Book on Biological Laboratory penalty 16
Page 122
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\46\v01 Page 1 of 2
Section: Technical Manual Subject Title: Thermonuclease Test
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
THERMONUCLEASE TEST
Principle
Staphylococcus aureus contains a heat-stable thermonu clease and coagulase negative
staphylococcus does not. This is a rapid test to differentiate between the two organisms.
Materials
Toluidine blue-O DNA plate (Q-Lab)
13x100 mm tube with white cap
pasteur pipettes
Procedure
1. Dispense 2 - 3 mL of blood broth from BacT/Alert bottle showing gram po sitive cocci in
clusters in the direct Gram stain into a sterile capped 13x100 mm tube.
2. Place tube in heating block, 100
0
C for 15 minutes.
3. Let cool to room temperature.
4. Centrifuge at approximat ely 2500 rpm for 3 minutes.
5. Inoculate a pre-warmed (35
o
C for 1 hour) toluidine blue-O DNA plate by filling wells (cut
well with the end of a pasteur pipette) with 2 drops of the supernatant.
6. Incubate the plate at 35
o
C in the upright positio n (agar side down).
7. Inspect the plate at, 1 hour, 2 hours and 4 hours and again after overnight incubation if
negative at 4 hours.
8. Always run negative and positive cont rol wells with each plate each day.
Interpretation
Positive: Pink zone of clearing at the edge of the well with a darker blue ring at the outer
periphery of the zone; indi cates thermonuclease activity
Negative: No zone or a small clear zone around the well
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 123
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\46\v01 Page 2 of 2
Technical Manual
Quality Control
1. Inoculate 5 day negative patient BacT/Alert bottles with 0.5 mL of a slightly turbid
suspension of (a) S. aureus (ATCC 25923) and (b) S. epidermidis (ATCC 12228) in
trypticase soy broth.
2. Incubate the bo ttles overnight at 36
o
C on the shaker.
3. Remove 3 - 6 mL of the broth-blood from the bottles and process in the same manner as the
patient specimens (steps 1 to 4). Always QC new controls before use with patient specimen.
4. Supernatants may be kept refriger ated for up to 1 month for use as controls.
Reference
1. Rafner, H.B., & Stretton C.W. 1985. Thermonuclease test fo r same day identification of S.
aureus in blood cultures. J. Clin. Microbiol. 21:995-996.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 124
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\47\v01 Page 1 of 1
Section: Technical Manual Subject Title: Tributyrin Test
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
TRIBUTYRIN TEST
Principle
A rapid chromogenic test for the identification of M. catarrhalis.
Reagents
Prolab Tributyrin (TRIB) tablets.
Store at room temperature.
Sterile saline.
Other Materials
Sterile tubes (13 x 100 mm)
Procedures
1. Suspend the growth from C HOC in 0.25 mL (6 drops) saline to achieve the turbidity >#2
McFarland standard.
2. Add 1 tablet to the tube.
3. Incubate at 35
0
C x 4 hours.
4. Examine the tube for development of a yellow colour.
Interpretation
Positive: Yellow/yellow orange colour
Negative: Red
Quality Control
Test the following organism weekly:
Positive: M. catarrhalis (ATCC 8176)
Negative: N. gonorrhoeae (ATCC 43069)
References
1. Prolab package insert, February 1985.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 125
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy #MI\TECH\48\v01 Page 1 of 3
Section: Technical Manual Subject Title: TSI (Triple Sugar Iron)
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
TSI (TRIPLE SUGAR IRON)
Principle
To determine the ability of an organism to attack a specific carbohydrate incorporated in a basal
growth medium, with or without the production of gas, along with the determination of possible
hydrogen sulfide (H
2
S) production. This test is used, in conjunction with others, for the
identification of enteric pathogens.
Materials
TSI Slant
Inoculating wire or sterile glass pasteur pipette.
Procedure
1. Using the an inoculating wire, dip into the previously inoculated TSB.
2. Stab the butt of the TSI to within 1/4 inch from bo ttom, draw out and fishtail over slant. Do
not tighten cap.
3. Incubate O
2, 35
o
C X 18-24 hours.
Interpretation
Carbohydrate utilization:
1. Fermentation of glucose only
(a) slant: red colour (alkaline reaction)
(b) butt: yellow colour (acid reaction)
2. Fermentation of glucose and sucrose and/or lactose
(a) slant: yellow colour (acid reaction)
(b) butt: yellow colour (acid reaction)
3. Neither glucose nor lactose nor sucrose fermented
(a) slant: red colour (alkaline reaction)
(b) butt: (i) aerobic organism
(a) No growth
(b) No colour change
(ii) facultative organism red colour (alkaline reaction)
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\48\v01 Page 2 of 3
Technical Manual
Gas production:
1. Aerogenic:
(a) Gas production: CO2 and H
2
(b) Evident by one of the following:
(i) a single gas bubble
(ii) bubbles in the medium
(iii) splitting of medium
(iv) complete displacement of the medium from bottom of the tube
leaving a clear area
(v) slight indentation of medium from the side of the tube
2. Anaerogenic:
No gas production
H2
S production:
The presence of a black precipita te (ferrous sulfid e) is evident by:
(i) A black colour spread throughout the entire butt masking the
acidity; may even be a slight evidence on the slant
(ii) A black ring near the top of the butt area
(iii) A black precipitate scattered throughout the butt but not entirely
masking the acidity present
Summary:
The ways of recording the TSI reactions are list ed below. Reme mber that the slant is first,
followed by the butt reaction.
acid/acid +/+
acid/acid/gas +/+ with gas
acid/acid/gas/H2S +/+ with H 2
S
alkaline/acid -/+
alkaline/acid/gas -/+ with gas
alkaline/acid/gas/H2S -/+ with gas and H2
S
alkaline/acid/H2S -/+ with H 2
S
alkaline/alkaline -/-
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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