The API 20E System — Comprehensive Notes | Harley-Prescott, Exercise 35 | Microbiology
THE API 20E SYSTEM
Comprehensive Study Notes
Standardized miniaturized biochemical identification of Enterobacteriaceae and other gram-negative
bacteria
1. Overview
127 22 18–24h 7-digit
Taxa Identifiable Biochemical Tests Incubation Time Profile Number
Gram-negative bacteria In 20 chambers At 35–37°C Identifies organism
What is the API 20E System?
The API 20E System is a standardized, miniaturized version of conventional biochemical procedures
used in the identification of Enterobacteriaceae and other gram-negative bacteria. A total of 127 taxa can
be identified with this system.
It is a ready-to-use, microtube system that performs 22 standard biochemical tests on pure bacterial
cultures from appropriate, primary isolation media.
System Components
• Strip: Contains 20 chambers, each with a microtube and a depression called a cupule
• Substrates: Dehydrated inside each tube; rehydrated by adding a bacterial saline suspension
• Anaerobic conditions: Created by adding sterile mineral oil to specific microtubes (ADH, LDC,
ODC, H2S, URE)
• Reading: Color changes indicate positive/negative results; some tubes require added reagents
• Identification: Achieved by determining a seven-digit profile index number and consulting the
API 20E Profile Recognition System or Index Booklet
The 20 Tests at a Glance
Standard tubes (fill tubes only): ONPG, TDA, IND, GLU, MAN, INO, SOR, RHA, SAC, MEL, AMY, ARA
Anaerobic tubes — underfill then add mineral oil (underlined on strip): ADH, LDC, ODC, H2S, URE
Both tube and cupule filled (bracketed on strip): CIT, VP, GEL
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The API 20E System — Comprehensive Notes | Harley-Prescott, Exercise 35 | Microbiology
2. Procedure
First Period
Step 1 — Oxidase Test (Performed First)
Using aseptic technique, select a well-isolated colony from the unknown streak plate or pure culture slant.
Smear a small amount on Whatman No. 2 filter paper and add several drops of oxidase reagent. Note the
color change. Oxidase test strips or disks may also be used.
Why oxidase first?
The oxidase test determines if the organism belongs to the Enterobacteriaceae (oxidase-negative)
BEFORE committing to the API strip. This helps guide interpretation of downstream results.
Step 2 — Prepare Bacterial Suspension
Transfer a loopful of bacteria to a test tube containing 5 ml sterile 0.85% saline. (If using an applicator
stick, load the tip with bacteria from the colony.) Recap and shake the tube. The turbidity should match
the McFarland No. 3 (BaSO4) reference standard. Add more bacteria if necessary.
Step 3 — Prepare Incubation Tray
Label the elongated flap of the incubation tray with your name and date. From the wash bottle, add 5 ml
of water to the bottom of the tray. This provides a humid atmosphere during incubation.
Step 4 — Place Strip in Tray
Remove the plastic API strip from the sealed pouch and place it in the incubation tray. Reseal the pouch
to protect the remaining strips.
Step 5 — Inoculate the Strip
Shake the bacterial suspension, remove the cap, and fill the 5-ml Pasteur pipette. Tilt the strip to avoid
forming bubbles. Inoculate as follows:
Method Tubes Notes
Fill tubes only ONPG, TDA, IND, GLU, MAN, INO, SOR, Standard fill
RHA, SAC, MEL, AMY, ARA
Slightly underfill ADH, LDC, ODC, H2S, URE (underlined Then fill cupule with mineral oil for
on strip) anaerobic conditions
Fill tube AND cupule CIT, VP, GEL (brackets on strip) Both sections must be completely filled
Step 6 — Incubate
Place the lid on the incubation tray and incubate at 35°C for 18 to 24 hours. If the strip cannot be read
after 24 hours, place it in a refrigerator at 2° to 8°C until the reactions can be read.
Step 7 — Purity Check
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The API 20E System — Comprehensive Notes | Harley-Prescott, Exercise 35 | Microbiology
Make an isolation streak on a TSA plate with a portion of the bacterial suspension to ascertain the purity
of the suspension.
Second Period — Reading Results
After 18 hours and before 24 hours incubation, record all reactions not requiring the addition of reagents
first. Do NOT read TDA, VP, and IND at this stage.
Record results: + for positive reaction, - for negative reaction. Also record the observed colors.
Reagent Additions
Tube Reagent to Add Positive Result Key Note
TDA 1 drop 10% ferric chloride Brown-red color Immediate reaction; golden
orange from indole =
negative
IND 1 drop Kovacs' reagent Red ring Read within 2 minutes;
brownish-red after delay =
negative
VP 1 drop 40% KOH + 1 drop 6% alpha- Red after 10 minutes Wait full 10 min; immediate
naphthol pale pink = negative
GLU 2 drops 0.8% sulfanilic acid + 2 Red color Check for bubbles before
(nitrate) drops 0.5% N,N-dimethyl-alpha- adding; confirm negative
naphthylamine with zinc dust
MAN/INO/ 1 drop 1.5% H2O2 Bubbles Best if no gas from
SOR fermentation present
(catalase)
3. Biochemical Reactions Reference
Chart I: Summary of All Tests
Tube What It Tests Positive Negative Key Notes / Pitfalls
ONPG Beta-galactosidase Yellow Colorless Any shade of yellow = positive. VP tube
(before reagents) serves as negative
control
ADH Arginine dihydrolase Red or orange Yellow Orange at 36-48h should be interpreted as
converts arginine to (18-24h) NEGATIVE
ornithine, ammonia,
CO2 causing pH rise
LDC Lysine decarboxylase Red or orange Yellow Any orange within 18-24h = positive.
converts lysine to (18-24h) Orange at 36-48h = negative
cadaverine (basic
amine) causing pH
rise
ODC Ornithine Red or orange Yellow Orange at 36-48h = negative
decarboxylase (18-24h)
converts ornithine to
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The API 20E System — Comprehensive Notes | Harley-Prescott, Exercise 35 | Microbiology
Tube What It Tests Positive Negative Key Notes / Pitfalls
putrescine causing
pH rise
CIT Citrate as sole carbon Turquoise or Light green or Fill both tube and cupule. Read in aerobic
source; utilization dark blue yellow (cupule) area only
causes pH rise
H2S H2S production from Black deposit No black Even a thin black line at tube bottom =
thiosulfate; reacts deposit positive. 'Browning' alone = negative
with iron salts forming
black precipitate
URE Urease releases Red or orange Yellow Klebsiella, Proteus, Yersinia routinely
ammonia from urea; (18-24h) positive. Lower sensitivity method
causes pH rise
TDA Tryptophan Brown-red Yellow Add 1 drop ferric chloride. Golden orange
deaminase forms (with FeCl3) from indole-positive organisms = negative
indolepyruvic acid
from tryptophane
IND Indole formed from Red ring (with Yellow Read within 2 minutes. HCl in reagent can
tryptophane Kovacs') cause false brownish-red after delay =
metabolism; reacts negative
with Kovacs' reagent
VP Acetoin (glucose Red (after 10 Colorless Wait full 10 min before calling negative.
metabolite) from min) Pale pink after 10 min = negative. Faster
sodium pyruvate; than conventional test (4 days vs API)
Creatine intensifies
color
GEL Gelatin liquefaction Pigment No diffusion Particles may spread after inoculation;
by proteolytic diffuses diffusion must occur. Any degree of
enzymes releases diffusion = positive
black pigment (Kohn
charcoal gelatin)
GLU Glucose fermentation Yellow Blue or blue- Fermentation: read bottom-up. Yellow only
produces acid green at bottom = weak positive.
causing pH drop Enterobacteriaceae ferment; non-
(blue-to-yellow Enterobacteriaceae oxidize
indicator)
MAN- Carbohydrate Yellow Blue or blue- Oxidative: read top-down. Yellow top +
ARA (8 utilization (Mannitol, green blue bottom = oxidative (positive for non-
sugars) Inositol, Sorbitol, Enterobacteriaceae only; negative for
Rhamnose, Sucrose, fermentative organisms)
Melibiose, Amygdalin,
Arabinose)
GLU Nitrate reduction; Red color Yellow Bubbles before reagents = N2 gas. Add
(Nitrate) nitrites form red (confirm with zinc to confirm true negative. Pink-orange
complex with zinc) with zinc = negative. Yellow after zinc =
reagents N2 reduction
Catalas Catalase releases Bubbles No bubbles Best results when tube has no gas from
e oxygen gas from fermentation. Add 1 drop 1.5% H2O2
hydrogen peroxide
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The API 20E System — Comprehensive Notes | Harley-Prescott, Exercise 35 | Microbiology
Abbreviation Key
Code Full Name Code Full Name Code Full Name
ONPG Beta-galactosidase CIT Citrate utilization GLU Glucose
ADH Arginine dihydrolase H2S Hydrogen sulfide MAN Mannitol
LDC Lysine decarboxylase URE Urease INO Inositol
ODC Ornithine decarboxylase TDA Tryptophan deaminase SOR Sorbitol
IND Indole production VP Voges-Proskauer RHA Rhamnose
SAC Sucrose MEL Melibiose AMY Amygdalin
ARA (L+) Arabinose GEL Gelatin hydrolysis
4. Determining the 7-Digit Profile Number
Scoring Method
The 20 tests are divided into groups of 3. Each test within a group is assigned a value:
• First test in group = 1
• Second test in group = 2
• Third test in group = 4
For each group, add the values of all positive results. The sum (0–7) becomes one digit of the profile
number. Seven groups produce the seven-digit code.
The Seven Groups
Group Test (value 1) Test (value 2) Test (value 4) Max sum Notes
1 ONPG ADH LDC 7
2 ODC CIT H2S 7
3 URE TDA IND 7
4 VP GEL GLU 7
5 MAN INO SOR 7
6 RHA SAC MEL 7
7 AMY ARA OXI* 7 *OXI = oxidase test
result
Example
If tests ONPG (+), ADH (-), LDC (+) in Group 1: score = 1 + 0 + 4 = 5. Repeat for all 7 groups to get
the complete 7-digit number, then look it up in the API 20E Profile Index Booklet or Recognition
System.
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The API 20E System — Comprehensive Notes | Harley-Prescott, Exercise 35 | Microbiology
Lookup Resources
• API 20E Profile Recognition System (primary)
• API 20E Quick Index Booklet
• Appendix G charts (alternative identification method)
5. Safety & Clinical Application
Safety Precautions
HAZARD WARNINGS
Nitrite test reagent B may be carcinogenic — wear disposable gloves, avoid skin contact and
aerosols. Nitrite test reagent A contains caustic acids — avoid skin contact. Ferric chloride solution is
an irritant — avoid skin contact, do not inhale vapors. Do not inhale zinc dust. No mouth pipetting. Be
careful with Bunsen burner flame.
Medical / Clinical Application
The API 20E system is used in clinical laboratories alongside the Enterotube II system to identify enteric
bacteria responsible for a range of human diseases:
Organism Associated Disease / Condition
Escherichia coli Opportunistic urinary tract infections
Proteus mirabilis Opportunistic urinary tract infections
Shigella dysenteriae Bacillary dysentery
Salmonella typhi Typhoid fever
Yersinia pestis Plague
6. Key Exam Points & Common Pitfalls
Must-Know Facts
• Oxidase test is performed FIRST — identifies oxidase-negative Enterobacteriaceae before strip
inoculation
• Turbidity must match McFarland No. 3 — incorrect turbidity gives unreliable results
• Platinum loops only — Nichrome wire loops must NOT be used
• Mineral oil creates anaerobic conditions in ADH, LDC, ODC, H2S, URE tubes
• CIT, VP, GEL: fill tube AND cupule (indicated by brackets on the strip)
• Fermentation reads bottom-up; oxidation reads top-down for carbohydrate tubes
• VP test: wait the full 10 minutes — pale pink immediately after reagent addition = negative
• IND test: read within 2 minutes — delayed brownish-red = negative (HCl artifact)
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The API 20E System — Comprehensive Notes | Harley-Prescott, Exercise 35 | Microbiology
• H2S: inspect tube bottom carefully — even a very thin black line = positive; 'browning' alone =
negative
• Orange reactions at 36-48h for ADH, LDC, ODC are interpreted as NEGATIVE
Learning Objectives Checklist
1. Understand why an oxidase test is first performed in the API 20E System
2. Correctly inoculate the API 20E System using proper filling techniques
3. Read and record the various biochemical reactions after 18 to 24 hours incubation
4. Determine a seven-digit profile number from the results
5. Look up the unknown bacterium in the API 20E Quick Index Booklet
Source: Harley-Prescott, Laboratory Exercises in Microbiology, Fifth Edition — Exercise 35: The API 20E System. BioMerieux Vitek,
Inc.
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