A Quantitative Evaluation of Three Blood
Culture Systems
RICHARD ROSNER,
M.S.
St. Joseph's Hospital, 703 Main Street, Paterson, New Jersey 07503
ABSTRACT
Rosner, Richard: A quantitative evaluation of three blood culture systems.
Amer. J. Clin. Path. 57: 220-227, 1972. Three separate blood culture systems.
were evaluated by parallel culture technic using clinical blood specimens.
The three systems under study were a plain brucella broth, the same base
medium with polyanethol sulfonate ® added in a final concentration of 0.05%,
and the same base broth with the polyanethol sulfonate plus sucrose in a
final concentration of 30%. After the test media had been inoculated and
allowed to incubate for 4 hr., pour plates were prepared from each. The
purpose of this step was to determine whether there was a differenc in a
blood culture system employing either polyanethol sulfonate or sucrose, or
both. The results indicated that specimens from only 74 of 121 patients demonstrated positive culture results in the flask containing only the base broth,
whereas 106 were positive when polyanethol sulfonate was added, and 121
were positive when both polyanethol sulfonate and sucrose were incorporated
in the base medium. T h e quantitative results indicated that there were 15
patients from whom recovery of organisms was accomplished only from the
polyanethol sulfonate/sucrose medium and an additional 33 patients from
whom the total number of organisms recovered from the polyanethol sulfonate/sucrose medium was at least 30% greater than from either the plain
broth or the polyanethol sulfonate broth. The results of this study indicate
clearly that polyanethol sulfonate has a beneficial effect on the recovery of
organisms, possibly by preventing phagocytosis and lessening the inhibitory
action of nonspecific antibodies present in the blood specimen. The results
also indicate the value of using an osmotic stabilizer such as sucrose to create
a better environment for bacterial cells with cell wall damage.
IN 1925, H. D. Wright 4 0 published a report
dealing with the then-known facts and concepts of subacute infective endocarditis and
the resulting bacteremia. He discussed
many of the problems that still face the
physician and the microbiologist today.
• The polyanethol sulfonate used in this report
was Liquoid, manufactured by Roche Diagnostics,
Nutley, New Jersey.
Received January 7, 1971; received revised manuscript March 15, 1971; accepted for publication
March 30, 1971.
The major areas covered by his report
were: how much blood to collect and how
often to collect it, the significance of the
organisms recovered from the blood culture, the significance of organisms recovered from cardiac blood at autopsy (indicating the limited success of therapy in
those days), the effects of various inhibitory
substances found in human blood on the
ability to recover organisms from the blood
culture, the effect of phagocytes on the
220
February
1972
EVALUATION OF BLOOD CULTURE SYSTEMS
ability to recover organisms, and the question of why there is often a significant
delay in the growth of organisms in the
blood culture. As to the last three areas
mentioned, Wright concluded: (1) failure
to recover organisms from the blood culture medium was frequently due to the
continued effects of both phagocytes and
inhibitory substances on the organisms in
the blood culture, and (2) the delay in
growth of organisms in many blood cultures was due to prolongation of their lag
phases. This prolongation appeared to be
due to certain "peculiarities" of the organisms, which seemed to vary from organism
to organism and from patient to patient.
In recent years, a great deal of work has
concerned itself with methods for obtaining
a higher percentage of positive cultures and
more rapid recovery of organisms from
positive cultures. The recent literature covers such areas as: the development of better
base media, new methods for collecting
the blood specimen, new methods such as
membrane filtration for processing the specimen, new methods for subculturing, the
effects of various atmospheres on the recovery of organisms, and the effects of various additives to the base medium on the
recovery of organisms. Not much work concerning the "peculiarities" mentioned by
Wright has appeared. T h e purpose of our
report is to describe the effects of two additives on the ability to recover organisms
from blood culture flasks and to attempt
to explain the reason for the prolongation
in the lag phases of organisms in many
cases. The two additives under study in
this report are polyanethol sulfonate and
sucrose.
Methods
Three separate blood culture systems
were evaluated by parallel culture technic.
Each of the test flasks contained 45 ml. of
brucella broth (Pfizer). Flask 1 contained
the base broth, flask 2 contained the base
broth plus polyanethol sulfonate in a final
221
concentration of 0.05%, and flask 3 contained the same mixture as flask 2 with
the addition of sucrose in a final concentration of 30%. Prior to starting this study
a small study was undertaken to determine
whether there was any difference in abilities to recover organisms from various
broths to which polyanethol sulfonate was
added. T h e broths examined were trypticase-soy broth, thioglycollate medium,
brain heart infusion broth, and brucella
broth. The results indicated that there was
no difference in the recovery rates when
polyanethol sulfonate was added to the
various broths, so the choice of brucella
broth for this study was a personal preference.
Since there were three flasks under study,
each blood culture required a 15 ml. sample of blood. This was accomplished by
using a 20 ml. syringe and inoculating 5
ml. of the blood sample into each of the
test flasks at the bedside. The three flasks
were then taped together and processed as
a single unit. Once inoculated, each set of
flasks was incubated in a carbon dioxide
incubator for exactly 4 hr., after which
duplicate pour plates were prepared from
each flask in the unit. The pour plates
were prepared by placing a 1 ml. sample
of the blood/broth mixture in a sterile
petri dish and adding 24 ml. of Columbia
blood agar base (BBL). One set of pour
plates was incubated for 24 hr. anaerobically and the other set was incubated in
a carbon dioxide incubator for 24 hr. T h e
blood culture flasks were returned to the
incubator immediately after the preparation of the pour plates and were processed
as previously described.28-20 After incubation, colony counts were determined on all
pour plates indicating growth. T h e process
of preparing pour plates exactly 4 hr. after
obtaining the blood culture was carried out
24 hr. a day, 7 days a week, to insure uniformity in the procedure. I n those few
cases in which the pour plates were prepared after either more or less than 4 hr..
222
ROSNER
Table 1. The Number of Positive Recoveries from
Each of the Test Media Based on the Quantitative
Results of the Pour Plates
Organisms/ml.
Flask 1
Flask 2
Flask 3
Group 1, < 20
Group 2, > 20
Group 3, > 100
1
22
51
16
31
56
22
39
60
the results were not included in this report. It was assumed that any organisms
present in a blood culture would still be
in the lag phase of growth, and therefore
the number of colonies times 10 (the blood
medium dilution factor) would indicate the
number of organisms present in the circulating blood of the patient at the time of
collection. This assumption was found to
be incorrect for those patients with Gramnegative enteric bacilli, because it was
found that these organisms grew equally
well in all of the test media and went into
the log phase of growth either almost immediately upon being placed in the blood
culture flask or while still in the circulating
blood of the patient. For this reason, those
patients with Gram-negative bacilli present in their blood cultures were not included in this study.
In order to be included in this study, a
patient had to have a minimum of three
separate blood cultures, all three flasks in
each unit had to be inoculated with 5 ml.
of blood, and the pour plates had to be
prepared after exactly 4 hr. In order to be
considered "positive" for this study, a patient had to have at least one of the test
flasks in each set indicate growth, and the
organism recovered had to be the same in
all positive flasks.
Results
A total of 4,816 blood culture sets (14,448
individual flasks) was evaluated during this
study. The blood specimens were collected
from 1,000 patients. According to the criteria set up for the study, 261 patients were
considered positive. Of this number, 140
were found to harbor Gram-negative bacilli
A.J.CP.—Vol
57
and were not included in the study, for
reasons previously mentioned. The remaining 121 patients were found to harbor
Gram-positive cocci, Gram-negative cocci,
minute Gram-negative bacilli, or anaerobic
organisms.
Based on the quantitative results obtained from the pour plates, each patient
was placed into one of three quantitative
groups. Group 1 consisted of 22 patients
having fewer than 20 organisms per ml.
of circulating blood. Group 2 consisted of
39 patients having more than 20 but fewer
than 100 organisms per ml. of blood. Group
3 consisted of 60 patients who had more
than 100 organisms per ml. of blood.
Table 1 indicates the number of positive
recoveries from each of the test media based
on the quantitative results and shows how
many patients from each of the quantitative groups had blood which was positive
in each of the test flasks.
Table 2 indicates the various organisms
recovered, the number of patients from
whom each organism was recovered, and
the quantitative groups in which the patients who had each of the organisms fell.
Table 3 indicates the frequency of isolation of each organism from each of the
test media and the total number of recoveries from each test medium.
Discussion
From the results of this study, if a single
flask containing only brucella broth were
used as the blood culture system, only 1 of
22 patients with fewer than 20 organisms
per ml. of blood would have had a positive
blood culture and only 22 of 39 patients
with more than 20 but fewer than 100 organisms per ml. of blood would have had
positive cultures. Only 23 of 61 patients
with fewer than 100 organisms per ml. of
blood would have had positive culture results even when three separate blood cultures were obtained for each patient. Comparing these results with the 100% (121 of
121) positive recoveries when both poly-
February
1972
EVALUATION OF BLOOD CULTURE SYSTEMS
223
Table 2. Frequency of Recovery of the Various Organisms Isolated and Numbers of
Patients Falling into the Quantitative Groups for Each Organism
Organism Recovered
Alpha hemolytic Streptococci
Diplococcus pneumoniae
Beta hemolytic Streptococci
Bacteroides species
Anaerobic Streptococci
Neisseria meningitidis
Haemophilus species
Staphylococcus aureus
Pasteurella-like organisms
Frequency of
Recovery
41
34
12
11
9
5
4
3
2
anethol sulfonate and sucrose were added
to the same base medium, it becomes obvious that these two additives play a major
role in the recovery of organisms from a
blood culture system. The question arises:
what does the polyanethol sulfonate do,
and what does the sucrose do, to cause such
a dramatic increase in the rate of recovery
and allow recovery within 24 hr.?
If we approach the question of culturing
blood with the idea that everything stops
once the blood is added to the medium,
with the exception of the eventual multiplication of the bacteria present, then there
should be no problems in recovering organisms. As indicated by this study, as well
as by a previous study,28 this is not the
case. In the circulating blood of the human
host, any given bacterial cell can be challenged by phagocytes, nonspecific inhibitory substances, the possible presence of
Frequency of Quantitative Recoveries
Group 1
Group 2
Group 3
0
0
2
3
7
4
4
0
2
12
6
10
8
2
1
0
0
0
29
28
0
0
0
0
0
3
0
specific antibody, and the possible presence
of antimicrobial agents.
The question now becomes: how much
damage to any given bacterial cell can any
or all of these factors do in the circulating
blood, and do they continue to act in the
blood culture system? Let us examine the
second part of the question first. T h e continued antagonism of many of the antimicrobial agents to organisms in a blood
culture system has been demonstrated many
times. The use of penicillinase in a blood
culture system will effectively neutralize
the actions of most penicillins. T h e use of
polyanethol sulfonate effectively inactivates
agents such as kanamycin sulfate,84 polymyxin B, colistin sulfate,84 and gentamicin.SB However, as pointed out by Bailey
and Scott, the simple dilution of the blood
1:10 with the base medium is usually sufficient to render the concentration of an
Table 3. Frequency with Which Each of the Various Organisms Was Recovered From the Three
Test Media and Total Number of Recoveries from each Test Medium
Organisms Recovered
Flask 3
Flask 1
Flask 2
Alpha hemolytic Streptococci
Diplococcus pneumoniae
Beta hemolytic Streptococci
Bacteroides species
Anaerobic Streptococci
Neisseria meningitidis
Haemophilus species
Staphylococcus aureus
Pasteurella-like organisms
37
24
10
0
0
0
0
3
0
41
34
12
6
6
2
1
3
1
41
34
12
11
9
5
4
3
2
Total recoveries
74
106
121
224
ROSNER
antimicrobial agent too low to affect bacteria.
Wright 4 0 and other investigators 36 ' 88
have demonstrated the continued ability of
phagocytes to destroy bacteria in a blood
culture system. This could present a major
problem in those patients in whom the
number of bacteria in the blood is small
to begin with. Von Heabler 3 8 and others,12, i, 11, 15-1T, 22, 28, 26, SS h a v e
S n 0
W n that
pOly-
anethol sulfonate prevents phagocytosis in
the blood culture system by disrupting the
phagocytes. For this reason alone, polyanethol sulfonate must be given serious
consideration as an essential additive in
any blood culture system. Von Heabler, 38
Wright, 40 and many others 3 - "-21- 2B-27-3037,38,41 have demonstrated the bactericidal
effects of nonspecific inhibitory substances
on bacterial cells. As with phagocytosis, this
can be a serious problem in those cultures
in which the original inoculum was small.
Several investigators 3 - 19 - 21 ' 23> 25~27'30'37- 88 - 41
have demonstrated the ability of polyanethol sulfonate to inactivate these substances in a blood culture system.
In addition, polyanethol sulfonate has
been found to be a very effective anticoagulant. 28 - 20 This is an important consideration when the base medium used is of the
type that allows blood to clot in the flask.
One such medium is thioglycollate medium. Once a clot is allowed to form, the
organisms entrapped in the clot produce
very small colonies which are easily overlooked. This is often the cause of negative
blood culture reports in laboratories that
subculture only those flasks that have visible growth. When using a blood culture
system containing either polyanethol sulfonate or sucrose, it is necessary to subculture all flasks, because polyanethol sulfonate will cause a cloudy broth layer in every
flask, and when sucrose is used there is
rapid lysis of the erythrocytes.
Hoare 1 4 and others s - 1S have reported
that polyanethol sulfonate is toxic to many
organisms, especially the anaerobic streptococci. The concentration of the chemical
A.J.C.P.—Vol. 57
used in those studies, however, was 10 times
the concentration suggested by the manufacturer. Our studies 28 - 29 and those of
others 8 - 10 - 12 - 1S - 18 - 24 - 3 °- 32 - 38 - 30 indicate that
polyanethol sulfonate in a concentration of
0.05% is nontoxic to all bacteria. In this
study and in the studies of others,6-10-12-18«
24,30-32,38,39 t n e addition of polyanethol
sulfonate resulted in a significantly higher
percentage of positive recoveries, indicating that it is beneficial rather than toxic.
If the presence of phagocytes and nonspecific inhibitory substances, and the possible presence of specific antibody or antimicrobial agents, or both, were the only
reasons for failure to recover organisms,
then the use of polyanethol sulfonate
should allow microbiologists to reach the
ultimate in recovery rate. The results of
this study indicate, however, that the addition of sucrose to the polyanethol sulfonate/broth mixture produced a significant
increase in the number of positive recoveries. It appears that the addition of sucrose produces a better environment for
the organisms. The question is: how?
There are two possible explanations. It
appears that when sucrose and polyanethol
sulfonate are combined in a good broth
base, either they act as a growth stimulant
for the organisms, or each additive has a
direct or indirect effect on the organisms
by producing a better environment. If both
additives act as a growth stimulant, then
it should be simple to demonstrate this in
artificially inoculated blood cultures in
which the number of organisms in the inoculum is quantitated.
For this purpose, 40 sets of flasks identical to the sets used in the study were
artificially inoculated with blood from
healthy donors and with various bacteria
in concentrations that produced 50 organisms per ml. of blood culture medium.
The 40 sets of flasks were divided into four
groups of 10 sets each. The flasks in the
first group were inoculated with alpha hemolytic streptococci from a 2-hr. broth
culture, the second group of flasks with a
Februmy
1972
EVALUATION OF BLOOD CULTURE SYSTEMS
2-hr. culture of Diplococcus
pneumoniae,
the third set with beta hemolytic streptococci, and the last set with anaerobic streptococci. All the flasks were then processed
exactly like the flasks in the study. The
quantitative results obtained from these
flasks indicated that there was no change
in the numbers of organisms recovered, regardless of organisms or test media used.
This test indicates that the combination
of additives does not act as growth stimulant for the organisms tested.
One question which does arise from this
study using artificially inoculated blood
culture flasks is, why does growth occur
equally well in the plain broth and in the
additive medium? Why does comparable
growth occur in plain and additive flasks
in artificially inoculated blood cultures but
not in clinical blood cultures with similar
organisms? This leads to questioning the
reliability of the results obtained from artificially inoculated blood culture systems.
In an artificially inoculated system, does
phagocytosis occur, does nonspecific inhibitory substance act, and is the presence of
antimicrobial agents a factor? These questions must be answered if we are to rely on
results from artificial systems. This is presently being studied in our laboratory.
Since it appears that sucrose does not act
as a growth stimulant, what does it do to
allow a higher recovery rate of organisms
from a blood culture system? One theory
which can be used to explain the action
of sucrose is taken from those investigators
working with the so-called transitional or
L-forms of bacteria. This concept deals
with the protection of cells which have cell
wall damage. This protection is in the
form of preventing changes in osmotic pressure. One factor often overlooked in the
development of any blood culture system
is the physical condition of the bacterial
cells as they are placed into the blood culture system. Bacteria, like any other living
cells, undergo various stages of physical and
biochemical deterioration in the process of
dying. The rate and method of death, to
225
a great extent, are determined by the environment in which the bacterial cell finds
itself. As previously mentioned, in the circulating blood of a human host the bacterial cell is confronted by several adverse
factors, any one of which is capable of destroying any bacterial cell quite rapidly.
It has been shown that many antimicrobial
agents, as well as both specific antibody and
nonspecific inhibitory substance, act on
bacteria by affecting the cell wall of the
organism either directly or indirectly. It is
fairly safe, therefore, to assume that in the
circulating blood of a host with bacteremia
there will be organisms in various stages of
cell wall destruction. Placing such a cell in
an artificial environment, such as a blood
culture system, can cause an additional
stress on the cell in the form of a change
in osmotic pressure. Investigators working
with L-forms have shown this stress to be
real, because the L-form cannot survive in
the absence of an osmotic stabilizer such
as sucrose. If one assumes that an L-form
probably represents a very advanced stage
in the death process of a bacterial cell and
thus has an absolute requirement for an
osmotic stabilizer, then it seems reasonable
to assume that a cell with less cell wall
damage will stand a better chance of survival in a system in which changes in osmotic pressure have been prevented. This
does not mean that a cell with little or
moderate cell wall damage requires an osmotic stabilizer, but merely that it stands a
better chance of surviving in a system
which includes one. This can be a very
important consideration in a case in which
the number of organisms is small and it
becomes necessary to recover as many individual bacterial cells as possible in order
to obtain a positive recovery.
In a previous study,20 we demonstrated
that the use of only sucrose without polyanethol sulfonate did not produce a higher
recovery rate than did the plain base broth.
This was probably due to the continued
action of phagocytes, nonspecific inhibitory
substance, and possibly specific antibodies
226
ROSNER
A.J.C.P.—Vol.
57
Table 4. Number of Patients for Whom Either Flask 1 or 2 Showed
at Least 30% Fewer Organisms Than Flask 3
Organism
Flasks 1 and 2
Flask 3
Alpha hemolytic Streptococci
Diplococcus pneumoniae
Beta Hemolytic Streptococci
Bacteriodes species
Anaerobic Streptococci
12 (all > 20 but < 100)
6 (all > 20 but < 100)
12 (2 < 20; 10 > 20 but < 100)
11 (3 < 20; 8 > 20 but < 100)
9 (7 < 20; 2 > 20 but < 100)
12 (all > 100)
4 (all > 100)
8 (2 > 50; 6 > 100)
2 (> 100)
7 (> 100)
which were not inactivated by polyanethol
sulfonate. From that study as well as from
this study, it would appear that the use of
any type of blood culture system which
does not include an osmotic stabilizer allows the recovery of only those organisms
which have no cell wall damage. It would
also appear that the use of any system
which does not include an antiphagocytic
anti-inhibitory substance will allow recovery from only those patients with large
numbers of organisms present. If this theory is correct, then not only should it be
possible to recover a higher percentage of
positive results from any given number of
patients, but it should also be possible to
recover a larger number of organisms from
a system which includes both polyanethol
sulfonate and an osmotic stabilizer such
as sucrose.
In a previous study,29 there were 20 patients from whom organism recovery was
accomplished only in the medium containing both additives, and in the present study
there were 15 patients from whom organism recovery was accomplished only in
the medium containing both additives. In
addition in the present study, as indicated
in Table 4, there were 33 patients from
whom organism recovery was accomplished
in more than one of the test media, but
the numbers of organisms recovered from
the flasks containing both additives were
at least 30% greater than from either of
the other two test media. All 20 patients
in the first study, as well as all 15 patients
in this study, from whom organism recovery was accomplished only in the medium
containing both additives, were either re-
ceiving or had recently received penicillin
prior to collection of the blood cultures.
Of the 33 patients from whom organism
recovery was greater in the medium containing both additives, only 11 had received
antimicrobial therapy. T h e remaining 22
patients had had no antimicrobial agents
for at least 3 days prior to obtaining the
blood cultures. This would indicate that
there are other factors in addition to antimicrobial agents in the circulating blood
of the host which cause some cell wall damage to the bacteria.
It appears from the results of this study
and a previous study,29 that in the circulating blood of some patients there are bacterial cells that either fail to survive or
cannot be recovered quickly from a blood
culture system that does not contain an
osmotic stabilizer such as sucrose or polyanethol sulfonate. It would appear that the
polyanethol sulfonate protects the cell from
antagonistic factors, whereas the sucrose
protects the cell from osmotic pressure
changes, thus allowing the cell an environment in which it can synthesize new cell
wall material quickly and become viable.
The prolongation of the lag phase, as described by Wright 4 0 as being due to "peculiarities" of some bacteria, might really
be due to prolongation of the time needed
by those cells with slight cell wall damage
to synthesize new cell walls in an environment not ideally suited for this purpose.
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