LABORATORY #9
Packaged Microbial Identification Systems
Laboratory #9
Packaged Microbial Identification Systems
Skills= 10 points
Objective:
At the completion of this lab, the student should be able to:
1. Demonstrate the reaction of Escherichia coli or Klebsiella pneumoniae with the API-20E
strip.
2. Summarize the principles of the tests of the API 20E test strip.
Materials:
TSA culture of Escherichia coli or Klebsiella pneumoniae
1-API-20E strip
Clean test tube
Sterile saline or deionized water
Mineral oil
Oxidase reagent
10% FeCl3
Kovac's reagent (Indole)
40% KOH
6% Alpha-naphthol
NIT 1 (API reagent)
NIT2 (API reagent)
References:
1. API-20E Biochemical Identification System (bioMerieux Products, Inc.) package insert,
API 20E Data Base CD, API website
2. http://www.jlindquist.net/generalmicro/102bactid2.html
Overview:
Rapid and multitest procedures represent ingenious, time and cost saving approaches to the
identification and classification of microorganisms, especially bacteria. These procedures
enable us to perform a larger number of biochemical tests on an organism than is feasible via
conventional means. Because of this feature, they not only identify an organism to its genus
and species, but also to its type.
MLAB 2534 – Laboratory 9 – 1
LABORATORY #9
Packaged Microbial Identification Systems
API-20E
Principle:
The API-20E kit includes a plastic strip with 20 miniaturized chambers containing
dehydrated substrates and a plastic incubation chamber with a loose fitting lid. Each
chamber is divided into two parts, the tube portion and the cupule (small cup) portion.
Each chamber has a small hole at the top through which the bacterial suspension can be
inoculated with a pipette. Once the bacterial suspension is added to the media, the media
becomes reconstituted. During incubation, metabolism of the substrates 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 manufacturer’s identification
software.
Procedure:
1. Perform an oxidase test using bacteria from a BAP plate, then record the result on
your result sheet to assist in determining the final bacterial identification.
2. Set up an incubation try and lid. Label the API strip with either the patient or
organism name, on the elongated flap of tray.
3. The lid is removed from the tray and approximately 5 ml of tap water is added to
provide a humid atmosphere during incubation.
4. An API-20E strip is removed from the sealed envelope and placed in the
incubation tray.
5. A bacterial suspension of the test organism is made by suspending the cells from a
well-isolated colony in 5 ml of deionized water. The colonies should come from an
18-24 hour old culture from the BAP plate.
6. Using a plastic pipette, fill the CIT, VP, and GEL CUPULE AND TUBE with the
bacterial suspension.
7. Using the same pipette, fill ONLY THE TUBE of the remaining tests.
8. Create an anaerobic condition in the tests ADH, LDC, ODC, H2S, and URE by
overlaying with mineral oil.
MLAB 2534 – Laboratory 9 – 2
LABORATORY #9
Packaged Microbial Identification Systems
9. Place the lid on the incubation chamber and incubate at 35-37 oC for 18-24 hours.
Reading the strip:
1. After the incubation period, read the strip by referring to the reading table below.
Procedure notes:
(1) A very pale yellow should also be considered positive.
(2) An orange color after 36-48 hours incubation must be considered negative.
(3) Reading made in the cupule (aerobic).
(4) Fermentation begins in the lower portion of the tube; oxidation begins in the
cupule.
(5) A slightly pink color after 10 minutes should be considered positive.
Tests
ONPG
ADH
LDC
ODC
Reactions or
Enzymes
ß-galactosidase
arginine dihydrolase
lysine decarboxylase
CIT
ornithine
decarboxylase
citrate utilization
H2S
H2S production
URE
TDA
urease
tryptophan
deaminase
IND
indole production
VP
acetoin production
(Vogues Proskauer)
GEL
gelatinase
GLU
fermentation
or oxidation (4)
MAN
fermentation
or oxidation (4)
fermentation
or oxidation (4)
fermentation
or oxidation (4)
fermentation
or oxidation (4)
fermentation
or oxidation (4)
fermentation
or oxidation (4)
fermentation
or oxidation (4)
fermentation
INO
SOR
RHA
SAC
MEL
AMY
ARA
Results
NEGATIVE
colorless
yellow
yellow
yellow
POSITIVE
yellow (1)
red / orange (2)
red / orange (2)
red / orange (2)
pale green / yellow
blue-green
/ blue (3)
colorless / greyish
black deposit / thin
line
yellow
red / orange (2)
TDA / immediate
yellow
reddish brown
KOVAC’S /immediate
colorless
pale green / yellow
pink
VP 1 + VP 2 / 10 min.
colorless
pink / red (5)
no diffusion
diffusion of
black pigment
blue / blue-green
yellow /
greyish yellow
blue / blue-green
yellow
blue / blue-green
yellow
blue / blue-green
yellow
blue / blue-green
yellow
blue / blue-green
yellow
blue / blue-green
yellow
blue / blue-green
yellow
blue / blue-green
yellow
MLAB 2534 – Laboratory 9 – 3
LABORATORY #9
Packaged Microbial Identification Systems
OX
Tests
ONPG
ADH
LDC
ODC
or oxidation (4)
cytochrome oxidase
Reactions or
Enzymes
ß-galactosidease
arginine diydrolase
lysine decarboxylase
CIT
ornithine
decarboxylase
citrate utilization
H2S
H2S production
URE
TDA
urease
tryptophane
deaminase
IND
indole production
VP
acetoin production
(Vogues Proskauer)
GEL
gelatinase
GLU
fermentation
or oxidation (4)
MAN
fermentation
or oxidation (4)
fermentation
or oxidation (4)
fermentation
or oxidation (4)
fermentation
or oxidation (4)
fermentation
or oxidation (4)
fermentation
or oxidation (4)
fermentation
or oxidation (4)
fermentation
or oxidation (4)
cytochrome oxidase
INO
SOR
RHA
SAC
MEL
AMY
ARA
OX
oxidase test -- See test package insert
Results
NEGATIVE
colorless
yellow
yellow
yellow
POSITIVE
yellow (1)
red / orange (2)
red / orange (2)
red / orange (2)
pale green / yellow
blue-green
/ blue (3)
colorless / greyish
black deposit / thin
line
yellow
red / orange (2)
TDA / immediate
yellow
reddish brown
KOVAC’S /immediate
colorless
pale green / yellow
pink
VP 1 + VP 2 / 10 min.
colorless
pink / red (5)
no diffusion
diffusion of
black pigment
blue / blue-green
yellow /
greyish yellow
blue / blue-green
yellow
blue / blue-green
yellow
blue / blue-green
yellow
blue / blue-green
yellow
blue / blue-green
yellow
blue / blue-green
yellow
blue / blue-green
yellow
blue / blue-green
yellow
oxidase test -- See test package insert
2. If three (3) or more tests are positive, record all the spontaneous reactions on the
results sheet, this will determine whch tests require the addition of other
reagents.
3. In the TDA well, add one drop of ferric chloride. A reddish brown color indicates a
positive reaction to be recorded on the results sheet.
4. In the VP well, add 1 drop of 40% KOH followed by 1 drop of 6% alpha naphthol.
MLAB 2534 – Laboratory 9 – 4
LABORATORY #9
Packaged Microbial Identification Systems
Wait 10 minutes. A pink or red color indicates a positive reaction to be recorded
on the result sheet. If a slightly pink reaction appears after 10 minutes, the
reaction should be considered negative.
5. In the IND well, add 1 drop of Kovac’s reagent. A pink color developed in the entire
cupule indicates a positive reaction to be recorded on the results sheet.
Interpretation of the strip:
1. On the result sheet, the tests are separated into groups of 3 and the value 1,2,or 4
is indicated for each. By adding together the values corresponding to the positive
reactions within each group, a 7-digit profile number is obtained for the 20 tests
for the API 20 strip. The oxidase reaction constitutes the 21st test and has a value
of 4 if it is positive.
2. Once the student has the 7-digit profile this can be converted to a bacterial
identification by using a profile register, which is accessed via the API CD. The
instructor will direct the student in the use of this reference.
3. If the code is not discriminatory enough, the following additional tests should be
performed.
4. Add 1 drop of Nitrate 1 and nitrate 2 reagents to the GLU tube. Wait 2 to 5
minutes. A red color indicates a positive reaction. This indicates that nitrates were
reduced to nitrites. A negative reaction (yellow) may be due to the reduction to
nitrogen. Add 2-3 mg of Zinc reagent to the GLU tube. After 5 minutes, if the tube
remains yellow this indicates a positive reaction which should be recorded on the
results sheet. If the tube turns orange-red, this is a negative reaction.
MLAB 2534 – Laboratory 9 – 5
LABORATORY #9
Packaged Microbial Identification Systems
Summary of Principles for the API 20E Identification Tests:
Tests
ONPG
ADH
LDC
ODC
CIT
H2S
URE
TDA
IND
VP
GEL
GLU
MAN
INO
SOR
RHA
SAC
MEL
AMY
ARA
Reactions
Hydrolysis of ONPG (colorless) by betagalactosidase releases orthonitrophenol
(yellow).
Arginine dihydrolase transforms arginine into ornithine, ammonium and CO2.
This causes a pH rise in the acid-buffered medium
and a change in the indicator from yellow to red.
Lysine decarboxylase transforms lysine into a basic primary amine, cadaverine.
This amine causes a pH rise in the acid-buffered
medium and changes the indicator from yellow to red
Ornithine decarboxylase transforms ornithine into a basic primary amine,
putrescine. This amine causes a pH rise in the acid-buffered
medium and changes the indicator from yellow to red.
Sodium citrate is the sole carbon source. Citrate utilization results in a pH rise
and changes the indicator from green to blue.
Hydrogen sulfide is produced from sodium thiosulfate. The H2S reacts with iron
salts to produce a black precipitate.
Urease releases ammonia from urea. Ammonia causes the pH to rise and
changes the indicator from yellow to red.
Trytophan deaminase forms indolepyruvic acid from tryptophan.
Indolepyruvic acid produces a brownish-red color in the presence of ferric
chloride.
Metabolism of tryptophan results in the formation of indole.
Kovac’s reagent forms a colored complex (pink to red) with indole..
Acetoin, an intermediary glucose metabolite, is produced from sodium
pyruvate and indicated by the formation of a colored
complex. Creatine intensifies the color when tests are
positive.
Liquification of Kohn’s charcoal impregnated gelatin by gelatinase
releases a black pigment which diffuses throughout the tube.
Utilization (fermentation/oxidation) of the carbohydrate results in acid
formation. Acid produces a drop in pH and changes the
indicator from blue to yellow. Respective reactive ingredients; glucose,
mannitol, inositol, sorbitol, rhamnose, sucrose,
melibiose, amygdalin, and (L+) arabinose. Fermentation
(Enterobacteriaceae) Read reaction from bottom of tube to the top.
Yellow color at bottom of tube only is a weak or delayed positive reaction.
Oxidation (not Enterobacteriaceae) Read reaction from top of tube to the
bottom. Yellow color in upper portion of the tube and blue on the
bottom.
MLAB 2534 – Laboratory 9 – 6
LABORATORY #9
Packaged Microbial Identification Systems
Name: _____________
Date:_____________________
Laboratory #9: Packaged Microbial Identification System
Result Form
Points = 10
Sample/patient name:_________________________________________________
I.D. Number (from worksheet below):
Organism Identification:
WORKSHEET
MLAB 2534 – Laboratory 9 – 7