550
Journal of Food Protection, Vol. 53, No. 7, Pages 550-554 (July 1990)
Copyright© International Association of Milk. Food and Environmental Sanitarians
Surface-adherent Growth of Listeria monocytogenes
is Associated with Increased Resistance to
Surfactant Sanitizers and Heat
JOSEPH F. FRANK* and ROSE A. KOFFI
Department of Food Science and Technology, University of Georgia, Athens, GA 30602
(Received for publication October 30, 1989)
ABSTRACT
Surface-adherent microcolonies of Listeria monocytogenes
were obtained by growing cells on glass slides immersed in a
low nutrient medium containing excess glucose. The susceptibil-
ity of the adherent populations to benzalkonium chloride (100,
400, and 800 ppm solutions), anionic acid sanitizer (200 and 400
ppm solutions), and heat (55 and 70°C) was determined. Adher-
ent microcolony cells decreased by 2 to 3 log cycles immedi-
ately after exposure to the sanitizers. The remaining population
of microcolony cells survived 20 min of exposure demonstrat-
ing resistance to both sanitizers at all concentrations. Adherent
single cells exhibited an initial 3 to 5 log decline in numbers and
reached undetectable levels after 12 to 16 min of exposure to the
sanitizers. Planktonic cells were reduced to undetectable levels
after 30 sec exposure to the lowest concentration of each sani-
tizer. Removing adherent cells from the surface increased their
sanitizer susceptibility to near that of planktonic cells. Heating
adherent microcolonies at 70°C for 5 min resulted in a less than
5-log decrease in population with a surviving population of over
10 cfu/sq cm. These results demonstrate the ability of L.
monocytogenes to develop resistance to inactivating agents when
exposed to specific growth environments.
Previous studies using conventional cell preparation
techniques have demonstrated the rapid inactivation of
Listeria monocytogenes by sanitizers commonly used in
the food industry for environmental sanitation (1,5,7).
However, Gilbert et al. (2) concluded that the preparation
of inocula for antimicrobial susceptibility testing should
reflect the growth conditions present in the environment in
which the organism is expected to be inactivated. Such
conditions often include slow growth rate, nutrient deple-
tion, and surface attachment. Each of these factors has the
potential to alter the composition and structure of the bacterial
cell envelope in such a manner that resistance to antimi-
crobial agents may be affected (2,14). This study was initiated
with the assumption that typical laboratory culture of L.
monocytogenes does not reflect its normal growth niche in
the food processing plant environment. A more relevant
inoculum for antimicrobial testing would include adherent
JOURNAL OF FOOD PROTECTION,
growth on an inert surface, depleted nutrient levels with
periodic exposure to fresh nutrients, and below optimum
growth temperatures. The objective of this study was to
prepare such an inoculum of L. monocytogenes and test
the ability of commonly used surface active sanitizers to
inactivate these cells.
MATERIALS AND METHODS
Cultures
L. monocytogenes Scott A was obtained from the culture
collection of the Department of Food Science and Technology,
Georgia Agricultural Experiment Station, Griffin, GA. The culture
was maintained on trypticase soy agar (TSA, Difco, Detroit, MI)
at 4°C. The culture was prepared for inoculation by growing in
trypticase soy broth (TSB, Difco) at 21°C with transfers every
24 h for 3 d.
Sanitizer and neutralizing solutions
The quaternary ammonia sanitizer used was n-alkyl (50%
C 40% C12, 10% C16) dimethyl dichlorobenzyl ammonium
chloride (benzalkonium chloride, BAC) obtained as a 10% solu-
tion (Roccal II, Lehn & Fink Industrial Products, Montvale, NJ).
The anionic acid sanitizer was dodecyl benzene sulfonic acid
(DBSA, Sigma, St. Louis, MO) made as a 5% solution in 30%
orthophosphoric acid (5). The diluted (200 ppm) DBSA solution
had a pH of 2.1. BAC was neutralized with a solution of 5.3 g
lecithin (purified grade, Fisher Scientific Co.), 37.5 g Tween 80
(Fisher Scientific Co.), and 0.5 g KH 2 P0 4 in 1000 ml of reagent
grade water. DBSA was neutralized with 3.6 mM NaOH. Sani-
tizers were diluted in 20 mM phosphate buffer solution (PBS,
pH 6.8) made with Type I reagent grade water.
Preparation of glass slides
Glass microscope slides were used to provide an attach-
ment surface for adherent growth. New slides were soaked in
20% NaOH for 5 d at room temperature to etch and clean the
surface, rinsed in .02 N HC1, rinsed with distilled water, boiled
for 1 h in 2% Micro detergent (International Products Corp.,
Trenton, NJ), and rinsed with Type I reagent grade water. Slides
were wiped with acetone immediately before use.
VOL. 53, JULY 1990
 SURFACE ADHERENT GROWTH OF LISTERIA
Preparation of broth-grown (planktonic) cells
Broth-grown (planktonic) cells were obtained using a growth
medium consisting of 2 g/1 dehydrated TSB supplemented with
8 g/1 glucose. This medium (diluted TSB) provided a nutrient
level of 1.32 g/1 hydrolyzed protein and 8.16 g/1 of glucose. The
sterile medium (200 ml) in 250 ml flasks was inoculated with 2
ml of a 24 h TSB culture. Incubation was at 21°C for 48 h with
shaking. Cells were removed from the medium by centrifugation
(2100 rpm for 15 min in a Damon/IEC bechtop centrifuge) and
resuspended in PBS to a concentration of 108 cfu/ml. This
suspension was used in subsequent inactivation studies.
Preparation of adherent microcolony cells
Biofilm cells were grown using diluted TSB (previously
described). Prepared slides were placed in 250 ml flasks con-
taining 200 ml of medium and autoclaved. The sterile medium
was inoculated with 2 ml of a 24 h TSB culture of L. monocytogenes.
After 3 d of incubation at 21°C, each slide was aseptically removed
from its flask, rinsed with sterile PBS using a 500 ml squeeze-
type wash bottle to remove unattached cells, and placed in a
flask of fresh sterile media. Microcolony formation was consid-
ered complete after 14 d of incubation at 21°C.
Preparation of adherent single cells
Sterile prepared slides were placed in a TSB culture (grown
at 21°C for 48 h) and incubated for 4 h at 21°C with shaking.
The slides were removed and rinsed with sterile PBS to remove
unattached cells.
Microscopic observations
Microscopic observations of adherent (4 h and 14 d) popu-
lations were made after staining the cells with either crystal
violet or acridine orange.
Sanitizer inactivation of planktonic cells
Five milliliters of planktonic cell suspension were mixed
with 45 ml of sterile PBS. Three milliliters of this mixture were
removed and mixed with 3 ml of neutralizer solution. This was
the "0" time sample. Enumeration was accomplished using serial
dilutions in PBS and TSA pour plates. Forty-five milliliters of
the remaining solution were mixed with 5 ml of chemical sani-
tizer diluted sufficiently to produce the required concentration
(100 to 800 ppm). The cell/sanitizer solution was continuously
mixed with a magnetic stirrer. Samples (3 ml) were removed
after 30 sec and 5 min and immediately mixed with neutralizing
solution. Samples were then enumerated using TSA pour plates.
Plates were incubated at 32°C for 48 h before colony counting.
The experiment was replicated three times.
Sanitizer inactivation of adherent cells
Slides containing adherent cells were rinsed with sterile
PBS to remove unattached cells. The slides were then submerged
in 200 ml of sanitizer at the appropriate dilution (100 to 800
ppm) for the appropriate time (30 sec to 20 min). The solution
was continuously stirred throughout the experiment. At the end
of the reaction time, the slides were placed in the appropriate
neutralizing solution. Control ("0 time) slides were placed di-
rectly in the neutralizing solution without exposure to sanitizer.
Cells were removed from the neutralized slides by scraping with
a teflon scraper, followed by swabbing with a calcium alginate
swab, followed by rinsing with PBS. The swab and rinsings
were placed in a sterile test tube and the volume adjusted to 4.5
ml with sterile PBS. The swab was partially dissolved by adding
JOURNAL OF FOOD PROTECTION, VOL. 53, JULY 1990
551
0.5 ml of sterile 10% sodium hexamethaphosphate and vortexing
for 1 min. Cells in the resulting suspension were enumerated
using TSA pour plates incubated at 32°C for 48 h. These experi-
ments were replicated 3 to 5 times.
Sanitizer inactivation of detached and dispersed/filtered cells
Adherent microcolonies were scraped and swabbed from
the slides as previously described. These are referred to as detached
cells. Additional cells were obtained by the same procedure but
were also vortexed with glass beads for 1 min and then filtered
through sterile glass wool. These cells are referred to as filtered/
dispersed cells. Both cell preparations were treated with sanitiz-
ers as described for the planktonic cells.
Cell inactivation and sanitizer effectiveness at various tempera-
tures
Data on the effect of temperature on sanitizer effectiveness
were obtained at 4 ± 0.5, 25 + 2, 55 + 0.5, and 70 ± 0.5°C using
5 min exposure times. Exposure at 4°C was in sanitizer solution
(200 ppm DBSA and 400 ppm BAC) and PBS tempered in an
incubator; exposure at 25°C was at ambient room temperature;
and exposure at 55 and 70°C was in solutions tempered in a
circulating water bath. Planktonic cells were suspended in either
tempered sanitizer or PBS at an initial concentration of 5 x 106
to 1 x 107/ml. For experiments at 55 and 70°C, the suspensions
were placed in sterile screw capped test tubes (16 x 125 mm)
which were completely submerged in the water bath using a
beaker of tempered water as a weight. After the treatment, cells
were cooled if necessary and enumerated as previously described.
Slides containing 14 d microcolony growth were submerged directly
into sanitizer or PBS tempered at the appropriate temperature.
After exposure, slides were scraped and cells enumerated as
previously described. An additional treatment consisted of bi-
ofilm-containing slides exposed to the various temperatures for
5 min in PBS and then exposed to DBSA or BAC for an additional
5 min. These experiments were replicated three times.
RESULTS
Adherent microcolonies of L. monocytogenes formed
on glass slides over a period of 14 d at 21°C. Surface cfu/
cm 2 approached 10 million during this time period. Micro-
scopic observation showed that adherent cells were in both
single and microcolony form after the 14 d incubation,
whereas after 4 h of incubation in a TSB culture, mostly
single cells were observed on the slide with a few adherent
small clumps.
Inactivation with bemalkonium chloride
Inactivation curves of adherent microcolony popula-
tions of L. monocytogenes were similar when using 100,
400, and 800 ppm BAC (Fig. 1). In each case, there was
a 2 to 3 log decrease in cell numbers during the first 30
sec after which a resistant population remained for at least
20 min. Broth-grown planktonic cells were rapidly inactivated
in 30 sec in all cases. Adherent single cell populations
exhibited a 3 to 5 log decrease in number during the first
30 sec, with a greater decrease observed when using 800
552 FRANK AND KOFFI

U
T cell populations were over 105 but always under 106 cfu/
100 ppm cm 2 , whereas microcolony populations were always over
1\ 106 and usually just under 107 cfu/cm2. When a second
microcolony-containing slide was treated for 20 min with
2-
previously used BAC, microcolony cell concentrations
^•^
declined by an amount similar to the decline observed
a V l\l
r-\_ •-
^ ^ \.
using fresh solution (data not shown), indicating that survival
a •' \ T• .
a ^ ^ ^ v of the adherent cells is a result of resistance to the BAC
4-
1\ rather than loss of BAC activity.
s b-
A^
^^^
•
1
Inactivation with acid anionic sanitizer
fi- G 1— 1 1 -==* 1 Inactivation of adherent populations of L. monocytogenes
12 16 20 24
TIME (MIN) by DBSA was similar to that observed by BAC (Fig. 2).

(J
UJ o
a UJ
a Q
o O
O

0 12 16 8 12 16 24
TIME (MIN) TIME (MIN)
0 II

1
800 ppn-l
1
1
400 ppm

<S1 2 ] iS
2-

3
• N •— IX.
o 3- f—f- —•—
i
a i 1 Q
o
Q 4 1
" •—— -4.
!
O
O 4-
- » \
^ '
.^
"A->.
1 ^H i

5-
^^
^^A,
J
^A _
*~A
-e 1— —1 -^^•A — i 1i R- e h- =-A 1 11
—1
8 12 16 20 24 12 16 20 24
TIME (MIN) TIME (MIN)

Figure 1. Inactivation (log decrease in cfu/cm2) of adherent

microcolonies, adherent single cells, and planktonic cells o/Listeria
monocytogenes by 100, 400, and 800 ppm benzylakonium chloride.
O—O planktonic cells; • — • adherent microcolonies; A—A
adherent single cells.
Figure 2. Inactivation (log decrease in cfu/cm2) of adherent
microcolonies, adherent single cells, and planktonic cells o/Listeria
monocytogenes by 200 and 400 ppm benzylakonium chloride.
O—O planktonic cells; • — • adherent microcolonies; A—A
adherent single cells.

ppm as compared to 100 ppm BAC. Adherent single cells
could not be recovered after 16 min when 100 ppm BAC
was used and after 12 min when 400 and 800 ppm BAC
were used (Fig. 1). Although adherence to the surface in
itself accounts for some BAC resistance, growth of the
adherent population resulted in substantially increased
resistance. Data comparing the inactivation of adherent
single cell populations and adherent microcolony popula-
tions should be interpreted with caution, since initial
population levels were different. Initial adherent single
A 2 to 3 log decrease in cfu was observed for adherent
microcolonies during the first 30 sec of exposure, after
which a resistant population remained for at least 20 min.
Adherent single cells were more rapidly inactivated than
the adherent microcolony population and could not be
recovered after 12 to 16 min. Increasing DBSA concentra-
tion from 200 to 400 ppm had no effect on survival of the
resistant microcolony population. Broth-grown (planktonic)
cells were rapidly and completely inactivated by a 30 sec
exposure to DBSA.

JOURNAL OF FOOD PROTECTION. VOL. 53, JULY 1990

 SURFACE ADHERENT GROWTH OF LISTERIA 553

Inactivation of detached microcolony cells

Listeria microcolonies detached from the slide were
susceptible to BAC inactivation, exhibiting a 5 log de-
crease in numbers after 30 sec of exposure (Table 1).
Dispersing and filtering the detached cells, so that large
clumps would be removed, further increased sanitizer 3
z
effectiveness, resulting in a greater than 6 log decline within o
30 sec of exposure.

TABLE 1. Inactivation of detached and dispersed/filtered sur-

face-grown Listeria monocytogenes by 400 ppm benzalkonium 4°c 25°c 55°c 70°c
chloride.
TEMPERATURE (°C)
Time (min) 1=1 PLANKTONIC, NO TREATMENT MBIOFILM, DBSA TREATMENT
Treatment 0 0.5 4 8 12 E a BIOFILM, NOTREATMENT C H BIOFILM.T°C, DBSA AT 25°C

(log cfu/cm2) Figure 4. Inactivation (log cfu/cm2) of adherent microcolony and

a
Detached 6.3±0.2 1.4+0.1 1.1+0.8 0.5+.7 <0.13 planktonic Listeria monocytogenes at different temperatures in
Dispersed 6.7±0.1 <0.13 <0.13 <0.13 <0.13 the presence of 200 ppm dodecyl benznes sulfonic acid. All
"Mean + standard deviation; N = 3. treatments were for 5 min. No treatment = no exposure to DBSA.
T°C = treatment at indicated temp, followed by exposure to
DBSA for 5 min.
Effect of temperature on sanitizer effectiveness
Planktonic cells were completely inactivated at 4°C not more effective in inactivating these cells. However,
exposure to 400 ppm BAC and 200 ppm DBSA for 5 min. heating for 5 min at 70°C followed by sanitizer exposure
Adherent microcolony cells, however, declined in number for 5 min at 25°C reduced the adherent population to nearly
only 1 to 2 log units when exposed to the sanitizers at this undetectable levels. Planktonic cells were reduced to
temperature (Fig. 3 & 4). undetectable levels by the 70°C heat treatment, a greater
than 7 log decrease in number.

6
\i DISCUSSION

5
Previous studies demonstrated the surface attachment
Ld
and adherent microcolony formation potential of L.
| 4
monocytogenes (4,6,7). Sanitizer resistance of microorgan-
o 3 isms attached to gasket surfaces has been reported by
Mosteller and Bishop (6). This study confirms that attach-

4°C 25°C 55°C

k 70°C
TEMPERATURE (°C)
• PLANKTONIC, NO TREATMENT BBBIOFILM, BAC TREATMENT
^ B I O F I L M , NO TREATMENT L5]BI0FILM, T°C, BAC AT 25°C
Figure 3. Inactivation (log cfu/cm2) of adherent microcolony and
planktonic Listeria monocytogenes at different temperatures in
the presence of 400 ppm benzalkonium chloride (BAC). All
treatments were for 5 min. No treatment = no exposure to BAC.
T°C = treated at indicated temp, followed by exposure to BAC
for 5 min.
Planktonic cells were slowly inactivated at 55°C even
in the absence of sanitizer, whereas the population of adherent
microcolonies declined only slightly at this temperature.
Treating the adherent cells with sanitizers at 55°C reduced
the population by only 2 log units in 5 min. In addition,
heating adherent cells to 55°C did not change susceptibil-
ity to subsequent sanitizer treatment.
Cells in adherent microcolonies were reduced in number
by less than 5 log units by heating at 70°C for 5 min (Fig.
3 & 4). A population of over 10 cfu/cm2 survived this heat
treatment. Treatment with sanitizer at 70°C for 5 min was
ment of cells to surfaces can provide protection against
chemical sanitizers. When attachment is followed by growth,
our data indicate that additional resistance develops. Ruseka
et al. (77) previously reported the ineffectiveness of BAC
in inactivating an undefined microbial biofilm population.
Mustapha and Liewen (7) reported the successful inactiva-
tion of adherent L. monocytogenes by QAC and hypochlorite.
However, their enumeration procedure after sanitizer treat-
ment may have excluded active adherent cells, since the
growth surface was not physically scraped or swabbed.
This study confirms the importance of method of inocula
preparation for antimicrobial sensitivity assays as discussed
by Gilbert (2). In addition, our data indicate that the adherent
form of growth has a remarkable effect on heat resistance.
Although our technique for cell growth may be more relevant
to food industry sanitation than those previously used, it
is probable that the physiological state of L. monocytogenes
in the food processing plant is highly variable, ranging
from planktonic growth in rich media to slow growth in
nutrient depleted multispecies biofilms. Additional study
on the prevalent growth forms of Listeria spp. in food
plants needs to be completed to assess the true importance
of data presented in this study."

JOURNAL OF FOOD PROTECTION, VOL. 53, JULY 1990

554 FRANK AND KOFF1
Mechanism of resistance
The adherent cells used in this study exhibited a slow
growth rate, as a 14 day incubation was required to de-
velop the microcolonies. Slow growth has been associated
with sanitizer resistance in Escherichia coli (14). A por-
tion of the cells attached to the surface would be single
cells originating from the planktonic population. We
hypothesize that the initial rapid inactivation of the adher-
ent cells was due to these single cells, with the micro-
colony cells remaining as the resistant population.
Quaternary ammonium compounds (QAC) are hydro-
philic cationic molecules. Since the bacterial surface is
hydrophilic and negatively charged, QAC readily adsorb
to it, penetrate the cell wall, and disrupt the cytoplasmic
membrane (9). Cells in an adherent microcolony might be
protected from QAC action if layers of cells prevent
penetration of the chemical, or if cells produce a protec-
tive coating on the exposed cell surface (12). Gram-nega-
tive bacteria exhibit increased resistance to BAC associ-
ated with increased lipid content of the outer membrane
(11). Gram-positive bacteria can produce extracellular
lipoteichoic acids (3) which being lipophilic may prevent
penetration of sanitizers. Lipoteichoic acid production can
be stimulated by adherent growth (10). In addition, L.
monocytogenes produces a surface-bound lipopolysaccharide-
like substance (75) which could increase cell envelope
lipophilicity. The adherent cells used in this study were
grown with excess glucose in the medium to stimulate
production of extracellular substances (glycocalyx); how-
ever, no attempt was made to observe or measure these
substances.
Nickel et al. (8) studied the antibiotic resistance of
adherent Pseudomonas aeruginosa and found that the scraped
biofilm population lost its resistance to tobramycin. Scrap-
ing the cells from the surface and subsequent mixing in
sodium hexametaphosphate solution may disrupt a protec-
tive coating sufficiently to allow sanitizer penetration into
the cell. This would explain the apparent increase in sanitizer
sensitivity caused by the removal of the adherent cells
from the surface. Our results indicate a common mecha-
nism of resistance against anionic and cationic sanitizers.
The different pH values employed by the anionic and cationic
sanitizers (2.1 and 6.8, respectively) would indicate that
cellular surface charge does not play a role in the resis-
tance mechanism.
Practical implications
This research indicates that if after cleaning a surface,
adherent microcolonies of L. monocytogenes still remain,
the cationic and anionic surface active chemicals studied
may not be effective sanitizers. Removal of adherent
microcolonies is accomplished through standard cleaning
procedures when applied to smooth food contact surfaces.
JOURNAL OF FOOD PROTECTION.
Subsequent sanitizing will then leave a surface free of
pathogens. However, the effectiveness of cleaning proce-
dures for environmental surfaces is unknown, and certainly
in many cases, where surfaces are cracked, rough or dif-
ficult to reach, removal of adherent microbial populations
will not be complete, possibly resulting in survival of a
contaminating pathogen. Our results indicate that the same
cleanliness standards applied to food contact surfaces must
be applied to environmental surfaces if adequate Listeria
spp. control is to be maintained within the food plant.
ACKNOWLEDGMENT
This research was supported by State and Hatch funds allocated to
the Georgia Agricultural Experiment Stations. The authors wish to thank
Dr. William Whitman, Department of Microbiology, for his helpful con-
sultations regarding this project.
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VOL. 53. JULY 1990