Identification
Primary
of Enteric
Isolation
Bacilli Directly
Media
impregnated
Using
Paper
from
Reagent-
Strips
RICHARD F. ROSNER, M.S., M(ASCP)
St. Joseph's Hospital, Paterson, New Jersey 07503
ABSTRACT
Rosner, Richard F.: Identification of enteric bacilli directly from primary
isolation media using reagent-impregnated paper strips. Amer. J. Clin. Path.
54: 587-595, 1970. A new and rapid method for the identification of enteric
bacilli directly from Hektoen enteric agar plates is described. This procedure
is based on the use of reagent-impregnated paper strips and requires only
4 to 6 hr. to obtain data from 13 biochemical reactions. Six to 20 well isolated
colonies which are morphologically similar are required for this procedure.
Nine hundred forty-three clinical specimens were examined both by the
direct method from Hektoen enteric agar and by the conventional method of
selecting representative colonies from either eosin-methylene blue or Salmonella-Shigella agars, inoculating a triple sugar iron agar tube, and then
carrying out biochemical testing. A total of 1,037 organisms was isolated and
correctly identified by the direct method and 1,046 by conventional methods.
Each biochemical test carried out with paper strips was controlled by standard
media, with a 99.3% correlation between results. Of the 943 clinical specimens examined, 842 (89%) were acceptable for the direct method. The remaining 101 either had too few colonies present or were improperly streaked.
The average time required to obtain significant biochemical data by the direct
method was 5 hr. after isolation on the Hektoen enteric agar plates, while the
average time needed to obtain the same biochemical data using conventional
methods was 48 hr. There was less than a 0.05% failure of the direct identification system to allow either isolation or identification, compared with conventional systems described in the literature. 4
SEVERAL SCHEMATA l-6' "•" are available to negative bacilli. An excellent review can
aid the clinical microbiologist in identifi- be found in The Journal of Infectious Discation of the enteric and related Gram- eases.*
Received October 8, 1969; accepted for publica- cssed by conventional bacteriologic procedures of
tion February 12, 1970.
obtaining colonies from either eosin-methylene
' ~, ; ,. , " ,
, , , ., , . . . blue agar or Salmonella-Shigella agar plates, inocu••
Autnors Note. The method described in this , . . . . ,
•
. i_
• .•
report requires that from 6 to 20 well isolated, , a t , n S tnfle s u S a r . l r o n a S a r tub<;s a n d ' he , n ,d . cn ;
morphologically similar colonies be taken directly tifying the organisms by standard biochemical
from the primary isolation medium and bio- methods as described by Edwards and Ewing."
chemically tested. The author recognizes that the
With the recent introduction of Hektoen enteric
assumption that morphologically similar colonies agar (HE agar) it is now possible to differentiate
represent a single organism will be considered by many Gram-negative bacilli
by careful observation
many investigators to be improper technic. As a of colony morphology.13"" The method described
result of this potential controversial assumption, in this report is only usable when HE agar is
each clinical specimen processed by the method used as the primary isolation medium and when
described in this report was simultaneously proc- well isolated colonies are available for testing.
587
ROSNER
A.J.C.P.—V0I. 54
Sterile cotton swab which has been dipped into the
broth culture or directly into a stool specimen is
touched to agar surface to cover an area approximately the size o£ a nickel.
Sterile loop is placed in inoculated area and then
streaked over approximately half of the petri dish.
Individual streak lines should be isolated from
each other.
V-
Loop is flamed, cooled, and placed onto agar surface in the area already streaked. The remainder
of the dish is streaked perpendicularly to the previous line of streak. This streaking should not
enter the previously streaked area more than two
or three times.
FIG. 1. Method of inoculating and streaking the
primary isolation plate.
Until recently, the various biochemical
reactions needed to identify Gram-negative
bacilli were obtainable only by conventional
methods requiring 24 to 72 hr. for completion. While this time requirement may
not be important to the industrial or academic microbiologist, it is critical to the
clinical microbiologist in the hospital laboratory, since the utility of the data obtained is inversely proportional to the
speed with which it can be reported to
the clinician. Recently, several investigators 1-3> 7,8,10,15-17 have reported the use of
reagent-impregnated paper strips * to obtain significant biochemical data. These
investigators report a close correlation between the results obtained using the test
strips and those obtained by standard biochemical methods. The biochemical information can now be available 4 to 6 hr.
after isolation. However, even with the use
of these strips it still has been necessary
to inoculate a medium such as triple sugar
* PathoTec TM, General Diagnostics, Morris
Plains, New Jersey.
iron agar (TSI) and a trypticase-soy agar
slant. This additional step required overnight incubation and added an additional
18 hr. before the test strips could be used.
The purpose of this report is to describe
a method of identification using PathoTec
and
experimental
reagent-impregnated
strips with colonies taken directly from
Hektoen enteric agar (HE agar).
Materials and Methods
The method described in this report is
a complete system of isolation and identification and has been tested only on the
primary isolation medium described in this
report.
All patient specimens received for routine culture were plated onto a battery of
primary isolation media. Stool specimens
were plated directly onto the primary
media while all other specimens (urine,
sputum, material from wounds, etc.) were
first inoculated into trypticase-soy broth
for 4 hr. before being streaked onto the
primary plates. The battery consists of:
October 1970
A NEW METHOD FOR IDENTIFYING NEGATIVE RODS
1. Chapman-Stone agar for staphylococci.
2. Horse blood agar with sodium colistimethatet and nalidixic acidt for
streptococci.
3. Eosin-methylene blue agar for Gramnegative bacilli.
4. Salmonella-Shigella agar for differentiation of enteric bacilli.
5. Hektoen enteric agar for differentiation of enteric bacilli.
All primary plates were streaked as described in Figure 1. After streaking, all
plates were incubated for 18 to 24 hr. at
35 C. Following incubation, the EMB and
SS plates were examined carefully to determine the types of colonies present. Typical representative colonies were selected
and used to inoculate TSI agar tubes.
These tubes were incubated at 35 C. overnight, after which organism identification
was accomplished as described by the biochemical schemata of Edwards and Ewing.6
589
but more than six colonies. In these cases,
a battery of four paper strips was used to
give the microbiologist a good idea of the
general type of organism present. This
four-strip battery consists of PathoTec
strips for detecting oxidase, urease, phenylalanine deaminase, and indole production.
The basic morphologic colony types
which are readily determined on HE agar
are: flat, yellow, dry colony of Escherichia;
large, creamy, yellowish-red colonies of the
K-E-S group; flat, yellow colonies with
green or black centers of Citrobacter organisms; small, green, sticky colonies of
Pseudomonaceae; colorless or pale green
colonies of Shigella, Providencia, or Proteus; colorless or pale green colonies with
black centers of the Salmonella, Arizona,
Edwardsiella or Proteus types.
Cytochrome oxidase, nitrate reduction,
urease, Voges-Proskauer and citrate tests
were performed by using PathoTec reagent
Following incubation, the HE agar plates systems. Experimental test strips were used
were examined carefully to determine the for detecting indole production (W4692),
number of morphologic colony types pres- malonate utilization (W7594), sugar ferent. If all the colonies appeared identical, mentations (W6595), and phenylalanine
it was assumed that only a single type of deaminase (W4004-3). All data obtained
Gram-negative bacilli was present and di- from the test strip systems and experimenrect identification was started. If there tal strips were confirmed by conventional
were two or more distinct morphologic procedures. 6
colony types, direct identification of only
Direct identification was initiated by sethose morphologic types in which there were lecting a single, typical colony and testing
6 to 20 well isolated colonies was started. for the presence of cytochrome oxidase;
When more than one colony type was pres- the colony was rubbed into the reagent
ent, with more than 20 of one type and zone of the oxidase strip. The development
fewer than six of the other types, direct of a blue color on the inoculated portion
identification of the predominant type was of the reagent zone within 1 min. indicated
performed whereas a single colony of the a positive reaction. Oxidase-positive orother type was used to inoculate a lysine- ganisms were further tested for their ability
iron agar tube. This tube was incubated to reduce nitrate to nitrite; two colonies
overnight and identification using the were suspended in 5 to 7 drops of saline
paper strips carried out the following day.
solution in a 13 by 100 mm. test tube. A
Frequently, there were fewer than 20 nitrate strip was placed in the suspension
isolated colonies of a given colony type and incubated for 2 hr. at 37 C. After
incubation, the tube was gently tilted to
•(• Coly-Mycin Diagnostic, Warner-Chilcott Laboratories, Div. Warner-Lambert, Morris Plains, New allow the indicator zone halfway up the
Jersey.
strip to become wet. The formation of a
| Winthrop Laboratories, Div. Sterling Drug Inc.,
faint pink to deep red color indicated a
New York, New York.
590
ROSNER
positive test. Organisms which were oxidase-positive and those which were nitratenegative were not members of the Enterobacteriaceae and were identified on conventional media such as Sellers agar and
O-F broths.
Organisms which were oxidase-negative
were further tested as follows: nitrate reduction, indole production, phenylalanine
deaminase production, urease production,
acetylmethyl carbinol production, citrate
and malonate utilization, and the formation of acid from lactose, sucrose, mannitol, sorbitol, dulcitol, and salicin.
The sugar fermentation strips are rub-in
strips similar to the oxidase strip. For each
sugar reaction a single colony was rubbed
into the specific sugar zone on the test
strip. A drop of saline solution was added
to each inoculated zone and the strip
placed in a dry 13 by 100 mm. tube. T h e
tube was placed on its side in an incubator at 37 C. for 2 to 3 hr. The development
of a yellow color on a specific sugar zone
indicated acid production. All other tests
used in this method were performed by
suspending two colonies in 5 to 7 drops of
saline solution and placing the desired reagent strip in the suspension. All reagent
strips were incubated for 2 hr. except the
citrate strip which required an incubation
period of 4 hr. Although the manufacturer
recommends incubating the PathoTec VP
strip and the experimental indole strip
for 4 hr., we have found that 2 hr. are
sufficient.
A positive urease test was indicated by
the development of a very faint pink to a
deep pink color in the cell suspension. The
development of a green color in the reagent zone of the experimental phenylalanine deaminase strips indicated a positive
reaction.
Sugar strips usually were not inoculated
until after the data were obtained from
the other test strips, since it was frequently
unnecessary to test for sugar reactions.
However, when indicated, the sugar strips
A.J.C.P.—Vol. 54
were inoculated and the results obtained
within 3 hr.
When required, motility was determined
by suspending a portion of a typical colony
from the HE agar in a drop of saline solution, covering it with a cover glass, and
observing the colony under oil magnification. There was a 94% correlation between
motility in colonies obtained from a 24 hr.
HE plate and those obtained by suspending the organism in broth for 4 hr.
Hydrogen sulfide production was determined by two methods. T h e presence of
colonies with black centers on the HE
agar plates indicated hydrogen sulfide production. All other colony types were tested
by placing two colonies in 1 ml. of fluid
thioglycollate medium in a small screw-top
tube. A piece of lead acetate paper was
hooked over the top of the tube and held
there by screwing down the cover of the
tube. This was incubated for 2 to 3 hr.
Hydrogen sulfide was indicated by the development of a deep brown or black color
on the lead acetate paper inside the tube.
The author has found that 90% of all
organisms which produce hydrogen sulfide
in a medium such as TSI will do so on
a 24 hr. HE agar plate. T h e remaining
10% are identified by the fluid thioglycollate/lead acetate method.
In almost every case, the total time required to obtain the biochemical information just described was less than 4 hr. after
the first observation of the primary isolation medium. When all the biochemical
data were available, the identification
scheme presented in Figure 2 was utilized.
This scheme identifies the more common
enteric bacilli to the genus level. Although
the scheme indicates differentiation of Serratia from Enterobacter, a few Enterobacter are malonate-negative.6 Confirmation of
Serratia requires the inoculation of DNase
agar, with overnight incubation. In order
to determine species in a genus other than
Proteus, it is also necessary to utilize standard biochemical sugar media.
DULCITOL
MALONATE
LACTOSE
UREASE
SALMONELLA
CITROBACTER
ARIZONA
c+
VP-
PDU- +
I-
H 2 S+
-H2S+
EDWARDSIELLA
ESCHERICHIA
SHIGELLA (40%)
KLEBSIELLA (30%)
EDVVARDSIELLA
PDU- +
1+
H2S
I
C
all negative
ESCHERICHIA
SHIGELLA
—
+ (98%)
+
+
—
-(80%)
+ (80%)
—
+ (90%)
—
—
+ (70%)
SHIGELLA
ARIZONA
SALMONELLA
CITROBACTER
FIG. 2. Scheme for identification of enteric bacteria using PathoTec experimental reagent-impregnated paper strips.
MOTILITY
SALACIN
ANY ONE- -DULCITOL
LACTOSE
POSITIVE
SUCROSE
ESCHERICHIA
ALL
NEGATIVE
U, VP, C
%
w
H
EG
O
Z
VI
<3"
<
w
>s
o
d
Z
o
z
w
o
>
+
o
+
o
P. rellgeri
o
M
z
PD +
UPROVIDENCE
P. morganii
ESCHERICHIA
SHIGELLA
KLEBSIELLA
+
P. mirabilis
+
U+ (94%)
VP+ or C+
KLEBSIELLA
+
P. vulgaris
PD +
U+
PROTEUS
+
ENTEROBACTERIACEAE
I
U, PD, I, C, VP and MALONATE
all negative
I
including VP- -MOT.
+
+
and C
MAL.
+
+
U.
+ (50%)
+ (94%)
I
KLEBSIELLA SERRATIA ENTEROBACTER
SHIGELLA
MOTILITY
MALONATE
UREASE
PD- U- +
IVP or C+
I
ENTEROBACTER
SERRATIA
KLEBSIELLA (70%)
SHIGELLA (60%)
PSEUDOMONAS
FAMILY
+ FAMILY PSEUDOMONAS FAMILY
PSEUDOMONAS
AEROMONAS
* ENTEROBACTERIACEAE
CO
HEKTOEN ENTERIC AGAR
SALMONELLA SHIGELLA AGAR
592
ROSNER
Table 1. Frequency of Isolation of the
Organisms (N = 1,037) Identified by
the Direct Method
Organism
Frequency of
Isolation
Escherichia coli
Enterobacter species
Klebsiella species
Proteus mirabilis
Citrobacter freundii
Pseudomonas family
Proteus vulgaris
Serratia species
Salmonella species
Providencia
Shigella species
Arizona group
Proteus morganii
Edwardsiella tarda
Proteus rettgeri
221
187
119
118
91
91
58
58
21
16
14
14
11
11
7
Results
Of 943 clinical specimens, each containing at least one type of Gram-negative bacilli, which were evaluated, 842 (89%)
were found to be satisfactory for direct
identification methods. Of the remaining
101 specimens which were not acceptable
for direct identification, 73 contained fewer
than five colonies of the organism to be
identified and 28 were improperly streaked.
A total of 1,037 Gram-negative bacilli was
recovered from the HE agar plates, including the 28 specimens which had to be
restreaked. A total of 1,046 organisms was
recovered from the same specimens by the
conventional EMB/SS/TSI method. There
were only nine instances where the HA
agar method failed to allow isolation or
identification, or both, of an organism
which was found by conventional methods.
The types of organisms and the frequency
of isolation from the HE agar plates are
listed in Table 1. The reactions obtained
using the paper strips were controlled by
the use of standard biochemical media.
There was complete agreement with the
biochemical results in 99.3% of the cases.
A.J.C.P.—Vol.
54
The only disagreements between the strip
reactions and standard media reactions occurred with the sugar strips. All the differences were due to false-negative strip
reactions.
Discussion
The results of this experiment indicate
that reagent-impregnated paper strips provide rapid, accurate results with relatively
small inocula and allow the clinical microbiologist to obtain significant biochemical
data in short periods. In addition, the use
of these strips for bacterial identification
allows the development of flexible schemata, since most biochemical results are
obtained relatively rapidly. In the past, the
use of these strips with organisms obtained
directly from primary isolation media has
been prevented by two factors. T h e first is
the insistence of almost all microbiologists
upon using a sugar differential medium,
such as TSI. The use of such a medium requires an additional 24 hr. incubation period between isolation of the organism and
the commencement of many of the biochemical tests. The justification for using
a medium such as TSI has always been
that valuable information is obtained from
this medium. TSI medium supplies the
microbiologist with information regarding
gas production from dextrose, acid production from lactose or sucrose, or both, and
the production of hydrogen sulfide. Gas
production is extremely variable in many
organisms4> 6 and is also highly dependent
on the preparation of the medium itself.
Therefore, the production of gas from dextrose in this type of medium system cannot
be considered completely reproducible or
even completely reliable. The production
of acid from lactose is a critical test in almost every scheme used for identifying
Gram-negative bacilli. Lactose-fermenting
organisms can be differentiated from nonlactose-fermenting organisms by the type of
colony produced on HE agar and this test
is, therefore, merely duplicated on TSI medium. Until the introduction of HE agar,
it was necessary to use a medium such as
October 1970
A NEW METHOD FOR IDENTIFYING NEGATIVE RODS
593
TSI in order to determine the production Table 2. All of the Possible Reactions Which
of hydrogen sulfide. The data obtained in
can Occur on TSI Agar and the Various
this study indicate that HE agar in comOrganisms Which Can Produce Them
bination with the lead acetate/thioglycolA/AG H 2 S late method is as sensitive to H 2 S producEscherichia
tion as TSI medium. Table 2 indicates all
Klebsiella
of the possible types of reactions which can
Enterobacter
occur on a medium such as TSI and the
Serratia (small amount of gas)
various ogranisms which can produce each
type of reaction. From this table, it becomes obvious that TSI medium can often
NC/AGH2Sbe more confusing than helpful.
Escherichia
The second factor which has prevented
Enterobacter
direct identification with PathoTec strips
Proteus morganii
has been the inoculum requirements of
Providencia
Klebsiella
some of the commercially available strips,
Rare Salmonella
such as the PathoTec I and PD strips. The
PD strip indicates a positive reaction of
gray to blackish when a colony is rubbed
A/A H 2 S into the reagent zone. This color reaction
Escherichia
prevents the use of any H2S-positive coloSerratia
nies, since they are blackish to begin with.
Rare Proteus
The PathoTec I strip requires that a medium such as trypticase-soy agar be used
NC/A H 2 S as the source of inoculum. In practice,
Escherichia
therefore, a colony must be picked from
Shigella
the primary plate, inoculated into a tube
Serratia
of medium, and incubated overnight beSalmonella typhi
fore an indole or phenylalanine deaminase
Proteus morganii
test can be performed. The development
Proteus rettgeri
of an experimental indole strip which alProvidencia
lows the inoculum to be taken from any
medium eliminates the need for overnight
NC/A H 2 S+
incubation on special media. The developSalmonella typhi
ment of an experimental phenylalanine
deaminase strip which allows the use of
NC/AG H 2 S+
H2S-positive colonies eliminates the neSalmonella
cessity for using any special media for this
Arizona
test.
Citrobacter
A single colony is used to determine the
Edwardsiella
oxidase reaction of the organism. Any orProteus mirabilis
ganism which is oxidase-positive is not a
Proteus vulgaris
member of the Enterobacteriaceae. All organisms, regardless of oxidase reaction,
A/AG H 2 S+
were tested for nitrate reduction. All niCitrobacter
trate-negative organisms were also excluded
Arizona
from the Enterobacteriaceae. Organisms
Proteus mirabilis
which were oxidase-negative and nitrateProteus vulgaris
positive were tested for urease, indole, cit-
594
ROSNER
rate, malonate, and phenylalanine deaminase production, and by the Voges-Proskauer test. Those organisms which were
urease-positive and phenylalanine deaminase-positive were members of the genus
Proteus. The identification to species of
Proteus is accomplished by the indole, citrate, and H 2 S reactions. Those organisms
which are urease-negative but phenylalanine deaminase-positive were members of
Providencia. Organisms which are phenylalanine deaminase-negative belong to any
one of the three remaining groups shown
in Figure 2. Of the three remaining groups,
those which are indole-positive are Escherichia, Shigella, Edwardsiella, or Klebsiella.
Edwardsiella is immediately separated out
by its ability to produce H 2 S on HE agar.
Klebsiella, in addition to being indolepositive, is either Voges-Proskauer-positive
or citrate-positive and is separated from
Escherichia and Shigella on this basis. Our
data indicate that 30% of all Klebsiella
isolates are indole-positive. Differentiation
of nonmotile, slow lactose-fermenting Escherichia from Shigella is based on the almost
complete biochemical inactivity of the Shigella as compared with the moderate activity of Escherichia. T o differentiate the
two, sucrose, lactose, dulcitol, sorbitol, and
salicin sugar strips were inoculated. Since
Escherichia is more active than Shigella,
positive fermentation on any of these sugars was considered to indicate Escherichia.
In this study even those Escherichia which
required 48 hours to produce acid from
lactose on conventional media produced a
yellowish zone in the test sugar strip in
2 hr.
Organisms which were indole-negative,
H2S-negative and either Voges-Proskauerpositive or citrate-positive were Enterobacter, Klebsiella, Shigella, or Serratia. Once
again, Shigella was ruled out by its relative
biochemical inactivity. Enterobacter was
identified by its motility, utilization of
malonate, and failure to produce urease
in 2 hr. Serratia was identified by being
A.J.C.P.—Vol.
54
consistently malonate-negative, motile, and
urease-variable. Confirmation of Serratia
was accomplished by the inoculation of a
DNase agar plate, with overnight incubation. Klebsiella, on the other hand, was
consistently nonmotile, malonate-positive,
and urease-positive.
Organisms which were indole-negative
but H 2 S positive were Salmonella, Arizona,
or Citrobacter. Citrobacter was identified
by its ability to produce acid from lactose
(90%) and dulcitol (70%), and its failure
to utilize malonate. Arizona was identified
by its failure to produce acid from dulcitol, production of acid from lactose, and
utilization of malonate. Salmonella, on the
other hand, is consistently malonate-negative, lactose-negative, and dulcitol-positive
(96%). These three organisms were further
identified by serologic procedures.
In many instances fewer than 20 well
isolated colonies were available for biochemical testing. When there were fewer
than six colonies, it was necessary to inoculate a lysine-iron agar tube, incubate overnight, and carry out the biochemical testing from this tube the following day. If,
however, there were at least six well isolated colonies, it was possible to perform
four basic biochemical reactions using
paper strips. By determining the oxidase,
urease, phenylalanine deaminase, and indole reactions of the organism, it is possible to place it in one of the five groups
indicated in Figure 2. From the same six
colonies it is also possible to determine H 2 S
production and motility more than 90% of
the time and to determine the organism's
action on lactose.
Conclusions
The direct identification of many Gramnegative enteric bacilli cultured on Hektoen enteric agar can be accomplished by
the use of PathoTec Reagent Systems and
experimental reagent-impregnated strips.
Identification to genus level by this method
was found to have a 0.05% failure rate
October 1970
A NEW METHOD FOR IDENTIFYING NEGATIVE RODS
compared with conventional methods of
isolation and identification for the same
clinical specimens. The biochemical reactions obtained from the reagent-impregnated paper strips were found to be more
than 99% accurate when compared with
similar biochemical reactions obtained
from standard media. When careful attention is given to the streaking procedure
used for the primary isolation medium, it
is possible to apply direct identification
methods to 89% of all clinical specimens.
The time required to obtain the necessary
biochemical data using the direct method
is 4 to 6 hr. after isolation of the organism,
or approximately 30 hr. after obtaining
the original clinical specimen.
Addendum
Since submission of this paper for publication, a rapid method of determining lysine decarboxylase activity has been made
available by Difco Laboratories. The product is Bacto Differentiation Disks-Lysine,
1636-35. About 5 to 7 drops of saline solution are placed in a 13 by 100 mm. tube.
One or two colonies from the Hektoen enteric agar are suspended in the saline solution and a disk is added. Incubation is for
2 to 4 hr. A yellow color indicates a negative reaction, whereas green or blue indicates a positive reaction. These disks were
compared with standard lysine decarboxylase broth and were found to be 100%
accurate.
The Lysine Disk allows the rapid differentiation of Citrobacter from Arizona and
Salmonella, as all Citrobacter species are
lysine negative, whereas Arizona and Salmonella species are lysine decarboxylase
positive. These disks are also valuable in
differentiating Escherichia from Shigella,
as 84% of all Escherichia are lysine decarboxylase positive while all Shigella are
negative.
The use of the Lysine Disks eliminates,
in many cases, the necessity for inoculating
the various sugar strips.
595
References
1. Kclliveau, R. R., Grayson, J. W., and Duller,
T. J.: A rapid, simple method of identifying
Enterobacteriaceae. Techn. Bull. Regist. Med.
Techn. 38: 152-154, 1968.
2. Borchardt, K. A.: Scheme for screening and
identifying enteric and other gram negative
bacteria using reagent impregnated strips.
Techn. Bull. Regist. Med. Techn. 38: 155-157,
1968.
3. Borchardt, K. A.: Simplified method for identification of enteric and other gram negative
bacteria using reagent impregnated strips.
Techn. Bull. Regist. Med. Techn. 38: 98-100,
1968.
4. Dumoff, M.: How far do we go with the Enterobacteriaceae? J. Infect. Dis. 119: 205-208,
1969.
5. Diagnostic Research Inc., Instruction Booklet:
The R/B Enteric Differentiation System. Diagnostic Research Inc., 25 Lumbar Rd., Roslyn, New York 11576. 1969.
6. Edwards, P. R., and Ewing, W. H.: Identification of Enterobacteriaceae. Minneapolis, Burgess Publishing Co., 1964.
7. Gandelman, A. L., and Mann, P. H.: An evaluation of reagent-impregnated paper strips for
use in the process of identifying certain species of clinically important bacteria. Curr.
Ther. Res. 7: 130-138, 1965.
8. Gendelman, A. L.: The use of reagent impregnated paper strips as an aid in the identification of certain gram negative organisms.
Amer. J. Med. Techn. 32: 85-87, 1966.
9. Isenberg, H. D., and Berkman, J. I.: Recent
Practices in Diagnostic Bacteriology. Progress
in Clinical Pathology, pp. 279-290. Available
from General Diagnostics, Warner Lambert
Co., Morris Plains, N. J.
10. Jeans, B.: The use of PathoTec Strips in medical bacteriology. Canad. J. Med. Techn. 48:
114-120, 1967.
11. Johnson, J. G., Kunz, L. J., Barron, W., and
Ewing, W. H.: Biochemical differentiation of
the Enterobacteriaceae with the aid of lysine
iron agar. Appl. Microbiol. 14: 212-217, 1966.
12. King, S., and Metzger, W. I.: A new plating
medium for the isolation of enteric pathogens. 1. Hektoen enteric agar. Appl. Microbiol. 16: 577-578, 1968.
13. King, S., and Metzger, W. I.: A new plating
medium for the isolation of enteric pathogens. II. Comparison of Hektoen enteric agar
with SS agar and EMB agar. Appl. Microbiol.
16: 579-581, 1968.
14. Pfizer Diagnostics, Descriptive Pamphlet: Hektoen Enteric (HE) Agar. New York, Pfizer Diagnostics Inc., 1968.
15. Small, N. N.: Evaluation of PathoTec strips
in diagnostic microbiology. Amer. J. Med.
Techn. 34: 65-68, 1968.
16. Von Graeventiz, A.: Identification of non-fastidious gram negative rods with delayed or
absent lactose fermentation: A simplified system for the hospital laboratory. Amer. J. Med.
Techn. 8: 459-466, 1968.
17. Weaver, D. K., Lee, E. K. H., and Leahy, M. S.:
Comparison of reagent impregnated paper
strips and conventional methods for identification of Enterobacteriaceae. Amer. J. Path.
49: 494-499, 1968.