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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\04\02\v01 Page 7 of 7
Technical Manual
SUMMARY OF RESULTS - 18- 24 HOUR PROCEDURE (cont'd)
TUBE INCUBATION POSITIVE NEGATIVE COMMENTS
After reading GLU reaction, add 2 drops 0.8% sulfanilic acid
and 2 drops 0.5% N. N-dimethyl-alpha-naphthylamine
GLU
NO2
N2
gas
Red
Bubbles: Yellow
after reagents
and zinc
Yellow
Orange after
reagents and
zinc
(1) Before addition of reagents, observe GLU
tube (positive or negative) for bubbles.
Bubbles are indicative of reduction of
nitrate to the nitrogenous (N
2
) state.
(2) A positive reaction may take 2-3 minutes
for the red colour to appear.
(3) Confirm a negative test by adding zinc
dust or 20 mesh granular zinc. A pink-orange colour after 10 minutes confirms a
negative reaction. A yellow colour
indicates reduction of nitrates to the
nitrogenous (N
2
) state.
After reading carbohydrate reaction, add 1 drop 1.5% H2 O2
MAN
INO
SOR
Catalase
Bubbles
No bubbles
(1) Bubbles may take 1-2 minutes to appear.
(2) Best results will be obtained if the test is
run in tubes which have no gas from
fermentation.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 24
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\04\03\v01 Page 1 of 4
Section: Technical Manual Subject Title: API Test Strips - API 20NE
Issued by: LABORATORY MANAGER Original Date: August 3, 2003
Approved by: Laboratory Director
Revision Date:
IDENTIFICATION OF NON-ENTERIC GRAM-NEGATIVE RODS (API 20NE)
Principle
The API 20NE system facilitates the identificat ion of non-fastidious Gram-negative rods not
belonging to the Enterobacteriaceae within 48 hours.
The API 20NE strip consists of microtubes contai ning dehydrated media and substrates. The media
microtubes containing conventional tests are in oculated with a bacterial suspension which
reconstitutes the media. After incubation, the metabolic end produc ts are detected by indicator
systems or the addition of reagents. The substr ate microtubes contain assimilation tests and are
inoculated with a minimal medium. If the bact eria are capable of utilizing the corresponding
substrate, then they will grow.
Materials
API 20NE strips - store at 2-8
0
C
0.85% sterile saline
Mineral oil
Zinc dust
AUX Medium }
James Reagent }
Nitrate 1 - store at 2-8
0
C } Store at 2-8
0
C
Nitrate 2 - store at 2-8
0
C }
Oxidase Reagent
Procedure
1. Preparation of Inoculum
a) Add 2 ml. of 0.85% saline to a sterile test tube.
b) Using a sterile inoculating loop, carefully touch the centre of a well isolated
colony (2-3 mm. Diameter) and thoroughly em ulsify in the saline. The suspension
turbidity should be equal to a 0.5 McFarland standard.
2. Preparation of the Strip
a) An incubation tray and lid are supplied for each strip.
b) Dispense 5 ml of distilled water in to the tray.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 25
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\04\03\v01 Page 2 of 4
Technical Manual
3. Inoculation of the Strip
a) Remove the cap from the tube containing the bacterial suspension and insert a
sterile pipette.
b) Tilt the API 20NE incubation tray an d fill the TUBE section of the NO 3
to PNPG
microtubes by placing the pipette tip against the side of the cupule.
c) Open an ampule of AUX Medium and add 200 uL of the bacterial suspension to
the ampule. Mix well with a pipette while avoiding the formation of air bubbles.
d) Using the AUX Medium bacterial suspension, fill both the TUBE and CUPULE
section of [GLU ] to [PAC ]. Do not overfill the cupules. Fi ll to a flat or slightly
convex meniscus.
e) After inoculation, completely fill the CUPULE section of the 3 unde rlined tests,
GLU, ADH and URE tubes with mineral oil.
f) Using the excess bacterial suspension, i noculate an agar slant or plate (non-selective media such as nutrient agar, blood ag ar or tryptic (tryp ticase) soy agar is
suggested) as a purity check and for oxidase testing, and/or additional
biochemical testing. Incubate the slant or plate with the API 20NE strip.
4. Incubation of the Strip
a) After inoculation, place the plastic lid on the tray and incubate the strip for 24
hours at 30
0
C in a non-CO 2
incubator.
5. Reading the Strip
a) After 24 hours incubation, record all reactions not re quiring the addition of
reagents.
b) Perform the oxidase test.
A portion of the growth from the agar slate or plate, inoculated from the 20NE
bacterial suspension, should be rubbed onto filter paper to which a drop of
oxidase reagent (1% tetramethyl-p-phenylen ediamine dihydrochloride) has been
added. The area where the growth has been added will turn dark purple within 10
seconds if the reaction is positive and will be colourless or light purple if
negative.
Note: (a) Nichrome wire loops should NOT be used in performing
the oxidase test. Nichrome wire can cause a false positivereaction.
(b) The oxidase test should NOT be performed using bacterial
growth from selective media such as MacConkey, EMB, etc.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 26
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\04\03\v01 Page 3 of 4
Technical Manual
c) Assimilation tests are observed for bacterial growth. An opaque cupule indicates a
positive reaction.
d) Protect the assimilation tests with the incubation tray lid during the reading of the
Nitrate
and TRP tests.
e) Perform the Nitrate test.
i. Add one drop of Nitrate 1 and one drop of Nitrate 2 reagents to NO
3
cupule.
ii. After 5 minutes a red color indicates a positive reaction.
iii. A negative reaction may be due to th e production of nitrogen. Add Zinc
dust to the NO 3
cupule. After 5 minutes a colorless cupule indicates a
positive reaction. A pink-red cupule indicates a negative reaction.
f) Perform the TRP test.
i. Add one drop of JAMES Reagent.
ii. The reaction takes place immediately, producing a pink color in the entire
cupule if the reaction is positive.
Interpretation
1. Use the API 20NE analytical profile index.
2. The tests are separated into groups of three. The following numerical value is assigned to
each positive reaction recorded:
1 - positive reaction in the first test of the group
2 - positive reaction in the second test of the group
4 - positive reaction in the third test of the group
By adding the values corresponding to posit ive reactions in each group, a seven digit
number is obtained.
3. The strip must be reincubated in the following cases:
i. If the profile cannot be found in the Analytical Profile Index.
ii. If the following note is indicated for the profile obtained:
IDENTIFICATION NOT VALID
BEFORE 48-HR INCUBATION
iii. If the strip is to be reincubate d, remove the reagents from the NO3
and
TRP cupules and then cover these tests with mineral oil.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 27
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\04\03\v01 Page 4 of 4
Technical Manual
iv. Reincubate the strip for another 24 hours at 30
0
C in a non-CO 2
incubator.
v. Read all the tests again, except for NO3,
TRP and GLU.
READING TABLE
TESTS SUBSTRATES REACTONS/ENZYMES NEGATIVE
RESULTS
POSITIVE
RESULTS
NITrate reduction to
nitrites
NIT 1 + NIT 2 / 5 min
colourless pink-red
N03
Potassium
nitrate
NITrates to nitrogen Zn / 5 min
pink colourless
TRP tryptophane indole production JAMES / immediate
colourless / pink
pale green / yellow
GLU glucose Acidification blue to green yellow
ADH arginine arginine dihydrolase yellow orange/pink/red
URE urea Urease yellow orange/pink/red
ESC esculin hydrolysis ( β -glucosidase) yellow grey/brown/black
GEL gelatine
(with India ink)
hydrolysis (protease) no pigment
diffusion
diffusion of black
pigment
PNPG p-nitrophenyl- β -D-galactopyranoside
β -galactosidase colourless
MUG negative: Colourless
Indole positive: Red colour after addition of Kovac's
Indole negative: Kovac's remains yellow
MUG INDOLE INTERPRETATION / ACTOIN
+ + report as E. coli
- + set up VITEK Identification
+ - set up VITEK Identification
- - set up VITEK Identification
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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