Book on Biological Laboratory penalty 13
Page 96
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\37\v01 Page 1 of 1
Section: Technical Manual Subject Title: RapID ANA II System
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
RapID ANA II SYSTEM
Principle
The RapID ANA II System is a qualitative micromethod employing conventional and
chromogenic substrate for the identification of medically important anaerobic bacteria of human
origin.
The tests used in it are based upon the microbial degradation of specifi c substrate detected by
various indicator systems. The reactions are a combination of conventional tests and single-substrate chromogenic tests.
Materials
1. RapID ANA II panels
2. Suspension fluid
3. Kovacs spot indole reagent
4. RapID ANA II reagent
5. RapID ANA ID forms
Procedure
Make an equivalent McFarland #3 turbidity suspension of 18-24 hours AnO
2
culture (not more
than 72 hours) in the supplied suspension fluid. Mix it thoroughly - can be used up to 15
minutes. Inoculate an agar (BA FAA) plate for purity and incubate for 24 hours anaerobically.
Peel the lid off the panel marked "peel to inoculate". Using th e Pasteur pipette, transfer the
entire contents into the right uppe r corner of the panel. Seal the panel. Level the contents in the
panel and slowly tilt the panel so that every chamber receives an equal amount of suspension.
Incubate the panel at least four hour s (not more than six hours) in non-CO
2
incubator at 35-37
0
C.
After the incubation period, read the panel prior to adding the r eagents and write results on the
ID form. Add the reagents as per instructions. Allow 30 seconds but not more than two minutes.
Read it and score on the form.
Interpretation and Identification
Please follow the guidelines from the manufact urer and see the RapID ANA II ID Code Book.
See RapID ANA II System Insert #iii08-1/94 brochure which follows.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 97
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\38\v01 Page 1 of 2
Section: Technical Manual Subject Title: RapID MGP Test
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
RapID MGP TEST
Principle
Rapid MGP Medium is a 5 hour test for the differentiation of Enterococcus faecium and E.
faecalis from Enterococcus gallinarum and E. casseliflavus based on the ability to acidify the
carbohydrate methyl-glucopyranoside (MGP).
Reagents
Rapid MGP Medium (Hardy Diagnostics)
Bacteriology loop
Procedure
1. Using a sweep of colonies from an 18-24 hour pure culture of the organism to be tested,
stab the MGP media with the loop. There should be a visible cell paste on the loop as the
media is inoculated.
2. Incubate aerobically at 35
0
C for 5 hours.
3. Observe for the development of a yellow colour along the stab line indicating a positive
test.
4. Reincubate weak reactions for 24 hours.
Interpretation
Positive: yellow colour along stab line
Negative: colour remains blue
E. casseliflavus +
E. gallinarum +
E. faecalis -
E. faecium -
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 98
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\38\v01 Page 2 of 2
Technical Manual
Quality Control
Positive and negative controls are run each time the test is set up.
Positive: E. casseliflavus (ATCC 12755)
Negative: E. faecalis (ATCC 19966)
Reference
1. Hardy Diagnostics package insert 1999.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 99
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\39\v01 Page 1 of 1
Section: Technical Manual Subject Title: RapID VP Test
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
RapID VP TEST
Purpose
To aid in the identification of S. milleri.
Media
MR - VP broth
Procedure
1. Transfer approximately 0.2 ml of VP broth into a sterile 13 x 100 test tube.
2. Using a sterile inoculating wi re inoculate the test organi sm heavily into the broth.
3. Incubate the tube at 35
0
C for 5 hours.
4. After incubation add 1 drop of alpha-naphthol and 1 drop of 40% KOH.
5. Shake the tube gently for one minute to expose the medium to air. Allow 10-15 minutes
for reaction to develop.
Interpretation
Positive - Red colour
Negative - No colour change within 10-15 minutes
Precautions
The order of adding reagents is important; alpha-naphthol followed by 40% KOH.
Reference
Ruoff, K.L., Ferraro, M.J. 1986. Presumptive identification of S. milleri in 5h J Clin Microbiol
24:495-497.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 100
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\40\v01 Page 1 of 2
Section: Technical Manual Subject Title: RapID Yeast Plus Test
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
RapID YEAST PLUS TEST
Purpose
Used for the identification of yeast and yeast like organisms.
Materials
Rapid Yeast Plus Panel
Rapid Yeast Plus Reagent A and Reagent B
Rapid ID Inoculating fluid 2ml
Pasteur Pipettes
Cotton swabs
Procedure
1. Use a cotton swab suspend sufficient growth of the yeast in the Inoculating fluid to give a
suspension heavy enough to obliterate the black lines on the inoculation card.
2. Peel back the panel lid over the inoculation port by pulling the tab marked "Peel to
inoculate".
3. Using a Pasteur pipette transfer the entire contents of the inoculation fluid into the upper
right hand corner of the panel and then reseal the panel.
4. Tilt the panel back away from the biochemical wells at approx. a 45% angle.
5. While tilting back gently rock the panel from side to side to evenly distribute the
inoculum along the rear baffles.
6. Slowly tilt the panel forward toward the reaction cavities until th e inoculum flows along
the baffles into the biochemical wells.
7. Incubate panel at 30
0
C for 4 hours.
8. After incubation peel the label lid from over the reaction cavities.
9. Add 1 drop of reagent A to cavities 7 to 14 inclusive.
10. Add 1 drop of reagent B to cavities 16-18 inclusive.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 101
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\40\v01 Page 2 of 2
Technical Manual
11. Read results after 30 seconds but no more th an 1 minute after addi ng reagent. Record
results onto supplied report form, and look results up in the Rapid ID Yeast Plus code
book for interpretation.
Interpretation
Well # Positive Negative
1 to 5
Yellow Blue to green
6
Yellow Red, pink, orange, gold
7 to 14
Any yellow Clear or cream
15
Red or dark red-orange Yellow, yellow-orange or orange
16-18
Purple, red or dark pink Clear straw, orange, pale to medium pink
Quality Control
Control strains are set up for each new lot number of panels.
References
1. Rapid ID yeast plus package insert issue #7/98.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 102
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\41\v01 Page 1 of 2
Section: Technical Manual Subject Title: SIM (Sulfide-Indole-Motility)
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
SIM (SULFIDE-INDOLE-MOTILITY)
Principle
1. To determine the ability of an organism to liberate hydrogen sulfide (H
2
S) from sulphur-bearing amino acids pr oducing a visible, black colour reaction.
2. To determine the ability of an organism to split indole from the tryptophan molecule.
3. To determine if the organism is motile or non-motile.
This test is used, in conjunction with others, for the identification of Enterobacteriaceae when
unable to identify usin g VITEK or API system.
Reagents
Kovac's Reagent
Other Materials
SIM Medium.
Inoculating wire or sterile glass pasteur pipette.
Procedure
1. With a pasteur pipette, draw up a smal l amount of previously inoculated TSB.
2. Stab vertically into the medium to within 1/4 to 1/2 inch fr om bottom: withdraw inoculating
needle following lin e of inoculation.
3. Incubate O
2, 35
o
C X 18-24 hours.
4. Add a few drops of Kovac's reagent and observe for development of a red colour.
Interpretation
H2
S production
(a) Positive: any blackeni ng of the medium
(b) Negative: no blackening
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 103
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\41\v01 Page 2 of 2
Technical Manual
Motility
(a) Positive: motile organisms migrate from the stab line and diffuse into the
medium causing turbidity. They may exhibit fuzzy streaks of
growth. (Compare with an uninoculated tube.)
(b) Negative: bacterial growth accentuated along stab line; surrounding medium
remains clear.
Summary:
Results are recorded as follows. Remember that H
2
S is first, then indole and finally
motility.
-/-/- no H2
S, indole neg, non motile
-/-/+ no H2
S, indole neg, motile
-/+/- no H
2
S, indole pos, non motile
-/+/+ no H
2
S, indole pos, motile
+/-/- H2
S, indole neg, non motile
+/-/+ H2
S, indole neg, motile
+/+/- H2
S, indole pos, non motile
+/+/+ H2
S, indole pos, motile
Refer to Manual of Clinical Microbio logy for specific organism reactions.
Precautions
1. An H
2
S-producing organism may exhibit blackening on SIM medium, but none on TSI
medium.
2. Some H
2
S inhibition occurs when the temperature exceeds 34
o
C.
3. Many bacteria are motile at one temperature and non -motile when at another.
4. If a motility test is difficult to interpret, compare with an uninoculated motility tube. If still
in doubt, perform a wet prep or hanging drop pr eparation using a heavy loopful of an 18-24
hr culture.
Quality Control
Quality control must be performed on each new lot of SIM before bein g put into general use.
K. pneumoniae (ATCC 13883): -/-/-
P. vulgaris (ATCC 13315): +/+/+
References
1. MacFaddin JF, Bioche mical Tests for identifi cation of Medical Bacteria, 2nd ed., Williams
and Wilkins, Baltimore MD, 1980, p162-173, 173- 183, 214-218.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 96
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\37\v01 Page 1 of 1
Section: Technical Manual Subject Title: RapID ANA II System
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
RapID ANA II SYSTEM
Principle
The RapID ANA II System is a qualitative micromethod employing conventional and
chromogenic substrate for the identification of medically important anaerobic bacteria of human
origin.
The tests used in it are based upon the microbial degradation of specifi c substrate detected by
various indicator systems. The reactions are a combination of conventional tests and single-substrate chromogenic tests.
Materials
1. RapID ANA II panels
2. Suspension fluid
3. Kovacs spot indole reagent
4. RapID ANA II reagent
5. RapID ANA ID forms
Procedure
Make an equivalent McFarland #3 turbidity suspension of 18-24 hours AnO
2
culture (not more
than 72 hours) in the supplied suspension fluid. Mix it thoroughly - can be used up to 15
minutes. Inoculate an agar (BA FAA) plate for purity and incubate for 24 hours anaerobically.
Peel the lid off the panel marked "peel to inoculate". Using th e Pasteur pipette, transfer the
entire contents into the right uppe r corner of the panel. Seal the panel. Level the contents in the
panel and slowly tilt the panel so that every chamber receives an equal amount of suspension.
Incubate the panel at least four hour s (not more than six hours) in non-CO
2
incubator at 35-37
0
C.
After the incubation period, read the panel prior to adding the r eagents and write results on the
ID form. Add the reagents as per instructions. Allow 30 seconds but not more than two minutes.
Read it and score on the form.
Interpretation and Identification
Please follow the guidelines from the manufact urer and see the RapID ANA II ID Code Book.
See RapID ANA II System Insert #iii08-1/94 brochure which follows.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 97
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\38\v01 Page 1 of 2
Section: Technical Manual Subject Title: RapID MGP Test
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
RapID MGP TEST
Principle
Rapid MGP Medium is a 5 hour test for the differentiation of Enterococcus faecium and E.
faecalis from Enterococcus gallinarum and E. casseliflavus based on the ability to acidify the
carbohydrate methyl-glucopyranoside (MGP).
Reagents
Rapid MGP Medium (Hardy Diagnostics)
Bacteriology loop
Procedure
1. Using a sweep of colonies from an 18-24 hour pure culture of the organism to be tested,
stab the MGP media with the loop. There should be a visible cell paste on the loop as the
media is inoculated.
2. Incubate aerobically at 35
0
C for 5 hours.
3. Observe for the development of a yellow colour along the stab line indicating a positive
test.
4. Reincubate weak reactions for 24 hours.
Interpretation
Positive: yellow colour along stab line
Negative: colour remains blue
E. casseliflavus +
E. gallinarum +
E. faecalis -
E. faecium -
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 98
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\38\v01 Page 2 of 2
Technical Manual
Quality Control
Positive and negative controls are run each time the test is set up.
Positive: E. casseliflavus (ATCC 12755)
Negative: E. faecalis (ATCC 19966)
Reference
1. Hardy Diagnostics package insert 1999.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 99
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\39\v01 Page 1 of 1
Section: Technical Manual Subject Title: RapID VP Test
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
RapID VP TEST
Purpose
To aid in the identification of S. milleri.
Media
MR - VP broth
Procedure
1. Transfer approximately 0.2 ml of VP broth into a sterile 13 x 100 test tube.
2. Using a sterile inoculating wi re inoculate the test organi sm heavily into the broth.
3. Incubate the tube at 35
0
C for 5 hours.
4. After incubation add 1 drop of alpha-naphthol and 1 drop of 40% KOH.
5. Shake the tube gently for one minute to expose the medium to air. Allow 10-15 minutes
for reaction to develop.
Interpretation
Positive - Red colour
Negative - No colour change within 10-15 minutes
Precautions
The order of adding reagents is important; alpha-naphthol followed by 40% KOH.
Reference
Ruoff, K.L., Ferraro, M.J. 1986. Presumptive identification of S. milleri in 5h J Clin Microbiol
24:495-497.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 100
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\40\v01 Page 1 of 2
Section: Technical Manual Subject Title: RapID Yeast Plus Test
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
RapID YEAST PLUS TEST
Purpose
Used for the identification of yeast and yeast like organisms.
Materials
Rapid Yeast Plus Panel
Rapid Yeast Plus Reagent A and Reagent B
Rapid ID Inoculating fluid 2ml
Pasteur Pipettes
Cotton swabs
Procedure
1. Use a cotton swab suspend sufficient growth of the yeast in the Inoculating fluid to give a
suspension heavy enough to obliterate the black lines on the inoculation card.
2. Peel back the panel lid over the inoculation port by pulling the tab marked "Peel to
inoculate".
3. Using a Pasteur pipette transfer the entire contents of the inoculation fluid into the upper
right hand corner of the panel and then reseal the panel.
4. Tilt the panel back away from the biochemical wells at approx. a 45% angle.
5. While tilting back gently rock the panel from side to side to evenly distribute the
inoculum along the rear baffles.
6. Slowly tilt the panel forward toward the reaction cavities until th e inoculum flows along
the baffles into the biochemical wells.
7. Incubate panel at 30
0
C for 4 hours.
8. After incubation peel the label lid from over the reaction cavities.
9. Add 1 drop of reagent A to cavities 7 to 14 inclusive.
10. Add 1 drop of reagent B to cavities 16-18 inclusive.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 101
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\40\v01 Page 2 of 2
Technical Manual
11. Read results after 30 seconds but no more th an 1 minute after addi ng reagent. Record
results onto supplied report form, and look results up in the Rapid ID Yeast Plus code
book for interpretation.
Interpretation
Well # Positive Negative
1 to 5
Yellow Blue to green
6
Yellow Red, pink, orange, gold
7 to 14
Any yellow Clear or cream
15
Red or dark red-orange Yellow, yellow-orange or orange
16-18
Purple, red or dark pink Clear straw, orange, pale to medium pink
Quality Control
Control strains are set up for each new lot number of panels.
References
1. Rapid ID yeast plus package insert issue #7/98.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 102
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\41\v01 Page 1 of 2
Section: Technical Manual Subject Title: SIM (Sulfide-Indole-Motility)
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
SIM (SULFIDE-INDOLE-MOTILITY)
Principle
1. To determine the ability of an organism to liberate hydrogen sulfide (H
2
S) from sulphur-bearing amino acids pr oducing a visible, black colour reaction.
2. To determine the ability of an organism to split indole from the tryptophan molecule.
3. To determine if the organism is motile or non-motile.
This test is used, in conjunction with others, for the identification of Enterobacteriaceae when
unable to identify usin g VITEK or API system.
Reagents
Kovac's Reagent
Other Materials
SIM Medium.
Inoculating wire or sterile glass pasteur pipette.
Procedure
1. With a pasteur pipette, draw up a smal l amount of previously inoculated TSB.
2. Stab vertically into the medium to within 1/4 to 1/2 inch fr om bottom: withdraw inoculating
needle following lin e of inoculation.
3. Incubate O
2, 35
o
C X 18-24 hours.
4. Add a few drops of Kovac's reagent and observe for development of a red colour.
Interpretation
H2
S production
(a) Positive: any blackeni ng of the medium
(b) Negative: no blackening
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 103
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\41\v01 Page 2 of 2
Technical Manual
Motility
(a) Positive: motile organisms migrate from the stab line and diffuse into the
medium causing turbidity. They may exhibit fuzzy streaks of
growth. (Compare with an uninoculated tube.)
(b) Negative: bacterial growth accentuated along stab line; surrounding medium
remains clear.
Summary:
Results are recorded as follows. Remember that H
2
S is first, then indole and finally
motility.
-/-/- no H2
S, indole neg, non motile
-/-/+ no H2
S, indole neg, motile
-/+/- no H
2
S, indole pos, non motile
-/+/+ no H
2
S, indole pos, motile
+/-/- H2
S, indole neg, non motile
+/-/+ H2
S, indole neg, motile
+/+/- H2
S, indole pos, non motile
+/+/+ H2
S, indole pos, motile
Refer to Manual of Clinical Microbio logy for specific organism reactions.
Precautions
1. An H
2
S-producing organism may exhibit blackening on SIM medium, but none on TSI
medium.
2. Some H
2
S inhibition occurs when the temperature exceeds 34
o
C.
3. Many bacteria are motile at one temperature and non -motile when at another.
4. If a motility test is difficult to interpret, compare with an uninoculated motility tube. If still
in doubt, perform a wet prep or hanging drop pr eparation using a heavy loopful of an 18-24
hr culture.
Quality Control
Quality control must be performed on each new lot of SIM before bein g put into general use.
K. pneumoniae (ATCC 13883): -/-/-
P. vulgaris (ATCC 13315): +/+/+
References
1. MacFaddin JF, Bioche mical Tests for identifi cation of Medical Bacteria, 2nd ed., Williams
and Wilkins, Baltimore MD, 1980, p162-173, 173- 183, 214-218.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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