Harley−Prescott: V: Rapid Multitest Systems 35.

The API 20E System © The McGraw−Hill

Laboratory Exercises in Companies, 2002
Microbiology, Fifth Edition

E X E RC I S E
35
The API 20E System

test-tube rack
SAFETY CONSIDERATIONS
wax pencil
Be careful with the Bunsen burner flame. No mouth
pipetting. Since nitrite test reagent B might be carcino-
disposable gloves
genic, wear gloves and avoid skin contact or aerosols.
The acids in the nitrite test reagent A are caustic. The fer- Learning Objectives
ric chloride solution is an irritant. Avoid skin contact and Each student should be able to
do not breathe the vapors. Do not breathe the zinc dust.
1. Understand why an oxidase test is first performed
in the API 20E System
2. Correctly inoculate the API 20E System
Materials per Group of Students
3. Read and record the various biochemical
API 20E System strip, incubator tray, and cover reactions that occur after 18 to 24 hours of
(bioMérieux Vitek, PO Box 42016, incubation
Hazelwood, MO 63042–2395. Phone 4. Determine a seven-digit profile number
800–638–4835). 5. Look up the unknown bacterium in the API 20E
tryptic soy agar slant or plate culture of unknown Quick Index Booklet
member of the Enterobacteriaceae
5 ml of sterile 0.85% saline in test tube with cap Suggested Reading in Textbook
50-ml plastic squeeze bottle containing tap water
1. The Enterobacteriaceae, section 22.3.
5-ml Pasteur pipettes with pipettor
2. Rapid Methods of Identification, section 36.2; see
oxidase test reagent or disk/strip
also figures 36.7 and 36.8.
petri plate
TSA plate
Whatman No. 2 filter paper
10% ferric chloride Medical Application
Barritt’s reagents A and B
sterile mineral oil The API 20E system and Enterotube II system are used in the
tube containing McFarland No. 3 (BaSO4) clinical laboratory for the identification of enteric bacteria.
reference standard Among them are Escherichia coli (opportunistic urinary tract
platinum inoculating loop (Nichrome wire loops infections), Proteus mirabilis (opportunistic urinary tract in-
fections), Shigella dysenteriae (bacillary dysentery), Salmo-
should NOT be used) or a wooden applicator
nella typhi (typhoid fever), and Yersinia pestis (plague).
stick
Bunsen burner
Kovacs’ reagent
nitrite test reagents A and B solution
Principles
zinc dust or 20-mesh granular zinc The API 20E System is a standardized, miniaturized
1.5% hydrogen peroxide version of conventional biochemical procedures used
incubator set at 35° to 37°C in the identification of Enterobacteriaceae and other
API 20E Quick Index Booklet gram-negative bacteria. A total of 127 taxa can be

208
 Harley−Prescott: V: Rapid Multitest Systems 35. The API 20E System © The McGraw−Hill
Laboratory Exercises in Companies, 2002
Microbiology, Fifth Edition

Figure 35.1 The API 20E Manual Biochemical System for

Add several drops of the oxidase reagent. Note
Microbial Identification. (a) Positive results. (b) Negative results. the color change (see exercise 30, figure 30.1).
You may also use oxidase test strips or disks as
described in exercise 30.
2. Transfer another loopful of bacteria to the test tube
containing 5 ml sterile saline. (If an applicator stick
(a) All tests: positive
is used, load the tip with bacteria from the colony.)
Recap and shake the tube. The turbidity should
match that of the McFarlandNo. 3 (BaSO4)
standard. Add more bacteria if necessary.
3. Label the elongated flap of the incubation tray
with your name and date. From the wash bottle,
(b) All tests: negative
add 5 ml of water to the bottom of the tray. This
provides a humid atmosphere during incubation.
4. Remove the plastic API strip from the sealed
pouch and place it in the incubation tray. Reseal
identified with this system. It is a ready-to-use, micro- the pouch to protect the remaining strips.
tube system that performs 22 standard biochemical 5. Shake the 5 ml of bacterial suspension. Remove
tests on pure bacterial cultures from appropriate, pri- the cap and fill the 5-ml Pasteur pipette with the
mary isolation media. bacterial suspension. (Tilt the strip to avoid
This system consists of a strip containing 20 forming bubbles.) Inoculate the strip as follows:
chambers (figure 35.1), each consisting of a micro- a. Tilt the API 20E incubation tray and place
tube and a depression called a cupule. The tubes con- the pipette tip against the side of each
cupule. Fill the tubes of the ONPG, TDA,
Cupule IND, GLU, MAN, INO, SOR, RHA, SAC,
Tube
MEL, AMY, and ARA microtubes.
b. Slightly underfill the ADH, LDC, ODC,
H2S, and URE microtubes. Note that these
tain dehydrated substrates. The substrates are rehy-
microtubes are underlined on the test strip!
drated by adding a bacterial saline suspension. To
c. Fill both the tube and cupule section of the
create anaerobic conditions, sterile mineral oil is
 CIT ,  VP , and  GEL  tubes. Note that
added to several of the microtubes. The strip of
these microtubes have brackets on the test strip!
microtubes is then incubated for 18 to 24 hours at
d. After inoculation, completely fill the cupule
35° to 37°C so that the bacterium can act on the sub-
section of the ADH, LDC, ODC, H2S, and
strates. The strip is read by noting color changes
URE tubes with mineral oil. This creates
after the various indicator systems have been af-
anaerobic conditions.
fected by the metabolites or added reagents (table
6. Place the lid on the incubation tray and incubate
35.1). The identification of the unknown bacterium is
at 35°C for 18 to 24 hours. If the strip cannot be
achieved by determining a seven-digit profile index
read after 24 hours, place it in a refrigerator at 2°
number and consulting the API 20E Profile Recog-
to 8°C until the reactions can be read.
nition System or the API 20E Profile Index Book-
7. Make an isolation streak on a TSA plate with a
let. Charts can also be used to determine the un-
portion of the bacterial suspension to ascertain the
known bacterium (see appendix G).
purity of the suspension.
Second Period
Procedure
1. After 18 hours and before 24 hours incubation,
First Period record all reactions not requiring the addition of
1. Using aseptic technique (see figure 14.3) and an reagents (do not read TDA, VP, and IND). Table
applicator stick or inoculating loop, select a well- 35.1 summarizes these reactions. Record these
isolated colony from the unknown streak plate or results by placing a + for a positive reaction and a
from the pure culture slant. Smear a small amount – for a negative reaction in the 24-hour box for
of the colony over a small area of filter paper. the report for exercise 35. Also record the colors.

The API 20E System 209

 Harley−Prescott: V: Rapid Multitest Systems 35. The API 20E System © The McGraw−Hill
Laboratory Exercises in Companies, 2002
Microbiology, Fifth Edition

Table 35.1 Chart I. Summary of Results—18- to 24-Hour Procedure

Interpretation of Reactions
Tube Positive Negative Comments
ONPG Yellow Colorless (1) Any shade of yellow is a positive reaction.
(2) VP tube, before the addition of reagents, can be used as a negative control.
ADH Incubation Orange reactions occurring at 36 to 48 hours should be interpreted as negative.
18–24 h Red or orange Yellow
36–48 h Red Yellow or
orange
LDC 18–24 h Red or orange Yellow Any shade of orange within 18 to 24 hours is a positive reaction. At 36 to 48 hours, orange
36–48 h Red Yellow or orange decarboxylase reactions should be interpreted as negative.
ODC 18–24 h Red or orange Yellow Orange reactions occurring at 36 to 48 hours should be interpreted as negative.
36–48 h Red Yellow or orange
CIT Turquoise or dark Light green (1) Both the tube and cupule should be filled. (2) Reaction is read in the aerobic (cupule) area.
blue or yellow
H2S Black No black (1) H2S production may range from a heavy black deposit to a very thin black line around the
deposit deposit tube bottom. Carefully examine the bottom of the tube before considering the reaction
negative. (2) A “browning” of the medium is a negative reaction unless a black deposit is
present. “Browning” occurs with TDA positive organisms.
URE 18–24 h Red or orange Yellow A method of lower sensitivity has been chosen.
36–48 h Red Yellow or orange Klebsiella, Proteus, and Yersinia routinely give positive reactions.
TDA Add 1 drop 10% ferric chloride (1) Immediate reaction. (2) Indole positive organisms may produce a golden orange color
due to indole production. This is a negative reaction.
Brown-red Yellow
IND Add 1 drop Kovacs’ reagent (1) The reaction should be read within 2 minutes after the addition of the Kovacs’ reagent
and the results recorded. (2) After several minutes, the HCI present in Kovacs’ reagent may
Red ring Yellow react with the plastic of the cupule resulting in a change from a negative (yellow) color to a
brownish-red. This is a negative reaction.
VP Add 1 drop of 40% potassium hydroxide, then 1 (1) Wait 10 minutes before considering the reaction negative. (2) A pale pink color (after 10
drop of 6% α-naphthol. minutes) should be interpreted as negative. A pale pink color which appears immediately
after the addition of reagents but which turns dark pink or red after 10 minutes should be
Red Colorless interpreted as positive.
Motility may be observed by hanging drop or wet-mount preparation.
GEL Diffusion of the No (1) The solid gelatin particles may spread throughout the tube after inoculation. Unless
pigment diffusion diffusion occurs, the reaction is negative. (2) Any degree of diffusion is a positive reaction.
GLU Yellow Blue or Fermentation (Enterobacteriaceae, Aeromonas, Vibrio)
or gray blue-green (1) Fermentation of the carbohydrates begins in the most anaerobic portion (bottom)
of the tube. Therefore, these reactions should be read from the bottom of the tube to
the top. (2) A yellow color at the bottom of the tube only indicates a weak or delayed
Comments for all

positive reaction.
carbohydrates

MAN Yellow Blue or Oxidation (Other gram negatives)

INO blue-green (1) Oxidative utilization of the carbohydrates begins in the most aerobic portion
SOR (top) of the tube. Therefore, these reactions should be read from the top to the
RHA bottom of the tube. (2) A yellow color in the upper portion of the tube and a
SAC blue color in the bottom of the tube indicates oxidative utilization of the sugar.
MEL This reaction should be considered positive only for nonEnterobacteriaceae
AMY gram-negative rods. This is a negative reaction for fermentative organisms such
ARA as Enterobacteriaceae.
GLU After reading GLU reaction, add 2 drops 0.8% (1) Before addition of reagents, observe GLU tube (positive or negative) for bubbles.
sulfanilic acid and 2 drops 0.5% N,N-dimethyl-Ȋ- Bubbles are indicative of reduction of nitrate to the nitrogenous (N2) state. (2) A
naphthylamine positive reaction may take 2 to 3 minutes for the red color to appear. (3) Confirm a
Nitrate NO2 Red Yellow negative test by adding zinc dust or 20-mesh granular zinc. A pink-orange color after
reduction N2 gas Bubbles; Yellow Orange after 10 minutes confirms a negative reaction. A yellow color indicates reduction of nitrates to
after reagents reagents nitrogenous (N2) state.
and zinc and zinc
MAN After reading carbohydrate reaction, add 1 drop (1) Bubbles may take 1 to 2 minutes to appear. (2) Best results will be obtained if the test
INO 1.5% H2O2 is run in tubes that have no gas from fermentation.
SOR
Catalase Bubbles No bubbles

The following abbreviations are used: ONPG (ȋ-galactosidase), ADH (arginine dihydrolase), LDC (lysine decarboxylase), ODC (ornithine decarboxylase), CIT (citrate), H2S
(hydrogen sulfide), URE (urease), TDA (tryptophan deaminase), IND (indole), VP (Voges-Proskauer), GEL (gelatin), GLU (glucose), MAN (mannitol), INO (inositol), SOR
(sorbitol), RHA (rhamnose), SAC (sucrose), MEL (melibiose), AMY (amygdalin), ARA (arabinose).
Copyright BioMerieux Vitek, Inc., Hazelwood, MO. Reprinted by permission.

210 Rapid Multitest Systems

 Harley−Prescott: V: Rapid Multitest Systems 35. The API 20E System © The McGraw−Hill
Laboratory Exercises in Companies, 2002
Microbiology, Fifth Edition

Chart II. Symbol Interpretation of API 20E System Components

Tube Chemical/Physical Principles Reactive Ingredients Quantity
ONPG Hydrolysis of ONPG by beta-galactosidase releases yellow ONPG 0.2 mg
orthonitrophenol from the colorless ONPG; ITPG ITPG 8.0 Ȗg
(isopropylthiogalactopyranoside) is used as inducer.
ADH Arginine dihydrolase transforms arginine into ornithine, ammonia, and Arginine 2.0 mg
carbon dioxide. This causes a pH rise in the acid-buffered system and a
change in the indicator from yellow to red.
LDC Lysine decarboxylase transforms lysine into a basic primary amine, Lysine 2.0 mg
cadaverine. This amine causes a pH rise in the acid-buffered system and
a change in the indicator from yellow to red.
ODC Ornithine decarboxylase transforms ornithine into a basic primary amine, Ornithine 2.0 mg
putrescine. This amine causes a pH rise in the acid-buffered system and
a change in the indicator from yellow to red.
CIT Citrate is the sole carbon source. Citrate utilization results in a pH rise Sodium Citrate 0.8 mg
and a change in the indicator from green to blue.
H2S Hydrogen sulfide is produced from thiosulfate. The hydrogen sulfide Sodium Thiosulfate 80.0 Ȗg
reacts with iron salts to produce a black precipitate.
URE Urease releases ammonia from urea; ammonia causes the pH to rise and Urea 0.8 mg
changes the indicator from yellow to red.
TDA Tryptophan deaminase forms indolepyruvic acid from tryptophane. Tryptophane 0.4 mg
Indolepyruvic acid produces a brownish-red color in the presence of
ferric chloride.
IND Metabolism of tryptophane results in the formation of indole. Kovacs’ Tryptophane 0.2 mg
reagent forms a colored complex (pink to red) with indole.
VP Acetoin, an intermediary glucose metabolite, is produced from sodium Sodium Pyruvate 2.0 mg
pyruvate and indicated by the formation of a colored complex. Creatine 0.9 mg
Conventional VP tests may take up to four days, but by using sodium
pyruvate, API has shortened the required test time. Creatine intensifies
the color when tests are positive.
GEL Liquefaction of gelatin by proteolytic enzymes releases a black pigment Kohn Charcoal Gelatin 0.6 mg
which diffuses throughout the tube.

GLU Utilization of the carbohydrate results in acid formation and a consequent Glucose 2.0 mg
MAN pH drop. The indicator changes from blue to yellow. Mannitol 2.0 mg
INO Inositol 2.0 mg
SOR Sorbitol 2.0 mg
RHA Rhamnose 2.0 mg
SAC Sucrose 2.0 mg
MEL Melibiose 2.0 mg
AMY Amygdalin 2.0 mg
ARA (L +) Arabinose 2.0 mg
GLU Nitrites form a red complex with sulfanilic acid and N, N-dimethylalpha- Potassium Nitrate 80.0 Ȗg
naphthylamine. In case of negative reaction, addition of zinc confirms
Nitrate the presence of unreduced nitrates by reducing them to nitrites (pink-
Reduction orange color). If there is no color change after the addition of zinc, this
is indicative of the complete reduction of nitrates through nitrites to
nitrogen gas or to an anaerogenic amine.
MAN Catalase releases oxygen gas from hydrogen peroxide.
INO
SOR

Catalase

Copyright BioMerieux Vitek, Inc., Hazelwood, MO. Reprinted by permission.

The API 20E System 211