Comparison of a Commercial Identification Kit and
Conventional Biochemical Tests Used for the Identification
of Enteric Gram-negative Rods
LAURENCE R. MCCARTHY, PH.D., JOAN B. MAYO, B.S., ASCP, GERALDINE BELL, B.S.,
AND DONALD ARMSTRONG, M.D.
Infectious Disease Service, and the Diagnostic
Microbiology Laboratory, Memorial Sloan-Kettering
Cancer Center, and Cornell University Medical Center,
New York, New York
McCarthy, Laurence R., Mayo, Joan B., Bell, Geraldine, and
Armstrong, Donald: Comparison of a commercial identification kit and conventional biochemical tests used for the
identification of enteric gram-negative rods. Am J Clin Pathol
69: 161-164, 1978. A comparison between 11 Minitek biochemical tests and corresponding conventional tubed media
was undertaken with 1,089 isolates of enteric gram-negative
rods. Overall correlation between Minitek and conventional
biochemicals was 97.4%. Minitek proved to be a time- and
space-saving miniaturized biochemical system that can be used
effectively for the identification of enteric gram-negative rods.
(Key words: Minitek®; Gram-negative rods; Bacterial identification.)
inocula for conventional tubed biochemical media and
the Minitek inoculum broth (BBL). Through the use
of the Minitek pipetter (BBL), 0.05-ml volumes of
the seeded Minitek broth were used to inoculate six
to ten wells of Minitek plates containing substrateimpregnated disks. All wells except those containing
phenylalanine, ortho-nitrophenyl-B-D galactopyranoside (ONPG), Voges-Proskauer and citrate disks were
overlayed with sterile mineral oil. Inoculated Minitek
plates were then placed in a Minitek humidified
incubator at 35 C.
Minitek reactions were read after 24 and 48 hours
using the Minitek color comparator cards. The conventional biochemical tests used for the identification
of isolates were incubated at 35 C and were checked
daily for each of seven days. Table 1 lists the substrates
evaluated.
Indole production was detected with Kovac's
reagents; phenylalanine deaminase activity and acetylmethylcarbinol production (Voges-Proskauer test)
were detected using standard methods. 1 When results
obtained with conventional and Minitek biochemicals
differed, both were tested again and observed for four
to seven days.
RECENTLY, several commercial identification kits
and media have become available for the simple and
rapid identification of enteric gram-negative rods. One
of these new products, Minitek®, offers the microbiologist the ability to select from 35 biochemical
tests only those that he desires to use. Each Minitek
biochemical test consists of a paper disk impregnated
with a substrate and an appropriate indicator. Each
selected disk is placed in one of 12 wells in a sterile
plastic tray, and subsequently inoculated with 0.05 ml
of an inoculum broth seeded with the test organism.
This report describes an evaluation of 11 Minitek
biochemical tests with 1,089 isolates of enteric gramnegative rods, each of which was simultaneously
tested using Minitek and conventional tubed biochemical tests.
Materials and Methods
Recent clinical isolates or laboratory stock cultures
of enteric gram-negative rods were first subcultured
MacConkey agar plates (BBL), and then incubated for
18 to 24 h at 35 C. Well-isolated bacterial colonies
that appeared on the MacConkey medium were used as
Received September 3, 1976; received revised manuscript
December 16, 1976; accepted for publication December 16, 1976.
Dr. McCarthy's present address is Department of Hospital
Laboratories, North Carolina Memorial Hospital, Chapel Hill,
North Carolina 27514.
Address reprint requests to Dr. Armstrong: Diagnostic Microbiology Laboratory, Memorial Sloan-Kettering Cancer Center,
1275 York Ave., New York, New York 10021.
Results
Minitek biochemical tests demonstrated a 97.4%
overall correlation with conventional tests. A summary
of results obtained for each Minitek test evaluated
appears in Table 2. As shown, each Minitek test
with the exception of citrate yielded correlations of
94.8% or better when compared with conventional
biochemical tests.
Discrepant citrate reactions frequently were encountered with Proteus mirabilis, Proteus vulgaris and
Aeromonas hydrophila (Table 3). All 172 strains of
0002-9173-78-0200—0161$00.60 © American Society of Clinical Pathologists
161
A.J.C.P. • February 1978
MCCARTHY ET AL.
162
Table 1. Conventional Biochemical Tests Used for
Bacterial Identification and Corresponding
Minitek Disks Evaluated
Test
Conventional Medium
Minitek Disk
Dextrose
Kligler iron agar (BBL)
Dextrose/nitrate
Hydrogen sulfide
Kligler iron agar (BBL)
H2S/indole
Indole
SIM (BBL)
H2S/indole
Lysine decarboxylase
Moeller lysine decarboxylase (BBL)
Lysine
Ornithine decarboxylase
Moeller ornithine decarboxylase (BBL)
Ornithine
Urea
Christensen's urea
(BBL)
Urea
Citrate
Simmons citrate (BBL)
Citrate
ONPG
ONPG
ONPG
Phenylalanine
deaminase
Phenylalanine agar
(BBL)
Phenylalanine
Arabinose
1% arabinose in phenol
red broth base (BBL)
Arabinose
Adonitol
1% adonitol in phenol
red broth base (BBL)
Adonitol
Voges-Proskauer
MR-VP broth (BBL)
Voges-Proskauer
Inositol
1% inositol in phenol
red broth base (BBL)
Not tested
DNase
DNase agar with toluidine blue 0 indicator
(BBL)
Not tested
Motility
SIM (BBL)
Not tested
Oxidase
Oxidase strips
Not tested
P. mirabilis, 5 of 13 citrate-positive strains of P.
vulgaris, and 3 of 6 strains of A. hydrophila yielded
false-negative Minitek citrate reactions. It is significant
that these three organisms were responsible for 95.2%
of all observed discrepant citrate reactions. Other
discrepant citrate reactions were seen with Klebsiella
pneumoniae and Providencia stuartii (Table 3).
Minitek urease reactions demonstrated a 97.8%
correlation with those observed on Christensen's urea
agar (Table 2). As shown in Table 3, the poorest
correlation with Minitek urease test was observed
withEnterobacter cloacae a n d £ . agglomerans strains.
In each instance these discrepancies were encountered
with strains of E. cloacae and E. agglomerans that
showed a negative Minitek reaction and a delayed
and/or weak positive reaction on Christensen's urea
agar.
The Minitek ornithine decarboxylase test demonstrated a 98.8% correlation with Moeller ornithine
decarboxylase broth (Table 2). The poorest correlation
for the Minitek ornithine decarboxylase test was seen
with Proteus morganii, where five strains yielding
positive reactions in the conventional medium were not
detected by the Minitek disk (Table 3).
Of the 9,317 biochemical tests performed, the
Minitek yielded ten positive reactions (0.1%) that
were not found with conventional media (Table 2).
None of these positive reactions could be classified
as false-positive, as each reaction was appropriate for
the organism tested. These reactions were: one positive
hydrogen sulfide reaction with Proteus mirabilis; two
positive indole reactions and one positive lysine
decarboxylase reaction with Klebsiella
pneumoniae;
four positive ornithine reactions (two with Escherichia
coli and two with Serratia marcescens); two positive
adonitol reactions, (one each with Providencia stuartii
and Enterobacter cloacae).
A comparison between Minitek and conventional
biochemical tests to determine which of the two yielded
more rapid definite positive reactions, demonstrated
Table 2. Comparison of Reactions Obtained with Minitek and Conventional Biochemical Tests
Minitek and
Conventional
Minitek - ,
Conventional +t
Minitek +,
Conventional — §
(%)
(%)
Test
No.
Positive*
Dextrose
Hydrogen sulfide
Indole
Lysine decarboxylase
Ornithine decarboxylase
Urea
Citrate
ONPG
Phenylalanine deaminase
Arabinose
Adonitol
Voges-Proskauer
1,089
1,089
1,089
1,089
1,089
1,089
1,089
695
348
81
428
142
1,089
199
346
572
795
573
813
389
292
3
236
102
0
890
743
517
294
516
276
306
56
78
192
40
1,089
1,084
1,079
1,086
1,076
1,065
900
694
348
81
426
142
(100)
(99.5)
(99.1)
(99.7)
(98.8)
(97.8)
(82.6)
(99.9)
(100)
(100)
(99.5)
(100)
0
4
8
2
9
24
189
1
0
0
0
0
9,317
5,409
3,908
9,070
(97.4)
237
TOTAL
No.
Negative*
Samet
(%)
No.
Tested
* Number of positive or negative reactions observed with conventional biochemical tests.
t Minitek and conventional tests yielded same reactions.
(0.4)
(0.7)
(0.2)
(0.8)
(2.2)
(17.4)
(0.1)
(2.5)
t Minitek negative, conventional test positive.
§ Minitek positive, conventional test negative.
0
1
2
1
4
0
0
0
0
0
2
0
(0.1)
(0.2)
(0.1)
(0.4)
(0.5)
10 (0.1)
vol. 69. No. 2
IDENTIFICATION OF ENTERIC GRAM-NEGATIVE RODS
163
Table 3. Comparison of Biochemical Results Obtained with Minitek and Conventional
Biochemical Tests According to Organism Tested
Correlation (%)
Organism
Number DexTested trose
H2S
Escherichia coli
197
100
100
Salmonella spp.
Ortho-nitroLysine
Ornithine
Phenylphenyl-B-DDecarbox- Decarboxalanine
galactoVogesArab- AdonUrea Citrate pyranoside Deaminase inose
itol
Proskauer
Indole
ylase
ylase
96.4
14
100
100
Klebsiella
pneumoniae
100
197
100
100
Enterobacter
cloacae
173
100
100
100
Enterobacter
aerogenes
129
100
100
5
100
Citrobacter
freundii
23
Citrobacter
diversits
98.5
99.5
100
99.0
98.0
100
100
100
NT*
NT
NT
100
100
100
NT
NT
100
100
NT
NT
NT
NT
100
100
100
NT
NT
99.5
NT
NT
NT
100
99.5
100
100
87.9
100
100
100
100
100
100
NT
NT
100
NT
100
100
100
100
80
100
100
NT
NT
100
NT
100
100
100
100
100
100
100
NT
NT
100
100
3
100
100
100
100
100
100
100
100
NT
NT
100
100
50
100
100
100
100
100
98.0
100
100
100
100
NT
Proteus
mirabilis
172
100
100
100
100
100
0
100
100
NT
NT
NT
Proteus
morganii
65
100
100
100
100
100
100
100
100
NT
NT
NT
Proteus
vulgaris
20
100
100
100
100
100
100
100
100
NT
NT
NT
Proteus rettgeri
10
100
100
100
100
100
100
100
100
NT
NT
NT
Providencia
stuartii
25
100
100
100
100
100
100
96.0
100
100
100
96.0
NT
Aeromonas
hydrophila
6
100
100
100
100
100
100
50
100
100
100
Enterobacter
agglomerans
Serratia
marcescens
97.1
92.3
96.0
95.9
75.0
100
95.7
100
NT
* NT = not tested.
that Minitek biochemical tests for lysine decarboxylase
and adonitol fermentation yielded positive reactions
earlier than those observed with Moeller lysine
decarboxylase broth and 1% adonitol in phenol red
broth base (Table 4). A distinct advantage was
especially evident with the Minitek adonitol disk,
where 20.3% of positive reactions preceded those in
phenol red broth base by 24 to 48 h.
Positive Minitek urease reactions appeared later than
those observed on Christensen's urea agar isolates
(Table 4). As found earlier with discrepant Minitek
urease reactions, these delayed Minitek urease
reactions occurred with strains of enteric gramnegative rods that gave delayed and/or weak reactions
on Christensen's urea agar.
Both Simmons citrate agar and the Minitek citrate
disk showed delayed reactions of approximately the
same magnitude. With respect to speed of reaction,
no other difference between Minitek and conventional
chemical tests was found.
Discussion
For our evaluation of the Minitek, we elected to
compare 11 commonly used biochemical tests with
conventional substrates. Our observed overall correlation of 97.4% between Minitek disks and conventional
tubed media agrees well with other evaluations of
the Minitek system.2-3 Although the Minitek demonstrated excellent overall correlation with conventional
media, discrepant reactions with citrate and urea disks
were found to occur frequently with specific organisms.
False-negative Minitek citrate reactions were encountered with all strains of P. mirabilis, 38.4% of
citrate-positive strains of P. vulgaris, and 50% of
A. hydrophila strains. As a result, we do not recommend that the Minitek citrate disk be used for the
A.J.C.P. • February 1978
MCCARTHY ET AL.
164
Table 4. Comparison of Positive Reactions )ccurring in Both Minitek and Conventional
Biochemical Tests 1 ir Speed of Reaction
Test
No. Positive
Minitek Early (%)*
Conventional Early (%)t
Dextrose
Hydrogen sulfide
Indole
Lysine decarboxylase
Ornithine decarboxylase
Urea
Citrate
ONPG
Phenylalanine deaminase
Arabinose
Adonitol
Voges/Proskauer
1,089
199
346
572
784
549
630
388
292
3
236
102
0
0
0
25 (4.4)
0
9 (1.6)
11 (1.8)
0
0
0
48 (20.3)
0
0
0
0
5 (0.9)
0
34 (6.2)
16 (2.5)
0
0
0
0
0
* Minitek reaction earlier than conventional test.
t Conventional test reaction earlier than Minitek.
speciation of Proteus spp. It is important to note that
the citrate disk, while not suitable for the identification
of Proteus spp., may effectively be used for the
speciation of other enteric gram-negative rods.
False-negative Minitek urease reactions occurred
with organisms giving weak and/or delayed reactions
on Christensen's urea agar. This result was not
unexpected, as the high level of sensitivity of the
Christensen's medium has been described by Vuje and
Pijck.4 False-negative Minitek urease reactions occurred primarily with strains of E. cloacae and E.
aggloinerans. In each case, however, a positive urease
test is not considered essential for identification.
False-negative ornithine decarboxylase reactions occurred infrequently with the Minitek system. It is
significant that 8.0% of the P. morganii isolates tested,
which normally decarboxylate ornithine, 1 yielded falsenegative ornithine results. Such false-negative ornithine
decarboxylase reactions may result in the occasional
misidentification of P. morganii as P. rettgeri.
Only ten "false-positive" reactions (0.1% of all
reactions) were found during our study. Since each
of these positive reactions may occur for the individual
species tested, they may simply indicate increased
sensitivity of the Minitek disks rather than reflect true
false-positive results.
We did not observe the frequent Minitek falsepositive hydrogen sulfide reactions described by Kiehn
and co-workers. 3 This conflict in results was probably
caused by variations among the manufacturer's initial
lots of the H 2 S/indole disk. Subsequent to the evaluation of the Minitek system by Kiehn and co-workers,
we have used H 2 S/indole disks without encountering
false-positive hydrogen sulfide reactions.
The Minitek compared well with conventional
biochemical tests in speed of reaction, and appeared to
offer an advantage over the conventional lysine
Minitek, Conventional
Same (%)$
1,089
199
346
542
784
506
603
388
292
3
188
102
(100)
(100)
(100)
(94.7)
(100)
(92.2)
(95.7)
(100)
(100)
(100)
(79.7)
(100)
I Minitek and conventional test reactions occurred simultaneously.
decarboxylase and adonitol fermentation media employed. In addition to the precautions described by
Hansen and co-workers, 2 we found that the oil necessary for the overlay, when dropped inadvertently
between the wells of the inoculum plate, spread when
the cover was replaced. The spreading oil formed a
seal between the plate's cover and the rims of wells
containing disks. The oil seal resulted in false-negative
reactions for disks not requiring an oil overlay (i.e.,
citrate and phenylalanine deaminase). To eliminate
such false-negative reactions, users of the Minitek
system should remove oil dropped between the wells
prior to replacing the inoculum plate cover.
In general, we found Minitek reactions to be quite
clear and distinct when compared with other methods.
The Minitek system saved time by reducing inoculation
time by approximately half. We were also impressed
with the significant reduction of refrigerator storage
space required for Minitek biochemicals. The flexibility
of the Minitek system should prove useful for a variety
of microbiological assays. The availability of different
inoculum broths may make the Minitek system useful
for the identification of bacteria other than enteric
gram-negative rods.
Based upon our results, it appears that the Minitek
may serve as a reasonable alternative to conventional
biochemical tests.
References
1. Edwards PR, Ewing WH: Identification of Enterobacteriaceae.
Minneapolis, Burgess, 1972, pp 1-362
2. Hansen SL, Hardesty DR, Meyers BM: Evaluation of the
Minitek system for the identification of enterobacteriaceae.
Appl Microbiol 28:798-801, 1974
3. Kiehn TE, Brennan K, Ellner PD: Evaluation of the Minitek
system for identification of enterobacteriaceae. Appl Microbiol 28:668-671, 1974
4. Vuye A, Pijck J: Urease activity of enterobacteriaceae:
Which medium to choose. Appl Microbiol 26:850-854, 1973