The Method of Quantitative Burn-wound Biopsy

Cultures and Its Routine Use in the Care

of the Burned Patient
E D W A R D C. L O E B L , M.D., J A N E T A. MARVIN, R.N., M.N., E L L E N L. H E C K , B.S., MT
(ASCP), P. WILLIAM CURRERI, M.D., F.A.C.S., AND
C H A R L E S R. BAXTER, M.D., F.A.C.S.

Department of Surgery, University of Texas, Southwestern Medical School at Dallas,

and Parkland Memorial Hospital, Dallas, Texas

ABSTRACT

Loebl, Edward C , Marvin, Janet A., Heck, Ellen L., Curreri, P. William, and
Baxter, Charles R.: The method of quantitative burn-wound biopsy cultures
and its routine use in the care of the burned patient. Am. J. Clin. Pathol. 61:
20-24, 1974. A method for quantitative biopsy cultures of burn wounds has
been developed and evaluated. Specimens are obtained by full-thickness biopsy
of the burn wound using a scalpel. Specimens are processed by maceration,
suspension in physiologic saline solution, and plating of serial dilutions of the
suspension on appropriate media. Quantitative counts are performed and
expressed as the number of bacteria per Gm. of burn-wound tissue. Experience
with this technic in the care of 210 consecutive patients with burns of more than
20% total body surface area has shown it to be more reliable than surface-
culture technics. Patients were considered to have wound colonization when
104 or more organisms per Gm. of burned tissue were recovered from
different areas of the body. Appropriate antibiotic therapy was instituted
on the basis of these results. Routine use of this method was found to be
effective, simple, and practical as an aid in the early diagnosis of sepsis in
the burned patient. (Key words: Bacteriology; Burns; Burn wound biopsy;
Burn wound sepsis; Culture technics; Sepsis; Tissue bacteriology.)

SINCE THE DEMONSTRATION by Teplitz teriologic monitoring of the burned pa-

and associates9 of the pathogenesis of burn-
wound sepsis by progressive bacterial
colonization of the burn wound and subse-
quent invasion from nonviable tissue into
normal tissue, attempts have been made to
develop improved meuhods of clinical bac-
- : ; ~~ , . ,,. .
Received May 23, 1973; accepted tor publication
July 19, 1973.
This investigation was supported in part by National
Institutes of Health Fellowship Number 1 F02
GM54887-01 and Research Grant Number 5 ROl
HL08771-10.
20
tient. Quantitative wound-culture technics
appear to offer significant advantages over
other technics such as blood cultures and
wound-surface cultures. Blood cultures, al-
though helpful, are of only limited value
due to the frequent absence of bacteremia
in cases of massive life-threatening burn-
. . r . .
wound colonization. Surface-culture tech-
nics, including Swabs, contact plates, and
r a n j l i a r v _ a i l 7 e ferhnics fail to Dredirt ac-
capillary-gauze tecnnics, rail to predict ac
curately the presence or progression of

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 January 1974 QUANTITATIVE BURN-WOUND BIOPSY CULTURES 21

burn-wound sepsis, due to poor correlation

between the surface flora cultured and the
colonization of deep tissue. 2 3 5 6
T h e early diagnosis of burn-wound sep-
sis and the evaluation of the effectiveness of
therapeutic regimens require a method of
serial assessment of the concentration and
distribution of bacteria in the burn wound.
Review of the literature failed to reveal any
detailed description of a clinically applica-
ble method for quantitative evaluation of
the bacteriology of the burn site. Full-
thickness burn-wound biopsies have been
developed and evaluated as an adjunct to
the care of the thermally injured patient.
FIG. 1. The method of obtaining biopsy culture.
Parallel incisions are made through the burn wound
Materials and Methods with a scalpel (a), the specimen is elevated from sur-
rounding tissue with forceps (b), dissected free with a
Of 272 consecutive admissions to the scalpel (c), and placed in a dry sterile tube (d).
Burn Unit of Parkland Memorial Hospital,
210 patients with burns of more than 20%
total body surface area were studied by both they may inhibit growth in vitro of deep
wound-surface culture and quantitative tissue organisms), a biopsy is performed by
biopsy culture technics. making two parallel incisions, approxi-
Multiple surface cultures and full- mately 1 to 2 cm. in length and 1.5 cm. apart
thickness wound biopsies were obtained at (Fig. la). The specimen is then elevated
three-day intervals, from admission until with tissue forceps and cut from the sub-
eschar separation. All patients were treated cutaneous tissue at a sufficient depth to ob-
from the time of admission with topical tain a small portion of unburned underly-
silver sulfadiazine cream (Silvadene, Mar- ing fat (Fig. 1, b, c, d). T h e specimen ob-
ion Laboratories, Inc.). 1 Because as many as tained in this manner typically weighs 0.02
102 organisms per Gm. may be recovered to 0.05 Gm. This technic only rarely pro-
from normal unburned skin, patients de- duces bleeding that cannot be controlled by
veloping 104 or more organisms per Gm. of digital pressure. Local anesthesia is needed
burned tissue from different areas of the only occasionally in partial-thickness burns.
body were considered to have positive biop- (The use of these agents should be avoided
sies. because they are prepared in bacteriostatic
solutions that can inhibit bacterial growth in
Specimen Collection vitro.) Biopsy specimens are placed in dry,
sterile tubes without the addition of nu-
To obtain cultures of the burn surface,
trient broth for transportation to the
topical agents are first removed with sterile
laboratory. Ideally, processing of these
saline-soaked gauze pads. T h e wound is
specimens should be done within one to two
then cultured using a Rodac plate contain-
hours following their procurement.
ing blood agar which is applied to the burn
wound to obtain the specimen. After cleans-
Evaluation of Surface-contact Plates
ing the surface of the burn with isopropyl
alcohol to remove contaminants (iodine- T h e Rodac plates require no processing
containing solutions are avoided because other than incubation for 24 to 48 hours

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 22 LOEBL ET AL.
Table 1. Summary of Clinical Observations
Children Adults
Number of patients 92 180
Patients cultured 67 143
Positive surface cultures 17 (25%) 100 (70%)
Positive biopsy cultures 8(12%) 65(45%)
Clinical sepsis 7 41
Death from sepsis 2 13
prior to examination. Growth can be inter-
p r e t e d as light, m o d e r a t e , heavy, or
confluent. More precise quantitation of
colonies is made difficult by uneven dis-
tribution of the organisms on the plates and
by the inability to separate individual col-
onies for an accurate quantitative count.
Preliminary bacterial species identification
can be made on the basis of colonial mor-
phology a n d G r a m stains, while final
identification should be made by routine
biochemical technics.
Method of Quantitative Bacteriology
Biopsy specimens are processed by
weighing each specimen in a sterile pre-
weighed petri dish on a Torsion semi-micro
balance (Model DWL-3), macerating the
tissue specimen with a knife, and suspend-
ing it in 2 ml. of sterile physiologic (0.9%)
saline solution. Serial 1:10 dilutions of this
suspension are then prepared in sterile
saline solution. Specimens from patients
with no previous biopsy or previously nega-
tive biopsies are usually prepared in dilu-
tions from 1:10 to 1:105 initially, unless in-
fection is suspected clinically. Patients with
previously positive biopsies may have
further dilutions (to 1:1012 when necessary)
prepared in order accurately to quantitate
wound colonization without further dilu-
tions and incubations that lead to unneces-
sary delay.
A blood a g a r plate a n d an e o s i n -
m e t h y l e n e blue plate a r e inoculated
with 0.1 ml. of each of the dilutions, which is
spread evenly over the plate surface with an
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A.J.CP.Vol. 61
inoculating loop or glass spreading rod.
Plates are incubated at 37 C. for 24 hours
before colony counts are made. An appro-
priate plate with a dilution sufficient to pro-
duce nonconfluent colonial growth is
selected for colony counts. Each colony is
assumed to have grown from a single or-
ganism in the dilution. Wound colonization
may then be quantitated with the following
formula:
Organisms/Gm. of tissue
_NxDx2xlQ
W
where N = T h e number of colonies on the
plate chosen for colony counts
D = T h e dilution inoculated on the
plate, {i.e., 1:10, 1:102, 1:104,
etc.), and
W = Weight of the biopsy specimen
in Gm.
T h e constant factors (2 and 10) in the
numerator adjust for the preparation of
the original suspension in 2 ml. of saline
solution and for the inoculation of only
0.1 ml. of its dilution on t h e plate
evaluated.
For example, if 68 colonies are counted
on the plate of the 1:104 dilution of a
0.02-Gm. biopsy specimen,
N = 68 colonies
D = 104
W = 0.02 Gm.
organism/Gm. of tissue
_ 68 x 10" x 20
0.02
= 68 x 107
= 6.8 X 108 organisms/Gm.
As is the case with contact plates, species
identification of isolated organisms must be
accomplished by r o u t i n e bacteriologic
technics. As a s c r e e n i n g m e t h o d for
anaerobic species, 1 ml. of the original sus-
pension is routinely inoculated in thio-
 January 1974 Q U A N T I T A T I V E BURN-WOUND BIOPSY CULTURES
glycollate broth and examined for growth
at 24 and 48 hours.
Quantitation and preliminary bacterial
identification are available to the physician
24 hours after biopsy, while complete re-
sults of culture and sensitivity tests usually
require 48 hours.
Results
Pediatric and adult patients in this series
were evaluated as separate groups due to
the previous clinical observation of a lower
rate of infectious complications in the
younger patients (Table 1). In the pediatric
age group, 17 of 67 patients (25%) had posi-
tive surface cultures during their admis-
sions. Of these 17 patients, only eight (12%
of the total) had positive biopsies (104 or
more organisms per Gm. on biopsies from
different burn areas). Seven of these eight
patients (88%) developed clinical signs of
sepsis, as evidenced by three or more of
the following findings: disorientation,
hypothermia, hyperpyrexia, throm-
bocytopenia, l e u k o p e n i a , t a c h y p n e a ,
tachycardia, or ileus. Two of these seven
patients subsequently died of sepsis. The
single child who had positive biopsy cul-
tures but did not develop sepsis had a 30%
total body surface area partial-thickness
burn which was treated with subeschar an-
tibiotic infusion when biopsy cultures be-
came positive.
In the g r o u p of 143 adult patients
studied with surface and biopsy cultures,
100 (70%) had positive contact plates for
surface organisms during their admissions.
Of these 100 patients, 65 had positive biop-
sies (45% of the total). Forty-one of the 65
patients with positive biopsies (63%) subse-
quently developed clinical sepsis, and 13 of
these patients died of sepsis. All of the 24
patients with positive biopsies and no clini-
cal evidence of sepsis had been treated with
subeschar and/or systemic antibiotics, in-
itiated when biopsy cultures revealed 104 or
more organisms per Gm. of burned tissue.
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23
Table 2. Incidence of Clinical Sepsis in
Pediatric and Adult Patients with
Positive Surface or Biopsy
Cultures
Children Adults
Patients with positive
surface cultures 44% 41%
Patients with positive
biopsy cultures 88% 63%
No patient with sterile wound biopsy cul-
tures in either the pediatric or the adult
group developed signs of systemic sepsis
unless another source of infection (e.g.,
pneumonia, urinary tract infection or sup-
purative thrombophlebitis) was present.
Discussion
This study suggests that full-thickness
wound biopsy cultures more accurately
reflect burn-wound colonization than do
surface-culture technics. In the present
series of pediatric patients, only seven of 17
patients (44%) who had positive Rodac
plates developed signs of sepsis, while seven
of the eight pediatric patients (88%) with
positive biopsies developed clinical sepsis. A
similar correlation was seen in the adult
series, where evidence of sepsis was ob-
served in 41 % of those with positive contact
plates, compared with 63% of those with
positive biopsy cultures (Table 2).
Using the relatively simple bacteriologic
technics described, we have been able accu-
rately to detect, quantitate and evaluate the
progression of burn-wound colonization.
Many studies have reported erratic results
with the use of surface-culture technics to
detect burn-wound colonization. 35 8,? In the
present series, results of surface cultures
were influenced by such factors as recent
hydrotherapy or the time after application
of topical antibacterial agents. In a small
group of patients biopsied a number of
 24
times during the course of a single day, no
such variability was observed.
A possible objection to the biopsy culture
technic relates to the possibility that or-
ganisms recovered from the biopsy cultures
may represent surface contaminants. Some
investigators have suggested that the sur-
face of the specimen be sterilized by passing
it through the flame of a bunsen burner. 8
Twenty biopsy specimens from ten patients
were evaluated using both the flame technic
and the routine method described. Similar
results were obtained by the two methods,
but the flame technic was found to be tech-
nically difficult to perform. To evaluate the
effectiveness of alcohol cleansing in remov-
ing surface contaminants from the wound,
Rodac plate surface cultures were per-
formed following such preparation of the
wound surface on 60 occasions in 20 pa-
tients with positive biopsy cultures from the
same areas. This technic reduced surface
flora to insignificant levels, demonstrating
that organisms grown in biopsy cultures in
fact reflect deep tissue colonization.
Other methods of homogenization of the
biopsy specimen, including enzymatic
(trypsin) digestion 4 and tissue grinding
with an electric blender, 7 were evaluated in
tests of a small number of patients. These
technics were not found to offer appreci-
able advantages over the use of a knife
blade to macerate tissue, and the latter was
chosen for its simplicity.
Quantitative full-thickness burn-wound
biopsy cultures have been used by this and
other burn centers for some time, largely as
a research technic, or in the evaluation of
deteriorating patients. In our experience,
frequent bacteriologic monitoring of the
burn wound by this technic is invaluable in
the day-to-day assessment and treatment of
the thermally injured patient. Since the
completion of this study, more than 1,000
patients have been monitored routinely by
this technic. The simplicity and reproduci-
bility of this method have permitted us to
make it an integral part of our routine burn
care.
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LOEBL ET AL. A.J.C.P.Vol. 61
Conclusion
The method of quantitative burn-wound
biopsy cultures is viewed. This technic was
used to evaluate 210 of 272 patients admit-
ted to this burn center. Eight (12%) of the
pediatric patients developed positive biop-
sies, and seven of eight (88%) subsequently
manifested clinical signs of sepsis. Sixty-five
(45%) of 143 adult patients had positive
wound biopsy cultures, and 41 (63%) of
these patients with positive biopsies de-
veloped clinical signs of sepsis. All patients
with positive biopsies who did not develop
subsequent evidence of sepsis had been
treated prophylactically with subeschar
and/or systemic antibiotics initiated when
biopsy cultures revealed 10 4 or m o r e
organisms/Gm. None of those patients with
sterile biopsy cultures clinically manifested
invasive infection originating in the burn
wound. Quantitative burn-wound biopsy
cultures represent an effective, practical aid
to monitoring the burned patient on a
routine basis.
References
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3. Clarkson JG, Ward CG, Polk HC: Quantitative bac-
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