ENTEROPLURI-TEST

Identification system of Enterobacteriaceae and other gram negative, oxidase negative bacteria

A summary

Gascon, Priscilla Czraine Y.

BSMLS IV S.Y 2021-2022

I. Definition
EnteroPluri-Test allows the identification of Gram-negative bacilli belonging from the family
Enterobacteriaceae and other Gram negative, oxidase negative bacteria isolated from non-clinical samples. It
utilizes a 12-sector system consisting of special culture media with specific substrates that are simultaneously
inoculated, permitting the execution of 15 biochemical reactions. The identification of microorganism is done
after incubation for 18-24 hours at 36 ± 1°C by evaluating the color change of the different culture media and by
interpreting the biochemical reactions (positive and negative reactions) through a 5-digit number code obtained
using the Codebook.
II. Difference of “Traditional” methods from the EnteroPluri-Test in identifying the species under the Family
Enterobacteriaceae
a) Procedures/ Test included

TRADITIONAL METHOD ENTEROPLURI-TEST

Tests included:
A) Triple Sugar Iron Agar (TSIA) Reactions
Procedure:
1) Obtain colonies from the primary
isolation plate using an inoculating
needle. Observe aseptic technique.
2) Transfer the colonies to the TSIA tube
by stabbing the butt/deep within 2-3
mm from the bottom of the tube first.
3) After removing the inoculating needle
fro om the butt/deep, streak over the
agar surface/slant from the bottom to
the top with a back-and-forth motion.
4) Remove the inoculating needle from
the TSIA tube. Flame the mouth of the
tube then replace the cap/cotton plug.
5) Incubate the TSIA tube at 35-37°C for
18-24 hours. Loosen the screw caps (if
applicable) slightly to allow typical
reactions to occur.
6) Examine the results.
Interpretation:
K: Alkaline reaction (retention of original,
red-colored medium); non-fermentation of
the carbohydrates in the medium
A: Acidic reaction (color change from red
to yellow in the butt portion
only/throughout the medium); production
of acid from fermentation of
carbohydrates in the medium
Ag: Gas production; gas bubbles or agar
butt splitting
H2S production: blackening of the medium
that first appears or remains in the butt
portion
B) IMVIC Reactions
Procedure:
1) Obtain colonies form the primary
isolation plate then inoculate each of
Procedure:
o Perform Gram staining and
oxidase test on the
microorganism to be identified.
The microorganism must be
recently isolated (18-24 hours)
to ensure reliable results.
o Prepare the EnteroPluri-test
system from the package.
o Remove the cap on both sides
of the system.
o Obtain a well-isolated colony,
without flambing, from a
selective or nonselective agar
medium using the tip of the
inoculating wire (white cap)
without penetrating the agar.
o Inoculate the EnteroPluri-test
by twisting then withdrawing
the wire throughout the 12
compartments of the system
applying a turning motion.
o Reintegrate the wire into the
EnteroPluri-test with a turning
motion through the sectors of
the system until the breakage
notch on the wire is aligned
with the tube opening then
break the inoculating wire.
o The portion of the wire left in
the tube maintains the
anaerobic conditions needed
for the reactions of the
Glucose/Gas, Lysine and
Ornithine sectors.
o Using the broken portion of the
wire, punch holes through the
plastic film on the sectors of
Adonitol, Lactose, Arabinose,
 the tubes containing media such as Sorbitol, VP, Dulcitol/PA, Urea,
Tryptone broth, MR-VP broth and and Citrate as this would
Simmon’s citrate agar slant. Observe support aerobic growth.
aseptic technique. o Screw both caps again then
2) Incubate the tubes at 35-37°C for 24- incubate the EnteroPluri-test at
48 hours. The MR-VP broth culture 36 ± 1°C for 18-24 hours. Place
may be incubated up to 2-4 days. the tube vertically or on its flat
3) Examine the results. surface in a test tube holder;
Indole Test the sector Glucose/Gas must be
1) Add 15 drops of Ehrlich’s/Kovac’s pointing upward.
reagent on the tube. o Interpret and record all
2) Add 1 ml of xylene or chloroform if reactions aside from the Indole
Ehrlich’s reagent is used. and Voges-Proskauer.
Methyl Red Test o ADDITIONAL INSTRUCTIONS.
1) Add 5 drops of methyl red indicator a) H2S/Indole sector: Place the
directly to the MR broth (1st tube from EnteroPluri-test in an upright position
the equally divided MR-VP broth). (flat surface pointing upwards), punch
Voges Proskauer Test the plastic film then add 3-4 drops of
1) Add 0.6 ml of 5% alpha-naphthol. Kovacs’ reagent
2) Add 0.2 ml of 40% KOH. b) VP sector: add 3 drops of alpha-
3) Gently shake the tube to expose the naphthol and 2 drops of potassium
medium to atmospheric oxygen. hydroxid. Required if database with VP
4) Allow the tube to remain undisturbed is used; used as a confirmatory test if
for 10-15 minutes. database without VP is used.
Simmon’s Citrate Utilization Test o Construct the 5-digit code
1) Examine the agar slant for a color following the instructions
change in the medium. provided in the package insert
Interpretation: and identify the microorganism
Indole Test using the Codebook.
(+) bright fuschia red color at the interface Interpretation:
of the reagent of broth Color of culture media in different sectors
(-) yellow color or no color change SECTOR COLOR REACTIONS
Methyl Red Test (+) (-)
(+) red color on the surface of the medium Glucose/Gas yellow/ red/
(-) yellow to orange color lifted wax overlaying
Voges Proskauer Test wax
(+) red color Lysine violet yellow
(-) yellow color or no color change Ornithine violet yellow
Simmon’s Citrate Utilization Test H2S/Indole black- Beige/
(+) Prussian blue or deep blue color brown/pink- colorless
(-) no color change/retention of green red
color
Adonitol yellow red
C) Sulfide-Indole-Motility Test
Lactose yellow red
Procedure:
Arabinose yellow red
1) Obtain colonies from the primary
Sorbitol yellow red
isolation plate using an inoculating
VP red colorless
needle. Observe aseptic technique.
2) Transfer the colonies to a tube of SIM Dulcitol/PA Yellow/dark- green/
medium and inoculate by stabbing to crown green
2/3 of the distance to the bottom in Urea purple beige
the center of the butt/deep. Citrate blue green
3) Loosen the cap then incubate the tube Code number forming: Database with VP
at 35-37°C from 18-24 hours. o The 15 biochemical tests are
 4) Examine the results. divided into 5 groups wherein
Interpretation: each contains 3 tests.
Motility: turbidity throughout the medium Group 1 Glucose
or diffuse growth away from the stab line Gas
H2S production: blackening along the stab Lysine
line or of the medium Group 2 Ornithine
Indole production: pink or red color after H2S
addition of 3-4 drops of Ehrlich’s or Kovac’s Indole
reagent Group 3 Adonitol
D) Lysine Iron Agar (LIA) Reactions Lactose
Procedure: Arabinose
1) Obtain colonies form the primary Group 4 Sorbitol
isolation plate using an inoculating VP
needle. Observe aseptic technique.
Dulcitol
2) Transfer the colonies to the LIA tube
Group 5 PA
by stabbing the butt/deep twice from
Urea
the bottom of the tube first.
Citrate
3) After removing the inoculating needle
from the butt/deep, streak over the Each test is indicated with a positivity
value of 4, 2, 1.
agar surface/slant from the bottom to
the top with a back-and-forth motion. o Value 4: first test positive in
4) Incubate the LIA tube at 35-37°C for each group
18-24 hours. Loosen the screw caps (if o Value 2: second test positive in
applicable) slightly to allow typical each group
reactions to occur. o Value 1: third test positive in
5) Examine the results. each group
Interpretation: o Value 0: every negative tests
Lysine decarboxylation: alkaline (purple) o Add the number of positive
slant reactions in each group to form
Lysine deamination: acid (red) slant a 5-digit code. Use the
H2S production: black precipitate Codebook to identify the
microorganism under
examination.
Code number forming: Database without VP
o The 14 biochemical tests are
divided into 5 groups wherein
Group 1 consists of 2 tests,
while Groups 2-5 consist of 3
tests.
Group 1 Glucose
Gas
Group 2 Lysine
Ornithine
H2S
Group 3 Indole
Adonitol
Lactose
Group 4 Arabinose
Sorbitol
Dulcitol
Group 5 PA
Urea
Citrate
 Each test is indicated with a positivity
value of 4, 2, 1.
o Value 4: first test positive in
groups 2,3,4,5
o Value 2: first test positive in
first group and second test
positive in the remaining
groups
o Value 1: second test positive in
first group and third test
positive in the remaining
groups
o Value 0: every negative tests
2) Add the number of positive reactions in
each group to form a 5-digit code. Use
the Codebook to identify the
microorganism under examination.
Note: The presence of asterisk (*) beside the
identification represents the presence of rare
organism. In the case where the rare organism
is considered the best choice, the purity of the
isolate must be checked, and the inoculation
procedure must be repeated. Moreover,
confirmatory test must be done on rare
organism encountered as first choice with
common microorganism listed.
Procedure and interpretation adapted from
Liofilchem.

b) Component/s or Reagent/s of the tests

TRADITIONAL METHOD ENTEROPLURI-TEST

A) Triple Sugar Iron Agar (TSIA) Reactions System with 12 agar media
Formula Liter o Glucose/Gas
Beef Extract 3.0 g/L o Lysine
Yeast Extract 3.0 g/L o Ornithine
Peptone Mixture 20.0 g/L o H2S/Indole
Sodium Chloride 5.0 g/L o Adonitol
Lactose 10.0 g/L o Lactose
Sucrose 10.0 g/L o Arabinose
Glucose 1.0 g/L o Sorbitol
Ferric Citrate 0.3 g/L o VP
Sodium 0.3 g/L o Dulcitol/PA
Thiosulphate o Urea
Phenol Red 0.025 o Citrate
g/L Reagents included:
Agar 12.0 g/L KOVÁCS REAGENT
Adapted from NEOGEN® Culture Media Ingredient Amoun
B) IMVIC Reactions t
Indole Test Amyl or isoamyl alcohol, 150.0
TRYPTONE BROTH
 Ingredient Amount reagent grade (butyl alcohol ml
Tryptone 10.0 g may be substituted)
Sodium chloride 5.0 g p- 10.0 g
dimethylaminobenzaldehyd
EHRLICH’S REAGENT e (DMAB)
Ingredient Amoun HCl (concentrated) 50.0 ml
t Adapted from American Society for
Ethyl alcohol (absolute 95.0 ml Microbiology
p- 1.0 g VP REAGENTS
dimethylaminobenzaldehyd Ingredient Amount
e (DMAB Alpha-naphthol, 5%
HCl (concentrated) 20.0 ml Alpha-naphthol 5g
Absolute ethyl 100 ml
KOVÁCS REAGENT alcohol
Ingredient Amoun Potassium hydroxide, 40%
t Potassium 40 g
Amyl or isoamyl alcohol, 150.0 hydroxide
reagent grade (butyl alcohol ml Distilled water 100 ml
may be substituted) Adapted from American Society for
p- 10.0 g Microbiology
dimethylaminobenzaldehyd
e (DMAB)
HCl (concentrated) 50.0 ml
Adapted from American Society for
Microbiology
Methyl Red Test
MR-VP BROTH
Ingredient Amount
Buffered peptone 7.0 g
Dipotassium 5.0 g
phosphate
Dextrose 5.0 g

METHYL RED INDICATOR

Ingredient Amount
Methyl red 0.1 g
95% ethyl alcohol 300 ml
Distilled water 200 ml
Adapted from American Society for
Microbiology
Voges Proskauer Test
MR-VP BROTH
Ingredient Amount
Buffered peptone 7.0 g
Dipotassium 5.0 g
phosphate
Dextrose 5.0 g

VP REAGENTS
Ingredient Amount
Alpha-naphthol, 5%
Alpha-naphthol 5g
 Absolute ethyl 100 ml
alcohol
Potassium hydroxide, 40%
Potassium 40 g
hydroxide
Distilled water 100 ml
Adapted from American Society for
Microbiology
Simmon’s Citrate Utilization Test
Ingredients Gms / Litre
Magnesium 0.200
sulphate
Ammonium 1.000
dihydrogen
phosphate
Dipotassium 1.000
phosphate
Sodium citrate 2.000
Sodium chloride 5.000
Bromothymol blue 0.080
Agar 15.000
Final pH ( at 25°C) 6.8±0.2
**Formula adjusted, standardized to
suit performance parameters
Adapted from HiMedia Laboratories

C) Sulfide-Indole-Motility Test
SIM medium
Ingredients Amount
Peptone 30.0 g
Beef extract 3.0 g
Ferrous 0.2 g
ammonium
sulfate
Sodium 0.025 g
thiosulfate
Agar 3.0 g
Adapted from American Society for
Microbiology
D) Lysine Iron Agar (LIA) Reactions
DIFCO™ LYSINE IRON AGAR
Peptone 5.0 g
Yeast Extract 3.0 g
Dextrose 1.0 g
L-Lysine HCl 10.0 g
Ferric Ammonium 0.5 g
Citrate
Sodium 0.04 g
Thiosulfate
Bromcresol Purple 0.02 g
Agar 15.0 g
 Adapted from BD Legacy

c) Advantages

TRADITIONAL METHOD ENTEROPLURI-TEST

Convenient: It is a ready to use tube system.
Rapid identification of Enterobacteriaceae
Minimal equipment required
Time wise: Biochemical tests are inoculated at
once.

d) Disadvantages
TRADITIONAL METHOD ENTEROPLURI-TEST
Labour intensive: It is not easy to prepare, and it Prone to contamination:
is not suitable for use in urgent medical
diagnosis.
Time consuming: It requires manual preparation Difficulty of interpretation
of culture media such as MacConkey agar,
chocolate agar, blood agar etc.
Media carry-over from neighboring chambers

3) Personal reaction of insights on the presented article

mas onting culture media

mas lesser prep

4) Bibliography

American Society for Microbiology. (2016). Indole test protocol. Retrieved from:
https://asm.org/getattachment/200d3f34-c75e-4072-a7e6-df912c792f62/indole-test-protocol-3202.pdf
American Society for Microbiology. (2016). Methyl red and Voges-Proskauer test protocol. Retrieved from:
https://asm.org/getattachment/0c828061-9d6f-4ae7-aea3-66e1a8624aa0/Methyl-Red-and-Voges-Proskauer-
Test-Protocols.pdf
BD Legacy. (n.d). Lysine iron agar. Retrieved from:
https://legacy.bd.com/europe/regulatory/Assets/IFU/Difco_BBL/211363.pdf
HiMedia Laboratories Pvt. Limited. (2019, March). Simmon’s citrate agar. Retrieved from:
https://himedialabs.com/TD/M099.pdf
Kelly, M. T., & Latimer, J. M. (1980). Comparison of the automicrobic system with API, enterotube, micro-ID, micro-
media systems, and conventional methods for identification of Enterobacteriaceae. Journal of clinical
microbiology, 12(5), 659–662. https://doi.org/10.1128/jcm.12.5.659-662.1980
Liofilchem. (2021). EnteroPluri-Test. Retrieved from: https://www.liofilchem.com/featured-products/enteropluri-
test.html
Neogen Corporation. (2021). Triple sugar iron (TSI) agar. Retrieved from:
https://www.neogen.com/categories/microbiology/triple-sugar-iron-tsi-agar/