Book on Biological Laboratory penalty 14
Page 104
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\42\01\v01 Page 1 of 2
Section: Technical Manual Subject Title: Acid Fast Stain for
Mycobacteria (Kinyoun)
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
ACID FAST STAIN FOR MYCOBACTERIA (KINYOUN)
Principle
To stain Mycobacteria present in specimens and cultures.
Mycobacteria are different to stai n with common aniline dyes. However, they will stain with basic
fuschsin. Once stained, they retain the dye de spite treatment wi th mineral acid s i.e. HCl H
2SO4
.
This property of acid fastness may be due to a lipd fraction called mycolic acid. Mycobacteria also
exhibit degrees of resistance to decolourization with alcohol.
Materials
Kinyoun Carbol fuchsin
3% HCl in 95% ethanol
Brilliant green
Procedure
1. Prepare smear over an area of 2-3 sq. cm.
2. Heat fix smear on heating block (56
0
C/1 hr). Then hold to incinerator for 10 secs.
3. Place slide on stain rack and allow to cool. Flood with Kinyoun Carbol fuschsin for 5 min.
4. Rinse off stain with water.
5. Decolourize with 3% acid alcohol for 3 mins.
6. Rinse with water.
7. Repeat decolourization for 1-2 mins. or until no red appears.
8. Rinse with water.
9. Flood slide 3-4 mins. with Brilliant green.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 105
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\42\01\v01 Page 2 of 2
Technical Manual
10. Rinse with water.
11. Air dry. DO NOT BLOT.
Microscopy
Place a drop of oil between the specimen and coverslip and oil again on top. Smears are examined
with oil immersion lens. The coverslip prevents cr oss contamination. An average of 15 mins. is
spent on each slide. The total area of the spec imen must be examined.
References
1. Baker, F.J., Breach, M.R. 1980. Medical Microb iological Technique, p. 15
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 106
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\42\02\v01 Page 1 of 2
Section: Technical Manual Subject Title: Acid fast stain for Nocardia
(Modifed Kinyoun)
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
ACID FAST STAIN FOR NOCARDIA (MODIFED KINYOUN)
Principle
Nocardia species possess the unique characteristic of resisting d ecolorization with acid alcohol.
Reagents
1. Carbol-fuchsin
Basic fuchsin solution (3 g basic fu chsin in 100 mL 95% ethyl alcohol)
10 mL
Phenol 5% aqueous 90 mL
2. Decolourizer (1% sulfuric acid)
H2 SO4
(concentrated) 1 mL
Distilled water 99 mL
3. Methylene blue
Methylene blue 0.3 g
Distilled water 100 mL
Staining Procedure
1. Fix the smear by gentle heating.
2. Flood the smear with Carbol fuchsin solution.
3. Allow the slide to stand for 5 minutes.
4. Wash the smear with tap water.
5. Decolorize the smear with 1% sulfuric acid until no more colour appears in the washing
(approx. 1 min.).
6. Rinse with tap water.
7. Counterstain with methylene blue about 1 minute.
8. Rinse with tap water and air dry.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 107
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\42\02\v01 Page 2 of 2
Technical Manual
Interpretation
The filaments of Nocardia species and Rhodococcus appear red-stained against a blue
background.
Quality Control
A positive control slide of Nocardia species is stained simultaneously with the clinical
specimens.
References
1. Murray, PA. et al. Manual of Clinical Microbiology, 7
th
edition, 1999 ASM,
Washington, D.C.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 108
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\42\03\v01 Page 1 of 2
Section: Technical Manual Subject Title: Acridine Orange Stain
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
ACRIDINE ORANGE STAIN
Principle
Acridine orange is a fluorescent dye which will bi nd to the nucleic acid of bacteria and other cells.
It is recommended for use for the detection of microorganisms in direct smears. It is useful for the
rapid screening of specimens from normally sterile site s (eg. CSF) and blood smears, or smears
containing proteinaceous material where differentiation of organisms from background material
may be difficult.
Reagents
Acridine Orange spot test dropper. Stored at room temperature.
Absolute Methanol
Procedure
1. Prepare a smear of the specimen to be stained.
2. Allow to air dry.
3. Fix with methanol for 1 to 2 minutes.
4. Hold the dropper upright and squeeze gently to crush the glass ampoule inside the dispenser.
5. Flood the slide with the acridi ne orange and stain for 2 minutes.
6. Rinse thoroughly with tap water and allow to air dry.
7. Examine with a fluorescent microscope using low and oil immersion objectives.
Interpretation
Bacteria and fungus stain bright orange. The background appears black to yellow green.
Leukocytes will stain yellow, orange and red.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 109
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\42\03\v01 Page 2 of 2
Technical Manual
Quality Control
Stain a smear of Streptococcus pneumoniae (ATCC 6303) each time the test is performed.
References
1. Difco Spot Test Acridine Orange Stain package insert, 1984.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 110
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\42\04\v01 Page 1 of 2
Section: Technical Manual Subject Title: Bacto 3-Step Gram Stain
Procedure
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
BACTO 3-STEP GRAM STAIN PROCEDURE
Principle
To be used for problem smears to determine the Gram reaction of organisms.
Materials
3-Step Stabilized Iodine Technique
Bacto Gram Crystal Violet
Bacto Stabilized Gram Iodine
Bacto 3-Step Gram Safranin-S
3-Step Technical Iodine Technique
Bacto Gram Crystal Violet
Bacto Gram Iodine
Bacto 3-Step Gram Safranin-T
Microscope slides
Bunsen burner or methanol
Bacteriological loop
Swabs
Blotting paper
Microscope with oil immersion lens
Bactrol Gram Slide
Bactrol Disks
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 111
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\42\04\v01 Page 2 of 2
Technical Manual
Procedure
1. Flood the fixed smear with primary stain (B acto Gram Crystal Violet) and stain for 1
minute.
2. Remove the primary stain by gently washing with cold tap water.
3. Flood the slide with mordant (Bacto Stabilized Gram Iodine or Bacto Gram Iodine
(traditional formulation) and retain on the slide for 1 minute. (Refer to LIMITATIONS
OF THE PROCEDURE, #5)
4. Wash off the mordant with decolourizer / counterstain (Bacto 3-Step Gram Safranin-S or
Bacto 3-Step Gram Safranin-T). ( NOTE : Do not wash off iodine with water). Add more
decolourizer / counterstain solution to the slide and stain 20-50 seconds.
5. Remove the decolourizer / counterstain solution by gently washing the slide with cold tap
water.
6. Blot with blotting paper or pa per towel or allow to air dry.
7. Examine the smear under an oil immersion lens.
Interpretation
REACTION 3-STEP TECHNIQUE
using either Bacto Gram Safranin-S or Bacto
Gram Safranin-T
Gram-positive
Gram-negative
Purple-black
to purple cells
Red-pink to
fuchsia cells
Quality Control
Run controls daily using 18-24 hour cultures of known gram-positive and gram-negative
microorganisms.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 112
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\42\05\v01 Page 1 of 1
Section: Technical Manual Subject Title: Eosinophil Stain
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
EOSINOPHIL STAIN
Principle
A stain for the detection of eosinophils in clinical specimens.
Reagents
AJP Scientific Eosinophil stain:
Solution I - Eosin Y
Solution II - Buffer Ph 6.5
Solution III - Methylene Blue
Stored at room temperature
Procedure
1. Make a thin smear and spread evenly.
2. Fix slide by air drying or with gentle heat.
3. Cover slide with solution I and leave for 30 seconds.
4. Add solution II to cover s lide. Mix gently and allow to stain for 3 to 5 minutes.
5. Wash off with tap water and drain.
6. Cover slide with solution III and immediately wash off with tap wate r. Drain and air dry.
Interpretation
Eosinophils stain with red cytoplasm and bright red granules.
Reference
1. A.J.P. Scientific INC package insert.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 113
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\42\06\v01 Page 1 of 2
Section: Technical Manual
Subject Title: Fungi-fluor Stain
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
FUNGI-FLUOR STAIN
Principle
The Fungi-Fluor stain is used for the rapid identification of various fungal infections in fresh or
frozen clinical specimens.
The active, fluorescing dye in the staining solution is Cellufluor which is the disodium salt of
4,4'-bis[4-anilino-6-bis-(2-hydroxyethel) amino-s-tr iazin-2-ylamino]-2,2'-stilbenedisulfonic acid.
Fungi-Fluor staining solution is a 0.05% solution of this dye in deionized water with potassium
hydroxide added as a clearing agent. The Fungi-Fluor counter staining solution B is an
aqueous solution of Evans Blue dye used to re duce background fluorescence. Cellufluor binds
nonspecifically to beta-linked polysaccharides found in the cell walls of various organisms such
as chitin and cellulose.
Materials
Staining Solution A
Counterstaining Solution B
Absolute alcohol
Water
Fluorescent Microscope (250-400 nm filter)
Precautions
1. Store in a dark or opaque bottle, tightly sealed, at room temperature.
2. Avoid eye or skin contact: use gloves and protective glasses.
Procedure
1. Prepare smear of specimen and allow to air dry.
2. Fix on the rack with absolute alcohol for 5 mins. until dry. Fixed smears can be held
indefinitely until ready to stain and examine.
3. Add few drops of Fungi-Fluor solution A (Cellufluor) for 1 minute.
4. Rinse gently with tapwater.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 104
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\42\01\v01 Page 1 of 2
Section: Technical Manual Subject Title: Acid Fast Stain for
Mycobacteria (Kinyoun)
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
ACID FAST STAIN FOR MYCOBACTERIA (KINYOUN)
Principle
To stain Mycobacteria present in specimens and cultures.
Mycobacteria are different to stai n with common aniline dyes. However, they will stain with basic
fuschsin. Once stained, they retain the dye de spite treatment wi th mineral acid s i.e. HCl H
2SO4
.
This property of acid fastness may be due to a lipd fraction called mycolic acid. Mycobacteria also
exhibit degrees of resistance to decolourization with alcohol.
Materials
Kinyoun Carbol fuchsin
3% HCl in 95% ethanol
Brilliant green
Procedure
1. Prepare smear over an area of 2-3 sq. cm.
2. Heat fix smear on heating block (56
0
C/1 hr). Then hold to incinerator for 10 secs.
3. Place slide on stain rack and allow to cool. Flood with Kinyoun Carbol fuschsin for 5 min.
4. Rinse off stain with water.
5. Decolourize with 3% acid alcohol for 3 mins.
6. Rinse with water.
7. Repeat decolourization for 1-2 mins. or until no red appears.
8. Rinse with water.
9. Flood slide 3-4 mins. with Brilliant green.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 105
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\42\01\v01 Page 2 of 2
Technical Manual
10. Rinse with water.
11. Air dry. DO NOT BLOT.
Microscopy
Place a drop of oil between the specimen and coverslip and oil again on top. Smears are examined
with oil immersion lens. The coverslip prevents cr oss contamination. An average of 15 mins. is
spent on each slide. The total area of the spec imen must be examined.
References
1. Baker, F.J., Breach, M.R. 1980. Medical Microb iological Technique, p. 15
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 106
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\42\02\v01 Page 1 of 2
Section: Technical Manual Subject Title: Acid fast stain for Nocardia
(Modifed Kinyoun)
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
ACID FAST STAIN FOR NOCARDIA (MODIFED KINYOUN)
Principle
Nocardia species possess the unique characteristic of resisting d ecolorization with acid alcohol.
Reagents
1. Carbol-fuchsin
Basic fuchsin solution (3 g basic fu chsin in 100 mL 95% ethyl alcohol)
10 mL
Phenol 5% aqueous 90 mL
2. Decolourizer (1% sulfuric acid)
H2 SO4
(concentrated) 1 mL
Distilled water 99 mL
3. Methylene blue
Methylene blue 0.3 g
Distilled water 100 mL
Staining Procedure
1. Fix the smear by gentle heating.
2. Flood the smear with Carbol fuchsin solution.
3. Allow the slide to stand for 5 minutes.
4. Wash the smear with tap water.
5. Decolorize the smear with 1% sulfuric acid until no more colour appears in the washing
(approx. 1 min.).
6. Rinse with tap water.
7. Counterstain with methylene blue about 1 minute.
8. Rinse with tap water and air dry.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 107
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\42\02\v01 Page 2 of 2
Technical Manual
Interpretation
The filaments of Nocardia species and Rhodococcus appear red-stained against a blue
background.
Quality Control
A positive control slide of Nocardia species is stained simultaneously with the clinical
specimens.
References
1. Murray, PA. et al. Manual of Clinical Microbiology, 7
th
edition, 1999 ASM,
Washington, D.C.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 108
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\42\03\v01 Page 1 of 2
Section: Technical Manual Subject Title: Acridine Orange Stain
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
ACRIDINE ORANGE STAIN
Principle
Acridine orange is a fluorescent dye which will bi nd to the nucleic acid of bacteria and other cells.
It is recommended for use for the detection of microorganisms in direct smears. It is useful for the
rapid screening of specimens from normally sterile site s (eg. CSF) and blood smears, or smears
containing proteinaceous material where differentiation of organisms from background material
may be difficult.
Reagents
Acridine Orange spot test dropper. Stored at room temperature.
Absolute Methanol
Procedure
1. Prepare a smear of the specimen to be stained.
2. Allow to air dry.
3. Fix with methanol for 1 to 2 minutes.
4. Hold the dropper upright and squeeze gently to crush the glass ampoule inside the dispenser.
5. Flood the slide with the acridi ne orange and stain for 2 minutes.
6. Rinse thoroughly with tap water and allow to air dry.
7. Examine with a fluorescent microscope using low and oil immersion objectives.
Interpretation
Bacteria and fungus stain bright orange. The background appears black to yellow green.
Leukocytes will stain yellow, orange and red.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 109
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\42\03\v01 Page 2 of 2
Technical Manual
Quality Control
Stain a smear of Streptococcus pneumoniae (ATCC 6303) each time the test is performed.
References
1. Difco Spot Test Acridine Orange Stain package insert, 1984.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 110
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\42\04\v01 Page 1 of 2
Section: Technical Manual Subject Title: Bacto 3-Step Gram Stain
Procedure
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
BACTO 3-STEP GRAM STAIN PROCEDURE
Principle
To be used for problem smears to determine the Gram reaction of organisms.
Materials
3-Step Stabilized Iodine Technique
Bacto Gram Crystal Violet
Bacto Stabilized Gram Iodine
Bacto 3-Step Gram Safranin-S
3-Step Technical Iodine Technique
Bacto Gram Crystal Violet
Bacto Gram Iodine
Bacto 3-Step Gram Safranin-T
Microscope slides
Bunsen burner or methanol
Bacteriological loop
Swabs
Blotting paper
Microscope with oil immersion lens
Bactrol Gram Slide
Bactrol Disks
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 111
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\42\04\v01 Page 2 of 2
Technical Manual
Procedure
1. Flood the fixed smear with primary stain (B acto Gram Crystal Violet) and stain for 1
minute.
2. Remove the primary stain by gently washing with cold tap water.
3. Flood the slide with mordant (Bacto Stabilized Gram Iodine or Bacto Gram Iodine
(traditional formulation) and retain on the slide for 1 minute. (Refer to LIMITATIONS
OF THE PROCEDURE, #5)
4. Wash off the mordant with decolourizer / counterstain (Bacto 3-Step Gram Safranin-S or
Bacto 3-Step Gram Safranin-T). ( NOTE : Do not wash off iodine with water). Add more
decolourizer / counterstain solution to the slide and stain 20-50 seconds.
5. Remove the decolourizer / counterstain solution by gently washing the slide with cold tap
water.
6. Blot with blotting paper or pa per towel or allow to air dry.
7. Examine the smear under an oil immersion lens.
Interpretation
REACTION 3-STEP TECHNIQUE
using either Bacto Gram Safranin-S or Bacto
Gram Safranin-T
Gram-positive
Gram-negative
Purple-black
to purple cells
Red-pink to
fuchsia cells
Quality Control
Run controls daily using 18-24 hour cultures of known gram-positive and gram-negative
microorganisms.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 112
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\42\05\v01 Page 1 of 1
Section: Technical Manual Subject Title: Eosinophil Stain
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
EOSINOPHIL STAIN
Principle
A stain for the detection of eosinophils in clinical specimens.
Reagents
AJP Scientific Eosinophil stain:
Solution I - Eosin Y
Solution II - Buffer Ph 6.5
Solution III - Methylene Blue
Stored at room temperature
Procedure
1. Make a thin smear and spread evenly.
2. Fix slide by air drying or with gentle heat.
3. Cover slide with solution I and leave for 30 seconds.
4. Add solution II to cover s lide. Mix gently and allow to stain for 3 to 5 minutes.
5. Wash off with tap water and drain.
6. Cover slide with solution III and immediately wash off with tap wate r. Drain and air dry.
Interpretation
Eosinophils stain with red cytoplasm and bright red granules.
Reference
1. A.J.P. Scientific INC package insert.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 113
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\42\06\v01 Page 1 of 2
Section: Technical Manual
Subject Title: Fungi-fluor Stain
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
FUNGI-FLUOR STAIN
Principle
The Fungi-Fluor stain is used for the rapid identification of various fungal infections in fresh or
frozen clinical specimens.
The active, fluorescing dye in the staining solution is Cellufluor which is the disodium salt of
4,4'-bis[4-anilino-6-bis-(2-hydroxyethel) amino-s-tr iazin-2-ylamino]-2,2'-stilbenedisulfonic acid.
Fungi-Fluor staining solution is a 0.05% solution of this dye in deionized water with potassium
hydroxide added as a clearing agent. The Fungi-Fluor counter staining solution B is an
aqueous solution of Evans Blue dye used to re duce background fluorescence. Cellufluor binds
nonspecifically to beta-linked polysaccharides found in the cell walls of various organisms such
as chitin and cellulose.
Materials
Staining Solution A
Counterstaining Solution B
Absolute alcohol
Water
Fluorescent Microscope (250-400 nm filter)
Precautions
1. Store in a dark or opaque bottle, tightly sealed, at room temperature.
2. Avoid eye or skin contact: use gloves and protective glasses.
Procedure
1. Prepare smear of specimen and allow to air dry.
2. Fix on the rack with absolute alcohol for 5 mins. until dry. Fixed smears can be held
indefinitely until ready to stain and examine.
3. Add few drops of Fungi-Fluor solution A (Cellufluor) for 1 minute.
4. Rinse gently with tapwater.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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