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MICROBIOLOGY STANDARD OPERATING PROCEDURE

BACTERIAL IDENTIFICATION USING BIOMERIEUX API KITS
Document number / version: Reviewed and approved by:
Replaces document: Date of original: 15-May-2013
Applies to: Microbiology laboratory Date of revision:
Modified by: Date for review:

1 Aim
To identify bacterial isolates using commercial biochemical test kits (bioMerieux API).
2 Principle
API test strips consists of microtubes (cupules) containing dehydrated substrates to detect the
enzymatic activity or the assimilation / fermentation of sugars by the inoculated organisms. During
incubation, metabolism produces colour changes that are either spontaneous or revealed by the
addition of reagents. When the carbohydrates are fermented, the pH within the cupule changes
and is shown by an indicator. Assimilation tests are inoculated with a minimal medium (API AUX
medium) and the bacteria grow if they are able to utilize the corresponding substrate: a positive
result is indicated by growth. Test results are entered into an online database to determine the
bacterial identity.
Following presumptive organism identification using Grams stain, morphological features and other
simple tests, the appropriate API kit should be selected using the table below.
Presumptive Organism ID Which API strip to Additional notes
use
Gram negative bacillus
Oxidase positive Stenotrophomonas&Acinetobacter spp.
API 20 NE (oxidase negative) may also be identified
Non-fastidious
using API 20E.
Non-Enterobacteriaceae
Gram negative bacillus
Oxidase negative Vibrio spp. and Aeromonas spp. (oxidase
API 20 E positive) may also be identified using API
Enterobacteriaceae & other 20NE.
non-fastidious GNB
Gram positive cocci
Pairs or chains Streptococcus pneumoniae and groupable
Catalase negative API 20 Strep beta-haemolytic streptococci do not usually
Streptococci, Enterococci& require API testing.
related genera
Gram negative cocci in pairs Moraxella catarrhalis can be adequately
Pleomorphic nutritionally demanding identified using the trybutyrin test if isolated
Gram negative bacilli or coccobacilli API NH from a non-sterile site.
(e.g. Neisseria, Haemophilus, Haemophilus influenzae can be adequately
Moraxella) identified by XV-factor dependent growth.

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3 Method
The following are summaries from the kit inserts present in each box: refer to these inserts for
further details if required.
API strips should only be used to identify pure cultures of an unknown organism. Confirm Gram
stain (plus catalase and oxidase if appropriate) before inoculating a test strip. Inoculation of the
incorrect strip can result in misidentification.
3.1 General
3.1.1 Inoculation
This section applies to all APIs. Differences between each type of strip are described under the
appropriate heading.
1. Label the API carrier tray with specimen number and date.
2. Put 5ml of sterile distilled water into the tray to provide a moist atmosphere which prevent
drying of the strip.
3. Lay the strip in the tray.
4. Open an ampoule of suspension medium if required:
a. Insert the base of the ampoule into the protective plastic guard.
b. Hold the ampoule vertically in one hand with the white plastic cap uppermost.
c. Press the cap down as far as possible.
d. Apply thumb pressure in an outward direction to the flattened part of the cap to
snap off the top of the ampoule inside the cap.
e. Carefully remove the cap and discard it.
5. Make a suspension of the organism and inoculate the wells as described in the section for
the type of strip used. The structure of the wells is as follows:

Tube Cupule

6. Tilt the strip slightly to help the inoculum to run into the tube. Avoid the tendency to trap air,
which is most likely if the tubes are filled too rapidly, by carefully allowing the inoculum to
run down inside one edge of the tube. The wells are of several types, and must be filled as
shown in the diagrams:

a. Those requiring filling of only the tube, denoted by a plain test name, e.g.: GLU

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b. Those requiring filling of both tube and cupule, denoted by a border round the test
name, e.g.: GEL

c. Those requiring filling of the tube, which is then overlaid with liquid paraffin,
denoted by a line under the test name, e.g.: URE

Liquidparaffin

7. For strips that are to be incubated overnight or longer, inoculate a purity plate from the
organism suspension, using a non-selective medium appropriate for the type of organism.
8. Put the lid on the tray and incubate it for the prescribed time under the appropriate
condition

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3.1.2 Profile calculation and interpretation

9. Add any reagents as described for the type of strip used and make any other observations
24 1

required, e.g.: haemolysis, then construct the profile:

a. Mark each test as positive or negative on the lid of the tray
b. The wells are marked off into triplets by black triangles, for which scores are
allocated as follows:

c. Add up the scores for the positive wells only in each triplet. Supplementary tests,
e.g.: oxidase may also be included in the profile. The highest score possible for a
triplet is 7 (the sum of 1, 2 and 4) and the lowest is 0, e.g.:

d. The profile for this combination of reactions is therefore 5147306

10. Identify the organism using apiweb:
a. Start Internet Explorer or Firefox web browser
b. Go to: https://apiweb.biomerieux.com
i. Login: pturner
ii. Password: apiweb
iii. Select the correct test (e.g. API 20E).
iv. Enter the numerical profile to obtain the identity.
v. Record the identity along with comments (% ID and T value) on the results
sheet.

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3.1.3 Disposal
11. After reading the API strip, place the carrier and contents into the autoclave bag in the
plastic discard bin.
3.2 API 20E

1. Make a suspension of the test organism in 5ml saline.

2. From this suspension inoculate a sheep blood agar / Columbia agar purity plate.
3. Prepare and inoculate the test strip as described above and overlay with sterile liquid
paraffin where indicated.
4. Incubate overnight (18-24h) in air at 36C (+/- 2C).
5. Assess strip:
a. If there are less than threepositivereactions (GLU test + or -) after incubation,
do not add any reagents. Reincubate for a further 24 hours after checking the
purity plate to make sure the organism is growing.
b. If there are three or more positive reactions (GLU test + or -) examine the purity
plate to ensure the culture is pure then add the reagents as follows:

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Well Reagent
TDA One drop of TDA reagent
IND One drop of James reagent
VP One drop of VP1 then one drop of VP2
Also perform an oxidase on the purity plate

6. Read the results from the following table:

TEST REACTION NEGATIVE POSITIVE
ONPG -galactosidase Colourless Yellow (maybe pale)
ADH Arginine dihydrolase Yellow Orange or red
LDC Lysine decarboxylase Yellow Orange or red
ODC Ornithine decarboxylase Yellow Orange or red
CIT Citrate utilisation Light green Blue-green or blue
H2S H2S production Colourless Black
URE Urea hydrolysis Yellow Pink
TDA Tryptophan deamination Yellow Dark brown
IND Indole production Colourless Pink
VP Acetoin production Colourless Pink or red
GEL Gelatin hydrolysis Colourless Black diffuse pigment
GLU Glucose fermentation Blue Yellow
MAN Mannitol Blue Yellow
INO Inositol Blue Yellow
SOR Sorbitol Blue Yellow
RHA Rhamnose Blue Yellow
SAC Sucrose Blue Yellow

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TEST REACTION NEGATIVE POSITIVE

MEL Melibiose Blue Yellow
AMY Amygdalin Blue Yellow
ARA Arabinose Blue Yellow
Oxidase Cytochrome oxidase Colourless Purple

7. The tests on the strip plus oxidase are used to determine the first seven digits of the
profile number. This is usually sufficient to determine the identity using apiweb software,
but supplementary tests can be used to determine a further two digits if required (see kit
insert).

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3.3 API 20NE

1. Make a suspension of the test organism in 2ml sterile saline.

2. Prepare the test strip as described above.
3. Inoculate the NO3 to PNPG (the first 8 cupules) from the saline suspension.
4. Open an API AUX ampoule (provided with the kit) and add 4 drops (200l) of the saline
suspension to it. Use the pipette to mix well without creating bubbles.
5. Use this suspension to inoculate the remainder of the cupules.
6. Overlay wells with liquid paraffin where indicated.
7. Incubate overnight (24h +/- 2h) in air at 29C (+/- 2C).
8. Examine the purity plate to ensure the culture is pure then add the reagents:
Well Reagent
NIT One drop of NIT1 and one drop of NIT2 and wait 5 minutes. If there is no
reaction (still colourless), add zinc powder, wait 5 minutes and interpret
according to the table below
TRP One drop of James reagent

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9. Examine the assimilation tests for bacterial growth. An OPAQUE cupule indicates a
POSITIVE REACTION. Occasionally, a cupule may show weak growth. In this case the
results should be noted as +/- or -/+ by comparison to other tests on the strip. Once these
readings have been made, identification should be possible.
10. Read the results from the following table:
TEST REACTION NEGATIVE POSITIVE
NO3NO2 Reduction of potassium nitrate Colourless Red (NIT1+NIT2)
NO2N2 Red/pink Colourless (Zn)

TRP Indole production from tryptophan Yellow Pink

GLU Glucose fermentation Blue/green Yellow
ADH Arginine hydrolysis Yellow Orange/pink/red
URE Urea hydrolysis Yellow Orange/pink/red
ESC Aesculin hydrolysis Yellow Grey/brown/black
GEL Gelatin hydrolysis No pigment diffusion Diffusion of black
pigment
PNPG p-nitrophenyl-D-galactopyranoside Colourless Yellow
hydrolysis
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
Oxidase Cytochrome oxidase Colourless Purple

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11. Construct the numerical profile and look up the number using apiweb
a. In the following cases, the strip must be reincubated:
i. Low discrimation, unacceptable or doubtful profile on apiweb.
ii. If the following note is added to profile obtained from apiweb:
IDENTIFICATION NOT VALID BEFORE 48 HOURS INCUBATION.
b. In these events, immediately remove the contents of the NO 3 and TRP wells using
a pipette and fill with mineral oil so that a convex meniscus is formed, to prevent
escape of acidic vapour. Reincubate the strip at 29C (+/- 2C)for a further 18-24
hours and read all the tests once more excepting NO 3, TRP and GLU which must
be read once only, at 18-24 hours.
c. Reread the tests and construct a new profile.

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3.4 API NH

Unlike API 20E/20NE/Strep, API NH relies on detection of preformed enzymes (not growth).

Important: suspensions of suspected N. meningitidis must be prepared in the BSLII safety cabinet

1. Using a swab make a heavy suspension (McFarland 4) of the organism in 2ml of 0.85%
sterile saline provided with the kit.
2. Into wells PEN to URE dispense about 50 l of this suspension. Fill the tube and cupule of
the last 3 tubes (LIP/ProA, PAL/GGT, BGAL/IND).
3. Cover the first seven tests with mineral oil.
4. Incubate the strip in air at 36C (+/- 2C) for 2 hours.
5. Before adding any reagents record the primary results using the table below.
6. Add the reagents as follows:
Note: ZYM B is very light sensitive and loses activity within a few days of opening. Check
that the date the ampoule was opened is within the last two weeks. If you start a new ampoule,
write the date on the bottle.
Well Reagent
Wells 8 & 9 One drop of ZYM B

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(LIP/ProA & PAL/GGT)

Well 10 One drop of James reagent
(BGAL/IND)

7. Wait three minutes and read the results from the following table:
TEST REACTION NEGATIVE POSITIVE
PEN Penicillinase production Blue Yellow
GLU Glucose Red Yellow/orange
FRU Fructose Red Yellow/orange
MAL Maltose Red Yellow/orange
SAC Saccharose Red Yellow
ODC Ornithine decarboxylase Yellow Blue
URE Urease Yellow Pink/violet
LIP Lipase Colourless Blue (+ precipitate)
PAL Alkaline phosphatase Colourless Yellow
GAL Galactosidase Colourless Yellow

AFTER ADDITION OF REAGENTS (ZYMB / JAMES)

ProA Proline arylamidase Yellow Orange
GGT Glutamyl transferase Yellow Orange
IND Indole Colourless Pink

8. Construct a four digit profile from the results as described above, ignoring the PEN result
and starting with the GLU, FRU, MAL triplet. The third digit is derived from the upper three
tests in the bifunctional wells and the fourth from the lower three.
9. Determine the organism identity using apiweb.

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3.5 API Strep

The heavy density of the inoculum allows detection of preformed enzymes in some tests, allowing
identification within 4 hours.
1. Subculture a single colony of the organism to be tested onto sheep blood agar and
incubate for 24 or 48 hours until sufficient growth is obtained. Note the type of haemolysis.
2. Make a heavy (McFarland 4) suspension of the test organism in 2ml sterile distilled water.
3. Prepare the test strip as described above.
4. Using this suspension, inoculate VP to LAP with 100l suspension, and fill the tube portion
only of the ADH.
5. Open an API Strep Medium ampoule (provided with the kit) and transfer about 0.5 ml of
the suspension to it. Use the pipette to mix well without creating bubbles.
6. Distribute this into the remaining tests (Rib to GLYG).
7. Overlay wells with liquid paraffin where indicated.
8. Incubate in air at 36C (+/- 2C) for 4 hours to obtain the initial profile.
9. Add the reagents as follows:

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Note: ZYM B is very light sensitive and loses activity within a few days of opening. Check that
the date the ampoule was opened is within the last two weeks. If you start a new ampoule, write
the date on the bottle.
Well Reagent
VP One drop of VP1 and one drop of VP2 and wait 10 minutes.
HIP One drop of NIN and read after 10 minutes
PYRA to One drop of ZYM A followed by one drop of ZYM B and read after ten minutes.
LAP If necessary decolourise with intense light.

10. Read the results from the following table.

TEST REACTION NEGATIVE POSITIVE
VP Acetoin production Colourless Pink / red
HIP Hippurate Colourless Dark blue / violet
ESC Aesculin hydrolysis 4hrs - colourless 4hrs - grey / black
24hrs - pale grey 24hrs - black
PYRA Pyrrolidonylaryl-amidase Colourless / Orange
pale orange
GAL -galactosidase Colourless Violet

GUR -glucuronidase Colourless Blue

GAL -galactosidase Colourless / Violet

pale violet
PAL Alkaline phosphatase Colourless / Violet
pale violet
LAP Leucine arylamidase Colourless Orange
ADH Arginine dihydrolase Yellow Red
RIB Ribose fermentation 4hrs - Red 4hrs - Orange / yellow
24hrs - Red / orange 24hrs - Yellow
ARA Arabinose fermentation 4hrs - Red 4hrs - Orange / yellow
24hrs - Red / orange 24hrs - Yellow

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TEST REACTION NEGATIVE POSITIVE

MAN Mannitol fermentation 4hrs - Red 4hrs - Orange / yellow
24hrs - Red / orange 24hrs - Yellow
SOR Sorbitol fermentation 4hrs - Red 4hrs - Orange / yellow
24hrs - Red / orange 24hrs - Yellow
LAC Lactose fermentation 4hrs - Red 4hrs - Orange / yellow
24hrs - Red / orange 24hrs - Yellow
TRE Trehalose fermentation 4hrs - Red 4hrs - Orange / yellow
24hrs - Red / orange 24hrs - Yellow
INU Inulin fermentation 4hrs - Red 4hrs - Orange / yellow
24hrs - Red / orange 24hrs - Yellow
RAF Raffinose fermentation 4hrs - Red 4hrs - Orange / yellow
24hrs - Red / orange 24hrs - Yellow
AMD Starch fermentation 4hrs - Red 4hrs - Orange / yellow
24hrs - Red / orange 24hrs - Yellow
GLYG Glycogen fermentation 4hrs - Red 4hrs - Orange / yellow
24hrs - Red / orange 24hrs - Yellow
HAEM Haemolysis No haemolysis haemolysis present

11. Construct the numerical profile and look up the number using apiweb:
a. In the following cases, the strip must be reincubated overnight:
i. If the profile cannot be found in apiweb.
ii. If the following note is printed for the profile obtained: IDENTIFICATION
NOT VALID BEFORE 24 HOURS INCUBATION.
b. After 24hrs incubation, reread ESC, ADH and RIB to GLYG then construct a new
profile.

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4 Quality assurance
No specific procedures required, aside from using the kits as described above and in the
manufacturers manuals (kit inserts).
5 Limitations
API kits should only be used to identify organisms as specified in this SOP or the relevant kit insert.
Inoculation of an organism with inappropriate presumptive identification test results may result in
an incorrect result.
If an unexpected / no result is obtained, it is important to recheck basic tests such as Gram result,
catalase, oxidase, and ensure that the purity plate is satisfactory. Repeat the test with a pure
culture if the purity indicates a mixture.
6 References
1. bioMerieux API kit inserts (20 100 (API 20E); 20 050 (API 20NE); 10 400 (API NH); 20 600
(API 20 Strep)).
2. Procedure for the use of API Strips. Whittington Hospital SOP MB/040.04 (2005).
3. Which API to use. LOMWRU SOP BIP 005-01 (2012).

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7 Synopsis / Bench aid

API 20E

Negative

Positive

API 20NE

Negative

Positive

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API NH

Before addition of reagents After addition of reagents

Negative

Positive

API 20 Strep

Negative

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Positive

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8 Risk assessment

COSHH risk assessment - University of Oxford COSHH

Assessment Form
Description of procedure Substances used
Biochemical identification of bacteria 1. Pathogenic bacteria
2. API test strips and associated reagents
Quantities of chemicals Frequency of SOP use
used Daily
Small
Hazards identified Could a less hazardous
JAMES (HCl) is an irritant to the eyes, skin substance be used instead?
or other mucous membranes No
NIN (Dimethylsulfoxide (DMSO) and
methanol) is a severe irritant and causes
chemical burns if in contact with the eyes,
skin, ingested or inhaled. Methyl alcohol is
very flammable.
NIT1&2 contain acetic acid, which is a
severe irritant and causes chemical burns if
in contact with the eyes, skin, ingested or
inhaled.
TDA (Ferric chloride)
VP1 (potassium hydroxide) is a severe
irritant and causes chemical burns if in
contact with the eyes, skin, ingested or
inhaled.
VP2 (alpha naphthol and ethyl alcohol) is a
severe irritant and causes chemical burns if
in contact with the eyes, skin, ingested or
inhaled. Also very flammable.
Zinc powder is highly flammable.
ZYM A & B is flammable and toxic. There is
a danger of severe irreversible effects

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through inhalation, in contact with skin and if

swallowed.
Potential risk of infection from bacterial
suspensions.
What measures have you taken to control risk?
1. Training in good laboratory practices (GLP)
2. Appropriate PPE (lab coat, gloves, eye protection)
3. Use of biosafety cabinet for reading of plates / follow-up of BSL-3 organisms (e.g. B.
pseudomallei)
Checks on control measures
Observation and supervision by senior staff
Is health surveillance Training requirements:
required? GLP
No
Emergency procedures: Waste disposal procedures:
1. Report all incidents to Safety Adviser 1. Sharps discarded into appropriate rigid
2. Clean up spills using 1% Virkon or containers for incineration
chemical spill kit 2. Infectious waste (incl. API strips) discarded into
3. In the event of any solution going on the autoclave bags or 1% Virkon solution prior to
eyes, flush with eye was for 15 minutes. autoclaving and subsequent incineration
Wash hands if any solution gets on your 3. Chemical waste disposed of according to
manufacturers instructions
hands. If swallowed do not induce vomiting,
if conscious drink copious amounts of water.

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