.
Interpretation
A. Susceptible: Zone of inhibition of ≥ 10 mm
B. Resistant: Zone of inhibition of < 10 mm
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 8
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\02\v01 Page 3 of 5
Technical Manual
Quality Control
A. Test special potency antibiotic disks by lot when initially received and weekly thereafter.
B. Test Bacteroides fragilis (ATCC 25285), Clostridium perfringens (ATCC 13124), and
Fusobacterium necrophorum (ATCC 25286) as described below under Procedure. The
results should show the following:
1. B. fragilis: resistant to all three antibiotics
2. F. necrophorum: resistant to vancomycin; susceptible to colistin and kanamycin
3. C. perfringens : susceptible to vancomycin and kanamycin and resistant to colistin
C. Record the results on a QC log.
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