A book about the second penalty Biological Laboratory
yellow
[GLU ] glucose Assimilation transparent opaque
[ARA ] arabinose Assimilation transparent opaque
[MNE ] mannose Assimilation transparent opaque
[MAN ] mannitol Assimilation transparent opaque
[NAG ] N-acetyl-glucosamine Assimilation transparent opaque
[MAL ] maltose Assimilation transparent opaque
[GNT ] gluconate Assimilation transparent opaque
[CAP] caprate Assimilation transparent opaque
[ADI] adipate Assimilation transparent opaque
[MLT ] malate Assimilation transparent opaque
[CIT ] citrate Assimilation transparent opaque
[PAC] phenyl-acetate Assimilation transparent opaque
OX see oxidase test cytochrome oxidase colorless/
light purple
dark purple
PROCEDURE MANUAL
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Policy & Procedure Manual
Policy # MI\TECH\04\04\v03 Page 1 of 4
Section: Technical Manual Subject Title: API Test Strips - API NH
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: August 3, 2003
SYSTEM FOR IDENTIFICATION OF NEISSERIA & HAEMOPHILUS (API NH)
Principle
The API NH strip consists of 10 microtubes containing dehydrated substrat es, which enable the
performance of 12 identification te sts (enzymatic reactions or suga r fermentations), as well as the
detection of a penicillinase (particular interest in Haemophilus influenzae, Haemophilus
parainfluenzae, Branhamella catarrhalis (Moraxella catarrhalis) and Neisseria gonorrhoeae).
The reactions produced during incubation result in spontaneous color changes or are revealed by
the addition of reagents.
After a 2-hour incubation period at a temperature of 35-37
o
C, the reading of the reactions is
performed visually and identification is obtained by consulting the profile list.
Reagents
API NH strips
NaCl 0.85% Medium (2 ml)
JAMES reagent
ZYM B reagent
Swab
Incubation box
Result sheet
1 package insert
McFarland Standard, point 4 on the scale
Mineral oil
Pipettes
Ampule rack
Ampule protector
Procedure
1. Specimen Processing
The microorganisms to be identified must first be isolated as separate colonies by streaking the
specimen onto Blood agar, Chocolate agar or Martin-Lewis agar according to standard
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\04\04\v03 Page 2 of 4
Technical Manual
microbial techniques.
2. Preparation of Strip
Each strip is composed of 10 cupules. Each cupule has an open and closed area (cupule
and tube). An incubation tray is supplied for each strip. It serves as a support and
individual chamber while both protecting the strip from contaminants in the air and
assuring the humid atmosphere necessary to avoid dehydration during incubation.
• Remove the strip from its individual packaging
• Place the strip in the incubation box
• Discard the desiccant sachet
Record the specimen number on the flat portion of the tray (do not record the number on
the lid as it may be misplaced during handling).
3. Preparation of the Inoculum
• Open an ampule of NaCl 0.85% Medium (2 ml) with the ampule protector.
• Using a swab, pick up a few well-isolated colonies and prepare a suspension with a
turbidity equivalent to 4 McFarland, ensuring it is well mixed .
• The suspension should be used immediately after preparation.
4. Inoculation of the Strip
• Distribute the prepared bacterial suspension into the cupules, avoiding the formation of
bubbles (tilt the stri p slightly forwards and place the tip of the pipette or PSIpette
against
the side of the cupule):
- Only fill the tube part of the first 7 microtubes (PEN to URE ): about 50 µl.
- Fill tube and cupule of the last 3 microtubes LIP/ProA , PAL/GGT , β GAL/IND:
about 150 µl, avoiding the formation of a convex meniscus.
• Cover the first 7 tests (PEN to URE ) with mineral oil (underlined tests).
NOTE: The quality of the filling is very import ant: tubes which are insufficiently or
excessively full may cause false positive or false negative results.
• Close the incubation box.
• Incubate for 2 hours at 35-37
o
C in aerobic conditions.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\04\04\v03 Page 3 of 4
Technical Manual
5. Incubation
Incubate for 2 hours at 35-37
o
C in aerobic conditions.
6. Reading the Strip
Refer to the Reactions Table for a desc ription of how to read the reactions.
Note all spontaneous reactions (PEN to β GAL) and record them as + or -.
• Add 1 drop of ZYM B reagent to microtubes 8 and 9: LIP/ProA and PAL/GGT.
• Add 1 drop of JAMES reagent to microtube 10: β GAL/IND.
• Wait 2 minutes then read the reactions by referring to the Reading Table in this package
insert and record them on the result sheet.
- If the LIP reaction is positive (blue pigment), interpret the ProA reaction as
negative , whether the ZYM B reagent has been added or not.
- If, after a 2-hour incubation period, severa l reactions (fermentation, penicillinase)
are doubtful, re-incubate the strip for anot her 2 hours and read the reactions again
(the enzymatic tests should not be re-read in this case).
Reactions Table
RESULTS TESTS REACTIONS SUBSTRATES QTY
(mg)
NEGATIVE POSITIVE
1) PEN
PENicillinase
Penicillin G
1.36
Blue
(penicillinase absent)
Yellow
Yellow-green
Yellow-blue
(penicillinase present
2) GLU
3) FRU
4) MAL
5) SAC
GLUcose (Acidification)
FRUctose (Acidification)
MALtose (Acidification)
SACcharose/Sucrose
(Acidification)
Glucose
Fructose
Maltose
Sucrose
0.5
0.1
0.5
Red
Red-orange
Yellow
Orange
6) ODC
Ornithine DeCarboxylase
Ornithine
0.55
Yellow-green
Grey-green
Blue
7) URE
UREease
Urea
0.41
Yellow
Pink-violet
8a) LIP
LIPase
5-bromo-3-indoxyl-caprate
0.033
Colorless
Pale grey
Blue
(+precipitate)
9a) PAL
Alkaline Phosphatase
Para-Nitrophenyl-phosphate 2CHA
0.038
Colorless
Pale yellow
Yellow
10a) β GAL
Beta GALactosidaase
Para-Nitrophenyl-BD
galactopyranoside
0.04
Colorless
Yellow
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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Policy & Procedure Manual
Policy # MI\TECH\04\04\v03 Page 4 of 4
Technical Manual
Reactions Table (Cont'd)
RESULTS TESTS REACTIONS SUBSTRATES QTY
(mg)
NEGATIVE POSITIVE
ZYM B / 3 min
8b) ProA
Proline Arylamidase
If LIP is +. ProA is always -
Proline-4-methoxy-
β naphthylamide
0.056
Yellow
Pale orange
(brown if LIP +)
Orange
ZYM B / 3 min
9b) GGT
Gamma Glutamyl Transferase
Gamma glutamyl
4-methoxy-
β naphthylamide
0.049
Yellow
Pale orange
(yellow-orange if PAL +)
Orange
JAMES / 3 min
10b) IND
INDole
Tryptophane
0.036
Colorless
Pink
Quality Control
To be performed on receipt of every new lot of strip by the Q.C bench technologist.
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