JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1975, p. 498-503 Vol. 2, No.

6
Copyright C 1975 American Society for Microbiology Printed in U.SA.

Improved Transport System for Neisseria gonorrhoeae in

Clinical Specimens
DOROTHY A. SYMINGTON
Ontario Ministry of Health, Provincial Public Health Laboratory, Toronto, Ontario, M5W 1R5 Canada
Received for publication 10 September 1975

Protective transport media have to be used to preserve Neisseria gonorrhoeae

in clinical specimens during their transit to the laboratory. In this study, a C02-
environment chamber, the Jembec chamber, was used for transport of clinical
specimens requiring examination for gonococci. The survival of N. gonorrhoeae
present in clinical specimens when placed in Amies charcoal transport medium
was compared to their survival when inoculated into Jembec chambers contain-
ing either modified Thayer-Martin medium (MTM) or modified New York City
transport medium (MNYC). For a period of up to 2 days in transit, the three
systems were not significantly different. However, after 3 days in transit,
MNYC/Jembec chambers preserved significantly more gonococci than Amies
charcoal transport medium (P < 0.0001) or MTM/Jembec chambers (P = 0.006).
MNYC/Jembec chambers withstood 241 miles (386 km) of postal transit during
winter months; 80% of the gonococci present in clinical specimens remained
viable from 2 to 5 days under these conditions. The CO2 generated by the tablet
in the Jembec chamber was sufficient to support the growth of N. gonorrhoeae if
the chambers were incubated at 36 C immediately after inoculation. However, if
delayed in transit, the chambers had to be incubated in 5 to 10% CO2 to promote
the growth of N. gonorrhoeae. MNYC/Jembec chambers provide a selective
environment that will protect and maintain the viability of N. gonorrhoeae for
extended periods, allowing a reasonable time for postal transit of clinical speci-
mens to the laboratory.

Gonorrhea is now recognized as one of the
most prevalent communicable diseases in
North America. The causative organism, Neis-
seria gonorrhoeae, is a quite fastidious bacte-
rium that autolyzes rapidly. In clinical speci-
mens, the preservation and culturing of N. gon-
orrhoeae is further complicated by the presence
of numerous, faster-growing commensal orga-
nisms. Ideally, clinical specimens should be di-
rectly inoculated onto a suitable gonococcal cul-
ture (GC) selective medium and immediately
incubated at 36 C in 5 to 10% CO2. However,
the necessary facilities are not available in phy-
sicians' offices or in most venereal disease clin-
ics.
Protective transport systems have to be used
to preserve N. gonorrhoeae in clinical speci-
mens during transfer to the laboratory for cul-
turing. For many years the type of transport
system used was a nonnutrient, semisolid,
buffered-agar medium, initially described by
Stuart (16) and subsequently modified by other
authors (1, 15). More recently, the Transgrow
system used was a non-nutrient, semisolid,
a combined transport and culture medium for
N. gonorrhoeae and N. meningitidis, has been
498
used in some areas of North America. Although
Transgrow medium has proved to have some
value in the bacteriological diagnosis of gonor-
rhea, it does have several important shortcom-
ings. Commercial preparations of Transgrow
have been found to contain variable C02 concen-
trations (3), and different batches of the me-
dium vary in their ability to support growth of
N. gonorrhoeae. Condensation frequently accu-
mulates inside the bottles, blocking visibility of
growth on the slope. This moisture also contrib-
utes to the spreading of contaminants and inter-
feres with the formation of isolated colonies.
Inoculation and subculturing of colonies
through the narrow bottle neck is awkward.
Recently Martin et al. (9) described a new
system called "Gono-Pak" for cultivation of N.
gonorrhoeae. This system consists of a petri
plate containing modified Thayer-Martin me-
dium (MTM), which is inoculated, then sealed,
in a plastic bag along with a C02-generating
tablet. Holston et al. (8) evaluated this system
in a clinical trial and found it to be as efficient
as the CO2 atmosphere in a candle jar for sup-
porting growth of N. gonorrhoeae. In a pre-
vious study (17) we found that the Gono-Pak
VOL. 2, 1975
system with a petri plate containing modified
New York City transport medium (MNYC) (7)
was suitable for both transportation and incuba-
tion of N. gonorrhoeae.
A rectangular plastic plate with a small well
to accommodate a C02-generating tablet is now
marketed as a Jembec plate (Ames Co., Divi-
sion of Miles Laboratories, Elkart, Ind.). This
plate and a C02-generating tablet, sealed in a
plastic "zip-lock" pouch, provides a CO2 environ-
ment chamber that will be referred to as a
Jembec chamber (Fig. 1). Jembec plates con-
taining prepoured MTM medium are marketed
as Neigon plates by Flow Laboratories Inc.,
Rockville, Md. In this study, we compared the
efficiency of three different transport media for
preserving gonococci in clinical specimens, as
follows: (i) Amies charcoal transport medium;
(ii) Jembec chambers containing Neigon plates
of modified Thayer-Martin medium (MTM/
Jembec), and (iii) Jembec chambers containing
plates of modified New York City transport
medium (MNYC/Jembec).
MATERIALS AND METHODS
Media. Amies charcoal transport medium was
prepared according to the method ofAmies (1). Modi-
fied Thayer-Martin medium was purchased as Nei-
gon plates from Flow Laboratories Inc., Rockville,
Md. The manufacturer states that this medium has
the formula listed in Table 1.
MNYC was prepared according to the formula
shown in Table 1, modified from the procedure of
Faur et al. (5, 6, 7). The main modification was the
TRANSPORT SYSTEM FOR N. GONORRHOEAE 499
replacement of 3 ug of vancomycin per ml by 4 tg of
lincomycin per ml. This change was made because
recent reports have shown that 3 to 10% of strains of
N. gonorrhoeae are sensitive to vancomycin (2, 4, 13,
14). Additional minor changes made were in the
other manufactured ingredients and the agar con-
centration. MNYC selective medium was prepared
according to the same formula as the transport me-
dium, except the agar concentration was 1% and the
yeast concentration was 2.5%.
Quality control. Each batch of MNYC and MTM
medium was tested by the quality control method
recommended by the Center for Disease Control,
Atlanta, Ga. (11).
Collection of specimens. Clinical specimens (two
urethral swabs from each male and two endocervical
swabs from each female patient) were received from
St. Michael's Hospital, Toronto, Special Treatment
Clinic. Specimens from this clinic were delivered by
courier and reached the laboratory within 24 h. The
FIG. 1. The Jembec chamber.

TABLE 1. Formulae of MTM and MNYC transport media

MTM medium MNYC transport medium"
Ingredient Amount (per liter) Ingredient Amount (per liter)
Agar 12.5 g Oxoid agar no. 4 12 g
Peptone 15 g Proteose peptone no. 3 15 g
KH2PO4 1g (Difco)
K2HPO4 4g KH2PO4 1g
Sodium chloride 5g K2HPO4 4g
Corn starch 1g Sodium chloride 5g
Distilled water 1,000 ml Corn starch 1g
L-Cysteine hydrochloride 260 mg Distilled water 615 ml
L-Glutamine 100 mg Fresh yeast dialysate
L-Cystine 10 mg (Fleishmann 2040) 50 ml
Coenzyme 1 2.5 mg 3% Lysed horse erythro-
Hemoglobin 10 g cytes 200 ml
Cocarboxylase 1g Horse serum 120 ml
Dextrose 5.55 g 50 g% Dextrose 10 ml
Trimethoprim lactate 5 mg Trimethoprim lactate 5 mg
Colistin 7.5 mg Colistin 7.5 mg
Vancomycin 3.5 mg Lincomycin 4 mg
Nystatin 50,000 U Amphotericin B 1 mg
a After addition of distilled water, autoclave at 15 lb/in2 for 15 min, cool to 50 C, and add the remaining
ingredients.
500 SYMINGTON J. CLIN. MICROBIOL.
subsequent storage of these specimens was varied. MNYC transport medium prepared in our
Specimens were also received by mail from Sudbury media laboratory consistently inhibited the
Regional Health Unit, Special Treatment Clinic, growth of a standard inoculum of 50 to 100
located 241 miles (386 km) north of Toronto. This organisms of Escherichia coli, Staphylococcus
latter study was undertaken to test the ability of the
different transport systems to withstand the rigors aureus, Proteus sp., and Candida sp. N. gonor-
of Northern Ontario winter temperatures, postal rhoeae always grew on freshly prepared media,
handling, and prolonged periods in transit. and the number of colonies was comparable to
At St. Michael's Hospital, the first swab was inoc- the growth on the same medium without anti-
ulated onto an MTM/- or MNYC/Jembec plate and biotics. Occasionally the size of the colony was
then placed in Amies charcoal transport medium. smaller on one batch of medium, and this phe-
The second swab was used to make a smear for nomenon was traced to the quality of horse
Gram staining. The plates were sealed in the bag serum used. MNYC/Jembec plates, stored at
with the TABCO2 tablet in the well and transferred room temperature, had a shelf life of 1 month or
along with Amies medium to the laboratory. On re- 2 months when stored at 4 C. Amphotericin B
ceipt, the swabs in Amies medium were cultured by
our routine GC laboratory on a petri plate contain- was the most unstable ingredient in this me-
ing MNYC selective medium. The MTM/- or MNYC/ dium; inhibition of growth of Candida was the
Jembec chambers were stored at room temperature first quality control criterion to fail.
for the various periods, and then the plates were Conditions of incubation of Jembec plates.
incubated at 36 C in 5% C02. Two hundred fifty- We found that in Jembec chambers containing
eight specimens were received in MTM/Jembec either MTM or MNYC medium, the CO2 gener-
chambers and 377 in MNYC/Jembec chambers. ated by the tablet was adequate to support the
In Sudbury, the first swab was processed as de- growth ofN. gonorrhoeae in clinical specimens,
scribed above; the second swab was used to make a provided the chambers were incubated at 36 C
smear and then placed in a second Amies charcoal
transport medium. One of the swabs from Amies immediately after inoculation. However, we
medium was cultured immediately or within 24 h by thought it necessary to determine whether suffi-
Sudbury Regional Public Health Laboratory (located cient CO2 for support of gonococcal growth was
in the same building as the clinic) on a petri plate still present in the chambers after some storage
containing MNYC selective medium and incubated time. Our previous study (17) showed that a
in a candle jar at 36 C. The other swab in Amies Gono-Pak system was comparable to Amies me-
medium and the inoculated Jembec chamber were dium for recovery of gonococci from clinical spec-
mailed to Toronto. The swabs taken first or second imens up to 24 h, but neither reliably preserved
were alternated for culturing in Sudbury or mailing
to Toronto. On receipt in Toronto, the Jembec plates
N. gonorrhoeae for 72 h. However, for dura-
were incubated at 37 C in a 5% C02 incubator. The
tions between 24 and 48 h, the Gono-Pak sys-
swab received in Amies medium was plated on a tem was superior to Amies medium. We there-
petri plate containing MNYC selective medium and fore chose to store specimens in MTM/- or
incubated under the same conditions. Both plates MNYC/Jembec chambers for 48 h at room tem-
were read after 24 and 48 h of incubation. Sixty- perature and then either incubate them at 36 C
seven specimens were received as MTM/Jembec in their bags in a regular incubator or remove
chambers and 173 as MNYC/Jembec chambers. the plates from the bags and incubate them at
The transit time of specimens from Sudbury to 36 C in a 5% C02 incubator. The corresponding
Toronto was 1 to 5 days, with an average of 3 days.
N. gonorrhoeae was identified on the basis of colo- swabs from the same patients, which were re-
nial morphology, oxidase reaction, Gram stain, and ceived in Amies charcoal transport medium,
fluorescent antibody technique. were processed by our routine GC department
Statistical analysis. The results obtained were immediately after they were received in the
analyzed by a standard Z test for population propor- laboratory, which was within 24 h of sampling.
tions (12). The number of N. gonorrhoeae isolated by this
procedure was considered as the total number
RESULTS of positive specimens. On MTM/Jembec plates,
65% of the gonococci were recovered when the
Quality control and shelf life of media. plates were incubated in their bags in a regular
Four separate batches of MTM/Jembec plates incubator, but 83% were recovered from the
were checked. The first batch of plates was plates that were incubated in a 5% C02 incuba-
satisfactory for the quality control standards. tor (P = 0.2). On MNYC/Jembec plates, 38% of
Candida sp. grew on the other three batches. the gonococci were recovered from the plates
The size of the Candida colonies was smaller, incubated in a 5% C02 incubator (P < 0.0001)
but the number was comparable to the control (Table 2). Since these results showed that the
plate without antibiotics. No shelf-life control presence of 5% fresh C02 during incubation
was carried out on the Neigon plates since their improved the recovery of gonococci from both
exact preparation date was not known. MTM/- and MNYC/Jembec chambers, in all
VOL. 2, 1975
further studies these plates were incubated at
36 C in a 5% CO2 incubator.
Comparison of Amies charcoal transport
medium to MTM/- and MNYC/Jembec cham-
bers. The recovery of gonococci from clinical
specimens inoculated into these three transport
systems was recorded after 48 or 72 h in transit
and compared to the results obtained by our
routine GC laboratory with swabs received in
Amies medium (Table 3). These swabs were
cultured within 24 h of sampling. The survival
rates after 48 and 72 h in transit were 70 and
34%, respectively, from Amies; 84 and 33% for
MTMIJembec chambers; and 85 and 87% for
MNYC/Jembec chambers. After transit times
of 48 h or less, there was no significant differ-
TABLE 2. Influence of incubation conditions on growth of Neisseria gonorrhoeae after 48 h of storage in
MTMI- or MNYC/Jembec chambers
No. of speci- No. positive by
mens exam- routine examina-
ined tion
No.
TRANSPORT SYSTEM FOR N. GONORRHOEAE 501
ence in survival of gonococci in any of these
three transport systems. At 72 h or longer,
MNYC/Jembec chambers gave a significantly
better recovery rate than Amies medium (P <
0.0001) or MTM/Jembec chambers (P = 0.006).
MTMIJembec chambers grew more contami-
nants (31%) than did MNYC/Jembec chambers
(19%).
Field trial in Sudbury. The results of this
trial are presented in Table 4. Sixty-seven speci-
mens were mailed in MTM/Jembec chambers;
23 of these specimens contained N. gonor-
rhoeae, but only 9 (39%) grew on MTM/Jembec
plates after mailing and incubation in Toronto,
and only 5 (33%) grew from the corresponding
Amies medium. There was no significant differ-
Positive from Jembec chambers
MTM medium (Neigon) MNYC transport medium
C02 Without C02 C02 Without C02
% No. % No. 9c No. %

114 18 15 83
119 17 11 65
96 18 15 83
110 21 8 38

TABLE 3. Recovery ofNeisseria gonorrhoeae from clinical specimens transported in Amies charcoal transport
medium, MTMI-, or MNYCIJembec chambers
Positive after storage time of
48h 72h
No. of speci- No. positive by
mens exam- routine examina- Amies char- MTM/Jem MNYC/Jem Amies char- MTM/Jem- MNYC/Jem-
ined tion coal trans-
. bec chamber bec chamber coal trans-- bec chamber bec chamber
port medium port medium
No. % No. % No. % No. 9i No. % No. %

195 41 29 70
114 18 15 83
155 43 37 86
173 40 15 37
33 15 5 33
122 24 21 87

TABLE 4. Recovery of Neisseria gonorrhoeae from clinical specimens after transport from Sudbury to Toronto
Positive after transport to Toronto in
No. of specimens No. positive when cul- Amies charcoal trans- MTM/Jembec cham- MNYC/Jembec cham-
examined tured immediately in port medium bers bers
No. % No. % No. %

67 23 5 22 9 39
173 57 23 39 46 80
502 SYMINGTON
ence between these two transport systems (P =
0.18). One hundred seventy-three specimens
were mailed to Toronto in MNYC/Jembec cham-
bers; 57 of these specimens contained gonococci.
Of the MNYC/Jembec plates 46 (80.3%) were
positive after incubation in Toronto, but the
corresponding swabs mailed in Amies medium
yielded only 23 (39%) positives. The MNYC/
Jembec chambers gave a significantly higher
recovery rate than Amies medium (P < 0.0001)
or MTM/Jembec chambers (P < 0.0003). Sixty-
eight percent of the positives on MNYC/Jembec
plates could be identified after 24 h of incuba-
tion, but only 42.8% of those from Amies me-
dium grew in that time. Contaminants grew on
20 (11%) of the MNYC/Jembec plates, but 54
(31%) of the swabs plated from Amies medium
yielded bacterial or yeast contamination.
DISCUSSION
The results of this study demonstrated that
the MNYC/Jembec chamber was the most effi-
cient of the three transport systems evaluated
for preservation of gonococci present in clinical
specimens. The MTM/Jembec chambers gave
no significant improvement in recovery of N.
gonorrhoeae as compared to Amies charcoal
transport medium in a field trial (P = 0.26).
On the other hand, the number of gonococci
recovered from MNYC/Jembec chambers was
significantly greater than from Amies charcoal
transport medium (P < 0.0001) or from
MTM/Jembec chambers (P < 0.0003) in this
trial. Furthermore, the N. gonorrhoeae sur-
vival time was longer in the MNYC/Jembec
chambers than in the other two systems.
Fewer bacterial or yeast contaminants grew
on the MNYC/Jembec plates than on
MTM/Jembec plates, which confirmed our ob-
servation from quality control studies that
MNYC transport medium was more selective
than MTM medium. In particular, amphoteri-
cin B was a more effective fungicide than nysta-
tin. MNYC/Jembec plates also grew fewer con-
taminants from the same specimens when cul-
tured after transport in Amies charcoal trans-
port medium. About 30% of the MTM/Jembec
plates that were sent to Sudbury could not be
inoculated because the medium had dislodged
or broken. Initially, we had a similar problem
with MNYC/Jembec plates, but we were able to
eliminate this by increasing the agar concentra-
tion in the medium.
The results presented in this article have
shown that the Jembec chamber containing
MNYC transport medium was a good transport
and growth system for recovery of N. gonor-
rhoeae from clinical specimens. In these clinical
J. CLIN. MICROBIOL.
trials, over 80% of the N. gonorrhoeae present
survived for 72 h in transit in MNYC/Jembec
chambers. With a petri plate of MNYC in a
Gono-Pak system, we previously obtained relia-
ble survival of gonococci for up to 48 h (17). The
increase in survival time in the Jembec cham-
ber could possibly be attributed to the fact that
the Jembec plate has a better seal, and thus an
adequate CO2 concentration is maintained for
longer periods. In addition, there was little de-
hydration of medium in the Jembec plates.
However, although CO2 generated by the
TABCO, tablet was initially sufficient to sup-
port the growth ofN. gonorrhoeae, this concen-
tration did not remain constant during trans-
port. The organisms were viable after 48 h in
transit, but the Jembec plates had to be incu-
bated in a CO2 incubator or candle jar to facili-
tate growth.
From our experience, we believe that the
Jembec chamber is a major improvement over
the Transgrow system for the recovery of N.
gonorrhoeae from clinical specimens that are
delayed in transit. In our hands, MNYC trans-
port medium was more selective and more pro-
tective for gonococci than MTM medium. The
Jembec plates were much easier to handle
than Transgrow bottles both for inoculation
and subculturing. These plates should be stored
at 4 C and brought to room temperature before
use. At 4 C they had a shelf life of 2 months.
The Jembec chamber could be used in two
ways. For physicians who see only a few pa-
tients, the Jembec chambers should be sent to
the laboratory immediately after inoculation
and then incubated at 35 C in 5% CO2 for 48 h.
At clinics, where many patients are seen in the
evening, it would be advantageous to have a
36 C incubator and place the Jembec chambers
inside immediately after inoculation and then
leave them overnight before transfer to the lab-
oratory. On receipt at the laboratory the
plates would need only to be incubated for 24 h
at 36 C in 5% CO2. This overnight incubation at
the clinic would enable results to be available
24 h earlier than if Amies charcoal transport
medium were used. Small, inexpensive, porta-
ble incubators are marketed now by Flow Labo-
ratories, Inc., Rockville, Md., and are ideal for
this purpose.
The Jembec chamber is by no means the
complete solution to the quest for a foolproof
method for the bacteriological diagnosis of gon-
orrhea. Because N. gonorrhoeae is a fastidious
bacterial species present among many contami-
nants in clinical specimens, several other fac-
tors are critical. Of paramount importance is
the type of specimen collected in order to in-
crease the chance of recovering gonococci if the
VOL. 2, 1975 TRANSPORT SYSTEM FOR N. GONORRHOEAE
organisms are present. A major improvement
in the rates of gonococcal recovery could be
achieved by administrative measures to ensure
immediate transfer of specimens to the labora-
tory for inoculation onto GC selective medium.
However, although delays in transit continue
to exist, a transport outfit containing an
MNYC/Jembec chamber, a slide, sterile swabs,
and a data sheet is robust enough to withstand
winter temperatures and postal handling. Such
an outfit provides a selective environment that
will protect and maintain the viability of 80%
N. gonorrhoeae for up to 72 h. This allows a
reasonable time for postal transfer of specimens
to the laboratory.
ACKNOWLEDGMENTS
I wish to thank W.T.R. Linton and staff at St. Michael's
Hospital, Special Treatment Clinic; J.B. Cook and staff at
Sudbury Regional Health Unit for their cooperation; the
staffs of Sudbury Regional Public Health Laboratory and
the GC and media laboratories in Toronto for their assis-
tance; and S. Toma for constructive criticism of the
manuscript.
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