API 20E system
• The API 20E is a standardized identification system
for Enterobacteriaceae and other non-fastidious,
Gram negative rods.
• The API 20 E strip consists of 20 microtubes (each
one of microtubes have cupule, and a small tube )
containing dehydrated substrates.
• These tests are inoculated with a bacterial suspension
that reconstitutes the media.
• During incubation, metabolism produces color
changes that are either spontaneous or revealed by the
addition of reagents.
• The reactions are read according to the reading table
and the identification is obtained by referring to the
Analytical Profile Index or using the identification
software.
Reagents
• NaCl 0.85 % Medium, 5 ml or API Suspension
Medium, 5 ml
• API 20 E reagent kit
• TDA
• JAMES
• VP 1 + VP 2
• NIT 1 + NIT 2
• Zn reagent
• Oxidase
• Mineral oil
Preparation of the inoculum
• Open an ampule of API NaCl 0.85 % Medium
(5 ml) or use any tube containing 5 ml of sterile
saline or sterile distilled water, without
additives.
• Use colony from an isolation plate. It is
recommended to use young cultures (18-24
hours old).
• Carefully emulsify to achieve a homogeneous
bacterial suspension.
• This suspension must be used immediately after
preparation.
• Dispense about 5 ml of tap water into the tray
with a squeeze bottle.
• Note: that the bottom of the tray has numerous
depressions to accept the water.
• Vortex mix the saline suspension to get uniform
dispersal
• Inoculate all the tubes on the test strip with the
pipette by depositing the suspension into the
cupules as you tilt the API tray
Important
• Create anaerobiosis in the tests ADH, LDC, ODC,
H2S and URE. by overlaying with mineral oil.
• Since the media in |CIT|, |VP|, and |GEL|
compartments require oxygen, completely fill both
the cupule and tube of these compartments.
• Fill only the tube (and not the cupule) of the other
tests.
• Close the incubation box. Incubate at 36°C ± 2°C for
18-24 hours.
NOTE : The indole production test must be performed
last since this reaction releases gaseous products
which interfere with the interpretation of other tests
on the strip.
• The plastic incubation lid should not be replaced after
the addition of the reagent.
• If the number of positive tests (including the GLU
test) before adding the reagents is less than 3 :
• Reincubate the strip for a further 24 hours (± 2
hours) without adding any reagents.
• Reveal the tests requiring the addition of reagents
(see previous paragraph).
Interpretation
• Determination of the numerical profile :
• On the result sheet, the tests are separated into
groups of 3 and a value 1, 2 or 4 is indicated for
each.
• By adding together the values corresponding to
positive reactions within each group, a 7-digit
profile number is obtained for the 20 tests of the
API 20 E strip.
Reduction of nitrates to nitrites (NO2) and N2 gas (N2)
• add 1 drop each of NIT 1 and NIT 2 reagents to the
GLU tube. Wait 2 to 5 minutes.
• A red color indicates a positive reaction (NO2). A
negative reaction (yellow) may be due to the reduction
to nitrogen (as sometimes evidenced by gas bubbles):
• add 2 to 3 mg of Zn reagent to the GLU tube. After 5
minutes, if the tube remains yellow this indicates a
positive reaction (N2) to be recorded on the result
sheet. If the test turns orangered, this is a negative
reaction :
Results
• the nitrates still present in the tube have been reduced
by the Zinc.
• This reaction is useful when testing Gram-negative,
oxidase positive rods.
Animation