Book on Biological Laboratory penalty 15
Page 114
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\42\06\v01 Page 2 of 2
Technical Manual
5. Apply coverslip to wetted slide and examine with the fluorescent microscope using the
designated filter. If there is a delay, add distilled water to the coverslip just prior to
examination. Use a fresh tube of water daily.
6. Optional for thicker smears . Add few drops of the counter stain Fungi-Fluor solution B.
Rinse gently with tap water and th en proceed as in step 5 above.
Quality Control
Stain a smear of Candida albicans daily.
Interpretation
Use 20x or 40x objective.
Fungal elements will appear yellow-green agains t a red-orange background when counterstain is
used. Observe for characteristic morphology.
References
1. Manufacturers' Instructions (Data Sheet #316). Fungi-Fluor kit - Polysciences, Inc.,
July 1995
2. Clin. Micro. Newsletter 9:33-36, March 1, 1987.
K.L. McGowna. "Practical Approaches to Diagnosing Fungal Infections in
Immunocompromised Patients".
3. J. Clin. Micro. 28:393-394, Feb. 1990. V.S. Baselski et al. "Rapid Detection of
Pneumocystis carinii in Bronchoalveolar Lavage Sample s by Using Cellofluor Staining".
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 115
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\42\07\v01 Page 1 of 2
Section: Technical Manual Subject Title: Gram Stain
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
GRAM STAIN
Principle
Bacteria can be recognized as gram positive (blue-black/purple) if they retain the primary dye
complex of crystal violet and iodine in the face of attempted decolourization, or as gram negative
(pink) if decolourization occurs as shown by the cell accepting the counterstain safranin.
Generally the mechanism of the Gram stain is: The fixed bacteria are stained with the
triphenylmethane dye, crystal violet. Next the smear is flooded with Grams solution which
oxidatively forms an insoluble complex with the crystal violet. The smear is then flooded with
the organic solvent, acetone-alcohol. Dependi ng on cell permeability the crystal violet-iodine
complex will be washed from Gram negative bact eria in solvent but not from Gram positive
bacteria. Upon counterstaining with safranin, organisms which had been discolorized by the
ethanol (Gram negative) will stain pink. Gram positive organisms which retained the crystal
violet will appear blue-black/purple microscopically.
Materials
Crystal violet solution
Grams Iodine solution
Acetone alcohol
Safranin solution
Procedure
1. Prepare the film on the slide and allow to air dry.
DO NOT HEAT TO DRY FILM.
2. When film is dry, place slide on heating bloc k for several minutes. Slide should be just
warm to your hand.
DO NOT OVERHEAT.
3. Allow slide to cool - this will happen quickly - in just a few seconds.
DO NOT ADD STAIN TO HOT SLIDE.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 116
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\42\07\v01 Page 2 of 2
Technical Manual
4. Flood slide with crystal violet - leave 1 minute.
5. Wash gently with water.
6. Flood slide with Grams Iodine - leave 1 minute.
7. Wash iodine from slide with acetone-alcohol mixture. Add a few more drops of acetone-alcohol until no more colour comes from film - usually 30 seconds.
8. Wash gently with water.
9. Flood slide with safranin - leave 1 minute.
10. Wash gently with water. Clean back of slide with tissue and place slide in tray.
Precaution
1. At no time should the film (smear) be exposed to too much heat. When the specimen is
still wet, heat causes coagulation of the protein resulting in heavy overstaining which
cannot be removed by the decolourizer. A thic k smear will also show more tendency to
"lift off" during staining.
2. Rinsing the Grams Iodine off with the decolorizer gives more stability to the CV-GI
complex and false over decolorizing will not take place.
3. Flooding a hot slide with crystal violet will cause the stain to precipitate and make
decolourizing much more difficult.
Quality Control
It is recommended that controls be run concurrently with unknowns or at least run on a daily
basis using known smears containing Gram positive and Gram negative bacteria.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 117
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\43\v01 Page 1 of 2
Section: Technical Manual Subject Title: Staphaurex Test
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
STAPHAUREX TEST
Principle
A rapid slide latex test for the detection of clumping factor and prot ein A produced by most
strains of S. aureus.
Reagents and Materials
Staphaureux latex suspensi on (store refrigerated)
Disposable reaction cards
Culture loop or wooden applicator stick
Procedure
1. Confirm the identification of a suspect Staphylococcus 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 reagent onto the reaction card.
5. Add 1-3 colonies to the drop, mix well with a loop or wooden applicator stick.
6. Rock the slide for 20 seconds and look for clumping.
7. Discard the slide into a discard container.
Interpretation
Positive test: Clumping within 20 seconds with the sensitized latex particles.
Negative test: No clumping
Precautions
1. False positive results may occur after 20 seconds.
2. False positive agglutination can occur with E. coli and
C. albicans
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 118
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\43\v01 Page 2 of 2
Technical Manual
Quality Control
Test known positive and negative controls daily:
Positive: S. aureus (ATCC 25923)
Negative: S. epidermidis (ATCC 12228)
References
1. Staphaurex Package insert, July 1992.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 119
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\44\v01 Page 1 of 2
Section: Technical Manual Subject Title: Streptococcal Grouping
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
STREPTOCOCCAL GROUPING
Principle
This test is used to determine the Lancefield gro up of an isolate. Latex particles labelled with
specific group antisera will agglutin ate in the presence of the corre sponding antigen after extraction
with nitrous acid.
Reagents
Pro-lab Streptococcal grouping Latex kit.
Other Materials
Droppers
Disposable slides
Wooden stirring sticks
13x100 mm test tubes
Procedure
1. Label one test tube for each isolate.
2. Add one drop of Extracti on Reagent 1 to each tube.
3. Suspend 4 beta-haemolytic colonies in the Extraction Reagent 1.
4. Add 1 drop of Extraction Reagent 2 to each tube.
5. Shake the tube and inc ubate for 2 minutes at RT.
6. Add 7 drops of Extraction Re agent 3 to each tube. Mix th e reaction by vortexing the tube
for 10 - 15 seconds.
7. Dispense one drop of each la tex suspension to be tested onto separate circles on the test
card.
8. Using a pasteur pipette, add one drop of extract to th e latex suspension.
9. Mix the latex and extract wi th the wooden stick using the co mplete area of the circle.
10. Gently rock the card for 2 minutes and lo ok for agglutination.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 120
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\44\v01 Page 2 of 2
Technical Manual
Interpretation
Positive: Strong vi sible agglutination within 2 minutes.
Negative: Milky appearance without visible agglutination.
Precautions
1. False positive reactions have been known to occur with organisms from unrelated genera eg.
E. coli, Klebsiella sp., Pseudomonas sp.
Quality Control
Test reagents are checked weekly.
Each test should be tested with at least one extra grouping latex su spension as a negative control.
Reference
1. Pro-lab Streptococcal Grouping package insert.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 121
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\45\v01 Page 1 of 1
Section: Technical Manual Subject Title: Tetrazolium Reduction
Test (TTC)
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
TETRAZOLIUM REDUCTION TEST (TTC)
Principle
To differentiate between E. faecalis and E. faecium.
E. faecalis reduces the colorless compound (Tetrazolium- chloride) to an insoluble substance -
formozan which is red.
Material
BHI broth/Tetrazolium-chloride (TTC)
Procedure
Inoculate a loopful of an overn ight plate culture to 1 ml of TTC broth. Incubate at 35
0
C and
observe the reaction at 2 hours. If negative, reincubate up to 8 hours / overnight.
Interpretation
Positive - deep magenta
Negative - colourless or faint pink
Quality Control
Positive: E. faecalis (ATCC 29212)
Negative: E. faecium (ATCC 19434)
References
1. J. Gen. Microbiology (1965), 38, 279-287.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 114
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\42\06\v01 Page 2 of 2
Technical Manual
5. Apply coverslip to wetted slide and examine with the fluorescent microscope using the
designated filter. If there is a delay, add distilled water to the coverslip just prior to
examination. Use a fresh tube of water daily.
6. Optional for thicker smears . Add few drops of the counter stain Fungi-Fluor solution B.
Rinse gently with tap water and th en proceed as in step 5 above.
Quality Control
Stain a smear of Candida albicans daily.
Interpretation
Use 20x or 40x objective.
Fungal elements will appear yellow-green agains t a red-orange background when counterstain is
used. Observe for characteristic morphology.
References
1. Manufacturers' Instructions (Data Sheet #316). Fungi-Fluor kit - Polysciences, Inc.,
July 1995
2. Clin. Micro. Newsletter 9:33-36, March 1, 1987.
K.L. McGowna. "Practical Approaches to Diagnosing Fungal Infections in
Immunocompromised Patients".
3. J. Clin. Micro. 28:393-394, Feb. 1990. V.S. Baselski et al. "Rapid Detection of
Pneumocystis carinii in Bronchoalveolar Lavage Sample s by Using Cellofluor Staining".
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 115
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\42\07\v01 Page 1 of 2
Section: Technical Manual Subject Title: Gram Stain
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
GRAM STAIN
Principle
Bacteria can be recognized as gram positive (blue-black/purple) if they retain the primary dye
complex of crystal violet and iodine in the face of attempted decolourization, or as gram negative
(pink) if decolourization occurs as shown by the cell accepting the counterstain safranin.
Generally the mechanism of the Gram stain is: The fixed bacteria are stained with the
triphenylmethane dye, crystal violet. Next the smear is flooded with Grams solution which
oxidatively forms an insoluble complex with the crystal violet. The smear is then flooded with
the organic solvent, acetone-alcohol. Dependi ng on cell permeability the crystal violet-iodine
complex will be washed from Gram negative bact eria in solvent but not from Gram positive
bacteria. Upon counterstaining with safranin, organisms which had been discolorized by the
ethanol (Gram negative) will stain pink. Gram positive organisms which retained the crystal
violet will appear blue-black/purple microscopically.
Materials
Crystal violet solution
Grams Iodine solution
Acetone alcohol
Safranin solution
Procedure
1. Prepare the film on the slide and allow to air dry.
DO NOT HEAT TO DRY FILM.
2. When film is dry, place slide on heating bloc k for several minutes. Slide should be just
warm to your hand.
DO NOT OVERHEAT.
3. Allow slide to cool - this will happen quickly - in just a few seconds.
DO NOT ADD STAIN TO HOT SLIDE.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 116
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\42\07\v01 Page 2 of 2
Technical Manual
4. Flood slide with crystal violet - leave 1 minute.
5. Wash gently with water.
6. Flood slide with Grams Iodine - leave 1 minute.
7. Wash iodine from slide with acetone-alcohol mixture. Add a few more drops of acetone-alcohol until no more colour comes from film - usually 30 seconds.
8. Wash gently with water.
9. Flood slide with safranin - leave 1 minute.
10. Wash gently with water. Clean back of slide with tissue and place slide in tray.
Precaution
1. At no time should the film (smear) be exposed to too much heat. When the specimen is
still wet, heat causes coagulation of the protein resulting in heavy overstaining which
cannot be removed by the decolourizer. A thic k smear will also show more tendency to
"lift off" during staining.
2. Rinsing the Grams Iodine off with the decolorizer gives more stability to the CV-GI
complex and false over decolorizing will not take place.
3. Flooding a hot slide with crystal violet will cause the stain to precipitate and make
decolourizing much more difficult.
Quality Control
It is recommended that controls be run concurrently with unknowns or at least run on a daily
basis using known smears containing Gram positive and Gram negative bacteria.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 117
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\43\v01 Page 1 of 2
Section: Technical Manual Subject Title: Staphaurex Test
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
STAPHAUREX TEST
Principle
A rapid slide latex test for the detection of clumping factor and prot ein A produced by most
strains of S. aureus.
Reagents and Materials
Staphaureux latex suspensi on (store refrigerated)
Disposable reaction cards
Culture loop or wooden applicator stick
Procedure
1. Confirm the identification of a suspect Staphylococcus 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 reagent onto the reaction card.
5. Add 1-3 colonies to the drop, mix well with a loop or wooden applicator stick.
6. Rock the slide for 20 seconds and look for clumping.
7. Discard the slide into a discard container.
Interpretation
Positive test: Clumping within 20 seconds with the sensitized latex particles.
Negative test: No clumping
Precautions
1. False positive results may occur after 20 seconds.
2. False positive agglutination can occur with E. coli and
C. albicans
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 118
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\43\v01 Page 2 of 2
Technical Manual
Quality Control
Test known positive and negative controls daily:
Positive: S. aureus (ATCC 25923)
Negative: S. epidermidis (ATCC 12228)
References
1. Staphaurex Package insert, July 1992.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 119
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\44\v01 Page 1 of 2
Section: Technical Manual Subject Title: Streptococcal Grouping
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
STREPTOCOCCAL GROUPING
Principle
This test is used to determine the Lancefield gro up of an isolate. Latex particles labelled with
specific group antisera will agglutin ate in the presence of the corre sponding antigen after extraction
with nitrous acid.
Reagents
Pro-lab Streptococcal grouping Latex kit.
Other Materials
Droppers
Disposable slides
Wooden stirring sticks
13x100 mm test tubes
Procedure
1. Label one test tube for each isolate.
2. Add one drop of Extracti on Reagent 1 to each tube.
3. Suspend 4 beta-haemolytic colonies in the Extraction Reagent 1.
4. Add 1 drop of Extraction Reagent 2 to each tube.
5. Shake the tube and inc ubate for 2 minutes at RT.
6. Add 7 drops of Extraction Re agent 3 to each tube. Mix th e reaction by vortexing the tube
for 10 - 15 seconds.
7. Dispense one drop of each la tex suspension to be tested onto separate circles on the test
card.
8. Using a pasteur pipette, add one drop of extract to th e latex suspension.
9. Mix the latex and extract wi th the wooden stick using the co mplete area of the circle.
10. Gently rock the card for 2 minutes and lo ok for agglutination.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 120
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\44\v01 Page 2 of 2
Technical Manual
Interpretation
Positive: Strong vi sible agglutination within 2 minutes.
Negative: Milky appearance without visible agglutination.
Precautions
1. False positive reactions have been known to occur with organisms from unrelated genera eg.
E. coli, Klebsiella sp., Pseudomonas sp.
Quality Control
Test reagents are checked weekly.
Each test should be tested with at least one extra grouping latex su spension as a negative control.
Reference
1. Pro-lab Streptococcal Grouping package insert.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 121
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\45\v01 Page 1 of 1
Section: Technical Manual Subject Title: Tetrazolium Reduction
Test (TTC)
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
TETRAZOLIUM REDUCTION TEST (TTC)
Principle
To differentiate between E. faecalis and E. faecium.
E. faecalis reduces the colorless compound (Tetrazolium- chloride) to an insoluble substance -
formozan which is red.
Material
BHI broth/Tetrazolium-chloride (TTC)
Procedure
Inoculate a loopful of an overn ight plate culture to 1 ml of TTC broth. Incubate at 35
0
C and
observe the reaction at 2 hours. If negative, reincubate up to 8 hours / overnight.
Interpretation
Positive - deep magenta
Negative - colourless or faint pink
Quality Control
Positive: E. faecalis (ATCC 29212)
Negative: E. faecium (ATCC 19434)
References
1. J. Gen. Microbiology (1965), 38, 279-287.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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