A book about the biological laboratory third penalty
Reference
QC organisms to be used:
Neisseria gonorrhoea ATCC 31426
Haemophilus influenzae ATCC 10211
Branhamella catarrhalis ATCC 23246
Haemophilus paraphrophilus ATCC 49917
Reference Package Insert - api NH system for the identification of Neisseria and Haemophilus
bioMerieux Inc., Missouri USA.
PROCEDURE MANUAL
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Policy & Procedure Manual
Policy # MI\TECH\04\04\v01 Page 1 of 1
Section: Technical Manual Subject Title: API Voice Response
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
API VOICE RESPONSE
1. Dial (800) 645-7056 from a touch tone phone.
2. Enter product code access number (0-14).
3. Press * symbol.
4. Enter profile number as outlined below.
5. Press # symbol.
6. Press 1# to end the call.
Press 2# to repeat the identification.
Press 3# for the next profile.
Press 4# to speak to a technologist.
ACCESS CODES
Product Code Access Number Incubati on Time Profile Number Format
0 API 20E 24 hours Enter 7 digits
1 API 20E 48 hours Enter 9 digits only
6 API 20C 48-72 hours Enter 7 digits
7 Coryne 24 hours Enter 7 digits
9 STAPH-IDENT 24 hours Enter 7 digits
12 API NE (Rapid NE) 24/48 hours Enter 7 digits
35 20 Strep 24 hours Enter 7 digits
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\05\v01 Page 1 of 2
Section: Technical Manual Subject Title: Bacitracin Disk Test
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
BACITRACIN DISK TEST
Principle
This is a screening test for the presumptive identifi cation of Group A Streptococci which are
susceptible to 0.04U baci tracin. Other beta-haemolytic streptoc occi are usually resistant to this
concentration of bacitracin.
Reagents
Bacto Differentiation Disks Bacitracin (0.04U). Store refrigerated.
Blood Agar (BA)
Other Materials
Culture loop
Cotton swabs
Forceps
Procedure
1. Inoculate the surface of the BA with the suspect beta haemolytic Streptococcus. Streak for
confluent growth.
2. Using aseptic technique, place a bacitracin disk onto the inoculated surface.
3. Incubate in O
2 at 35
o
C X
18-24 hr.
Interpretation
Susceptible: any zone of inhibition around the disk (Presumptive Group A Stre ptococcus).
Resistant: growth up to the edge of the disk
Precautions
1. Other beta-haemolytic streptococci may be susc eptible to bacitracin. Therefore this test can
be used only for the presumptive identification of Gp. A Strep.
PROCEDURE MANUAL
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Quality Control
Test with known susceptible and resistant control strains weekly.
Susceptible: Gp.A Strep. (ATCC 19615)
Resistant: Gp.B Strep. (ATCC 13813)
Reference
1. Difco Differentiation Disks Bacitracin package insert.
PROCEDURE MANUAL
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\06\v02 Page 1 of 2
Section: Technical Manual Subject Title: Beta-Lactamase (Cefinase)
Test
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: November 1, 2002
BETA-LACTAMASE (CEFINASE) TEST
Principle
Cefinase discs are intended for use in rapid testing of isolated colonies of Neisseria gonorrhoeae,
Staphylococcus species, Enterococcus species, Hameophilus influenzae and anaerobic bacteria for
the production of beta-lactamase.
The Cefinase disc is impregnated with the chromogenic cephalosporin, Nitrocefin. This compound
exhibits a very rapid colour change from yellow to red as the amide bond in the beta lactam ring is
hydrolyzed by a beta-lactamase. When a bacterium produces this enzyme in signifi cant quantities,
the yellow-colored disc turns red in the area where the isolate is smeared.
Although other penicillins and ce phalosporins may be used as substrates for specific enzymes,
Nitrocefin has the wide spectrum of susceptibility and sensitivity of the comm ercially available beta
lactams. It is not known to react with other microbial enzymes.
Each disc is used to test one bacterial strain for the presence of beta-lactamase.
Materials
Cefinase discs impregnated with Nitrocefin.
Procedure
1) Using a single disk dispenser, dispense the required number of disks fr om the cartridge into
an empty petri dish or onto a microscope slide.
2) Moisten each disc with 1 drop of Sterile di stilled water.
3) With a sterilized loop or applicator stick remove several well-isolated similar colonies and
smear onto a disk surface.
4) Observe disk for colour change.
5) Alternate procedure: Using forceps moisten disk with one drop of Purified Water and then
wipe across colony.
PROCEDURE MANUAL
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Policy # MI\TECH\06\v02 Page 2 of 2
Technical Manual
Interpretation
A positive reaction will show a yellow to red colour change on the area where the culture was
applied. Note: colour ch ange does not usually develop over the entire disk. A negative result will
show no colour change on the disc.
For most bacterial strains a positive result will develop within 5 minutes. However, positive
reactions for some stap hylococci may take up to 1 hour to develop.
Organisms
Result
Approx.
Reaction Time
Interpretation
Staphylococcus aureus Positive 1 hr Resistant to penicillin,
ampicillin, carbenicillin.
Probably susceptible to
cephalothin, methicillin,
oxacillin, naficillin and
other penicillinase-resistant penicillins.
Enterococcus faecalis Positive 5 min Resistant to penicillin
and ampicillin.
Hameophilus influenzae Positive 1 min Resistant to ampicillin
Susceptible to
cephalosporins.
Neisseria gonorrhoeae and
Branhamella catarrhalis
Positive
1 min
Resistant to penicillin.
Anaerobic bacteria Positive 30 mins Probable identification is
Bacteroides species.
Probably resistant to
penicillin an d may be
resistant to
cephalosporins including
cefotaxime and rarely
cefoxitin.
Controls: Staphylococcus aureus (ATCC 29213): Positive
Haemophilus influenzae (ATCC 10211): Negative
Reference
1. Murray P.A., et al. Manual of Clinical Microbiology, 7
th
ed. 1999.
PROCEDURE MANUAL
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\07\v01 Page 1 of 1
Section: Technical Manual Subject Title: Bile Esculin Test
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
BILE ESCULIN TEST
Principle
This test determines the ability of an organism to grow in the presence of bile and to hydrolyze the
glycoside esculin to escu letin and glucose. The test is used to presum ptively identify Group D
Streptococci.
Materials
Bile esculin agar slant / plate
Culture loop
Procedure
1. Heavily inoculate a bile esculin slant / plate with the suspect organism.
2. Incubate in O
2 at 35
o
C for 18-24 hr.
Interpretation
Positive: Presence of a dark brown to black colour on the slant.
Negative: No blackening of the medium. Growth may occur, but this does not indicate esculin
splitting.
Quality Control
Each new lot of media should be te sted with known control strains.
Positive: E. faecalis (ATCC 29212)
Negative: Gp.B Strep. (ATCC 13813)
No Growth: Gp.A Strep. (ATCC 19615)
References
1. MacFaddin JF, Biochemical Tests for Identification of Medi cal Bacteria, 2nd ed., Williams
and Wilkins, Baltimore MD, 1980, p4-12.
PROCEDURE MANUAL
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\08\v01 Page 1 of 1
Section: Technical Manual Subject Title: Bile Solubility Test
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
BILE SOLUBILITY TEST
Principle
Tests the ability of alpha ha emolytic streptococci to lyse in the pr esence of bile salts. This test is
used for the identification of Streptococcus pneumoniae.
Reagents
BBL Spot Test dropper (10% sodium desoxycholate).
Procedure
1. Hold the dropper upright and squeeze gently to crush the glass ampoule inside the dispenser.
2. Place 1 drop of the reagent directly on isolated colonies of suspected S. pneumoniae .
3. Keep the plates very level to prevent the reagent from running and washing a non-
pneumucoccal colony away, producing a false positive result.
4. Incubate at room te mperature on the bench for 15-30 minutes until the reagent drys. Do not
invert the plate; leave the lid ajar.
5. Examine the colonies for lysis.
Interpretation
Positive (bile soluble): Lysis of the colonies.
Negative (bile insoluble): No lysis of colonies.
Quality Control
Test with known positive and negative cont rol strains weekly.
Positive: S. pneumoniae (ATCC 6303)
Negative: Viridans Streptococcus (LPTP 8610)
References
1. Murray PA, et al. Manual of Clinical Microbiology, 7
th
ed., 1999; p. 1665.
2. BBL Desoxycholate Reagent Dr oppers package insert, April 1991.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\09\v01 Page 1 of 2
Section: Technical Manual Subject Title: Catalase Test
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
CATALASE TEST
Principle
Detects the presence of the enzyme catalase which hydrolyzes H2O2 to produce H
2O and O2
. This
test is used to differentiate Staphylococci (catal ase positive) from Streptococci (catalase negative).
Reagents
Hydrogen peroxide (H 2O2
), 3%
Store in a dark bottle and av oid any undue exposure to light.
Keep refrigerated at a ll times when not in use.
Other Materials
Clean glass microscope slides
Plastic culture loop or wooden applicator stick
Procedure
1. Pick a colony from an 18-24 hr culture and pl ace it on a clean glass slide. Avoid carry over
of blood agar which can cause false positives.
2. Put one drop of 3% H
2O2
over the organism on the slide. Do not reverse the order of the
procedure as false pos itive results may oc cur. Do not mix.
3. Observe for immediate bubbling (gas liberation) and record the result.
4. Discard the slide into a discard container.
Interpretation
Positive test: Immediate bubbling, easily observed (0
2
formed)
Negative test: No bubbling
Precautions
1. Carry over of blood agar must be avoided.
2. Growth for testing must be from an 18-24 hr culture.
3. 3% H
2O2 is caustic - avoid exposure to skin. If H
2O2
does get on the skin, immediately
flood the area with 70% ethyl alcohol, not water.
PROCEDURE MANUAL
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Policy # MI\TECH\09\v01 Page 2 of 2
Technical Manual
4. Aerosols may be released by the bubbling of the O
2
.
5. H
2O2
is unstable and breaks down easily on exposure to light. The solution must be kept
refrigerated in the dark.
Quality Control
H2O2
is very unstable and should be tested daily or immediately prior to its use.
Positive: S. aureus (ATCC 25923)
Negative: Gp. A. Strep. (ATCC 19615)
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