STUDIES ON IMMUNITY IN ANTHRAX XI. CONTROL OF CELLULAR PERMEABILITY BY BICARBONATE ION IN RELATION TO PROTECTIVE ANTIGEN ELABORATION
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STUDIES ON IMMUNITY IN ANTHRAX
XI. CONTROL OF CELLULAR PERMEABILITY BY BICARBONATE ION IN RELATION
TO PROTECTIVE ANTIGEN ELABORATION
MILTON PUZISS AND MARY B. HOWARD
U.S. Army Biological Laboratories, Fort Detrick, Frederick, Maryland
Received for publication 20 August 1962
ABSTRACT and Wright, 1954; Wright and Puziss, 1957).
PuzIss, MILTON (Fort Detrick, Frederick, Sodium bicarbonate was one of the specific re-
Md.) AND MARY B. HOWARD. Studies on im- quirements for elaboration of antigen into the
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munity in anthrax. XI. Control of cellular perme- medium; this requirement was demonstrated by
ability by bicarbonate ion in relation to protec- Gladstone (1946), and has been confirmed re-
tive antigen elaboration. J. Bacteriol. 85:237-243. peatedly in both aerobic and anaerobic cultures.
1963.-No elaboration of Bacillus anthracis pro- Small amounts of antigen were demonstrated in
tective antigen was demonstrated after addition filtrates from anaerobically grown cultures in a
of bicarbonate to cultures incubated 42 hr in a bicarbonate-free medium, probably as a result of
bicarbonate-free medium. Antigen accumulation metabolic carbon dioxide retained in the closed
in culture filtrates was greater if the bicarbonate system used to obtain anaerobiosis (Puziss and
was added to these cultures early in the growth Wright, 1959). Thorne and Belton (1957) also
period. Cell-free extracts of washed cell suspen- considered retention of metabolic carbon dioxide
sions, prepared by hand grinding or lysozyme in an alkaline medium a factor in antigen accumu-
treatment, had no demonstrable intracellular lation in a bicarbonate-free culture. The bicar-
protective antigen. Sonic treatment released bonate could not be replaced by functionally
antigen in measurable amounts, but tended to related compounds (Puziss and Wright, 1954);
destroy or degrade the labile antigen. Freezing its mode of action in antigen elaboration has not
and thawing disrupted the cells and liberated the been defined. Bicarbonate had no influence on
antigen; protective antigen was shown to be of carbohydrate metabolism of the organism during
internal rather than of extracellular origin. growth and elaboration of protective antigen in
Maximal intracellular antigen concentration culture filtrates (Puziss and Wright, 1959). The
occurred before the peak concentration was metabolism of amino acids similarly was not
reached in the culture filtrates. Antigen also significantly affected by bicarbonate in aerobic or
was found in cell extracts from bicarbonate- anaerobic culture (Wright, Puziss, and Neely,
free culture media when none was present in 1962).
the corresponding culture filtrates. A high level This report describes the results of studies of
of internal protective antigen was present in the influence of bicarbonate on accumulation of
cells grown in a bicarbonate-free medium main- extracellular and intracellular protective antigen,
tained at a constantly alkaline pH; only a trace and presents a plausible hypothesis for the mode
of antigen was present in the corresponding pH- of action of bicarbonate.
adjusted culture filtrate. The hypothesis is
presented that bicarbonate functions to control MATERIALS AND METHODS
cellular permeability and release of antigen Cultures. The strains of B. anthracis were non-
from the cells into the ambient medium. encapsulated, nonproteolytic, avirulent mutants
of the virulent strains 107 and V770 (Wright et
Previous reports from this laboratory have al., 1962). Spore suspensions for inocula were
shown that a protective antigen was produced by adjusted to give a final concentration of 500
nonencapsulated, nonproteolytic mutants of spores per ml of medium. Immunized animals
Bacillus anthracis grown in a chemically defined were challenged by a standard spore suspension of
medium under certain cultural conditions (Puziss the virulent Vollum strain of B. anthracis.
237238 PUZISS AND HOWARD J. BACTERIOL.
JMedium and methods of culture. Preparation of The crude dried cell-free extracts were reconsti-
the chemically defined medium 1095 for anaerobic tuted at 25 mg/ml concentration. Guinea pigs
cultures has been described (Wright et al., 1962). (250 to 350 g) were immunized, each animal re-
The pH of the complete medium was approxi- ceiving a total of 62.5 mg of the cell-free extract,
mately 7.8. When sodium bicarbonate was given subcutaneously in five doses of 0.5 ml each
omitted, the pH of the medium was adjusted to on alternate days. Culture filtrates obtained from
the same level as the control by addition of sterile the complete medium were used as controls; these
1 N NaOH. Media for anaerobic growth were pre- were reconstituted at the routine 15 mg/ml con-
pared in volumes of 4 to 10 liters in glass aspira- centration. Cell extracts were assayed, in most
tor bottles, and were incubated under nitrogen cases, in parallel with their corresponding culture
as already described. filtrates. The extracts and filtrates were also
Harvest and preparation of cells. After the re- titrated in vitro for antigenic activity by the
quired incubation at 37 C, the culture was dis- complement-fixation method of McGann,
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pensed via the delivery tube of the aspirator Stearman, and Wright (1961). The Ouchterlony
bottle into sterile 600-ml centrifuge bottles. agar diffusion method of Thorne and Belton
Cells were readily collected by centrifugation at (1957) was used to identify and to titrate the in-
1,500 X g for 20 min. tracellular antigen.
When cells from larger volumes of anaerobic
culture (10 liters) were harvested, the culture RESULTS
vessel was placed in a cold bath (4 C); the cells Effect of bicarbonate additions to incubated cul-
sedimented satisfactorily in 3 to 4 hr at this tem- tures. Experiments were performed to determine
perature. Most of the supernatant was discarded, the effect on antigen elaboration of addition of
and the concentrated cells in suspension were bicarbonate to a mature cell culture previously
collected by centrifugation. grown in a bicarbonate-free medium. Bicarbonate
In the initial studies, the packed cells were was added aseptically, at double the normal con-
washed five times by suspension in 500-ml por- centration, to bicarbonate-free cultures after 42
tions of cold sterile distilled water. It was later hr of incubation (the normal harvest period for
found that one washing for the removal of soluble antigen-containing cultures). Part of the culture
surface antigen was as adequate as five washings; was filtered at once, along with a complete me-
this was the method used in the latter part of dium control. Another part of the culture was
this study. Washed cells were resuspended in reincubated anaerobically for an additional 3 hr
sterile buffer and adjusted to give an optical before filtration. A surface-active agent (Tween
density reading (with a blue filter) of 500 i 50 80) was also added to 42-hr bicarbonate-free
Klett-Summerson units. Buffers were either 0.05 cultures, in an attempt to remove any protective
M phosphate (pH 7.9) or tris(hydroxymethyl)- antigen that might have accumulated on the
aminomethane (tris), 0.04 M (pH 8.0). bacterial cell surface. This addition was without
Processing of cell extracts. After the completion effect on the accumulation of antigen in the cul-
of the cell extraction procedures (see Results), ture filtrate. Normal antigen accumulation oc-
the extracellular fluid and the cell debris were curred in the control culture filtrates, whereas
separated by centrifugation 4,000 X g for 30 only traces of antigen were detected in the fil-
min) at 4 C. The supernatants were drawn off and trates of cultures in which the bicarbonate was
were sterilized by filtration, tested for sterility, added to the 42-hr-old cultures. Other cultures
and stored either in the frozen or the lyophilized in which the bicarbonate was added after 42 hr of
state. incubation were reincubated with supplemental
Assay for protective antigen in culture filtrates glucose as an additional energy source, since the
and cell extracts. The in vivo assay method for initial glucose was exhausted after 42 hr. The
estimation of protective antigen by immunization presence of this extra source of energy, and the
and challenge of guinea pigs has been described addition of bicarbonate, did not influence the
by Puziss and Wright (1959). Cell-free extracts accumulation of protective antigen; only a trace
in the normal fluid state, or the lyophilized of antigen was found in the culture filtrates, if the
product, were stored at -20 C for use during bicarbonate was added after the 42-hr period.
the period required for animal immunization. Evidently, bicarbonate had no effect on antigenVOL. 85, 1963 CONTROL OF CELLULAR PERMEABILITY 239
elaboration when it was added to an already TABLE 1. Protective antigen elaboration in culture
mature culture. filtrates after addition of bicarbonate to
To determine at what point during growth the an incomplete medium
bicarbonate effect on antigen elaboration could Antigen assay
be demonstrated, bicarbonate was added to bi- Culture medium
Post-inoculation
bicarbonate Survival Av day Comple-
carbonate-free cultures grown for 20, 28, or 35 hr. addition ratio* of geath ment
fixationt
These cultures were then reincubated anaero-
bically, until harvest at 42 hr. Assay results hr
showed that the earlier the bicarbonate was added Complete 0 7/8 5.0 120
to the cultures, the more antigen accumulated HCO3-free No 0/8 3.7 5
in the filtrate. No antigen, or at most only trace addition
amounts, was detected if bicarbonate was added HCO3-free 20 3/8 5.3 30
after 35 hr of incubation (Table 1). General agree- HCO3-free 28 2/8 3.5 30
ment between animal assay and complement- HCO3-free 35 0/8 3.9 5
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fixation titers was obtained.
Preliminary studies on extraction procedures. Normal animals 0/4 3.9
In an effort to understand the factors controlling *
Ratio of the number of animals surviving
the accumulation of extracellular protective anti- over the total number challenged.
gen, an attempt was made to detect the presence t Expressed as 50% units per ml.
of intracellular antigen. Washed cells were ground
in a chilled mortar and extracted with 0.05 M cold theon Co., Waltham, Mass.) for 40 min. An
phosphate buffer, pH 8.0 (Mcllwain, 1948). After alcohol-Dry Ice mixture was circulated through
centrifugation, the supernatant was assayed for the jacket of the treatment cup in an attempt to
antigen activity by complement-fixation and agar minimize destruction of labile material liberated
diffusion methods. For a control in the agar during sonic treatment. Good cell disruption was
diffusion test, a highly immunogenic dried culture achieved, as evidenced by loss of turbidity and
filtrate was used at a concentration of 5 mg/ml. an increase in viscosity of the clarified super-
This control material formed a single precipita- natant from the disrupted cells. Cell breakage was
tion line with horse antispore serum that merged confirmed by microscopy. The cell-free extracts
with the line formed by the purified protective released by sonic treatment were too highly
antigen of Strange and Thorne (1958). Cell dis- anticomplementary for use in complement-
ruption by hand grinding did not release pro- fixation reactions. The sonically treated extracts,
tective antigen. filtered through a membrane filter, and ly-
Treatinent with lysozyme was investigated as ophilized and concentrated threefold, showed the
a means of liberating intracellular antigen. Cells presence of protective antigen; a line was evident
from a 23-hr culture were incubated at 37 C with on the agar diffusion plates that joined the line
lysozyme, at a final concentration of 0.5 mg/ml, produced by a lyophilized protective antigen
and sampled after 0.5, 1, 2, 3, and 5 hr. Optical control.
density measurements and stained smears were Sonic liberation of antigen was investigated
made; the suspensions were then filtered and the further. A 15-min sonic treatment was sufficient
supernatants frozen. Increasing the concentra- to liberate the maximal amount of protective
tion of lysozyme and incubating the treated cell antigen. Cultures incubated less than 44 hr
suspensions at different temperatures did not were then investigated, to determine whether
lead to liberation of antigen detectable by in vitro protective antigen within the cells was present
assay methods. A slight increase in optical density in amounts large enough to be detected without
of the lysozyme-containing suspension, and a concentration, and at what period during growth
decrease in that of the control, were noted. This it appeared. Volumes (10-liter) of culture media
was similar to the findings of Gladstone and were used to obtain cells at different periods of
Johnston (1955). time from a single culture source. Results of the
Ultrasonic disintegration of cell suspensions. serological assays on the sonic extracts of these
Washed cells suspended in buffer were subjected cells are presented in Table 2. The data indicate
to ultrasonic treatment in a 10-kc oscillator (Ray- that protective antigen was present in the cell ex-240 PUZISS AND HOWARD J. BACTERIOL.
TABLE 2. Protective antigen in cell extracts and remainder of the study, since the frozen-thawed
culture filtrates after ultrasonic treatment extracts possessed somewhat greater protective
of cell suspensions activity than the sonic extracts and had the
Antigen titers additional advantage of not being anticomple-
Incubation
time Antigen Complement
ntigenentAsourcet
Agar plate mentary.
fixation iffusion Protective antigen in extracts from a complete
medium. Prior to cellular disruption and release
hr 50% units/ml
of intracellular contents, it was necessary to de-
16 Filtrate 5 Neg termine whether the soluble protective antigen
Extract AC* 1:1 could be removed from the cell surface by
23 Filtrate 30 Neg
Extract AC 1:4 washing. All the washings from the cell suspen-
28 Filtrate 70 1:2 sions were pooled, lyophilized, and reconstituted
Extract AC 1:2 at a 40-fold concentration. No protective antigen
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34 Filtrate 80 1:8 was demonstrated in the concentrated washings
Extract AC Neg by either in vitro or in vivo assay. The negative
47 Filtrate 120 1:8 results of these assays indicated that no soluble
Extract AC Neg protective antigen was present on the cell surface.
64 Filtrate 80 1:4 The extracts of cells incubated for 42 hr in a
Extract AC Neg complete medium contained only small amounts
*
Anticomplementary; no complement-fixation of antigen; antigen in the corresponding culture
titer could be obtained. filtrate, however, was at the usual level. This ob-
servation indicated that the optimal time for
tracts in measurable amounts before a comparable antigen elaboration into the medium was not the
concentration was detectable in the culture optimum for accumulation of intracellular anti-
filtrate. gen. To clarify this point, 10-liter batches of
There were indications that sonic treatment of culture grown in complete medium were sampled
culture filtrate degraded the protective antigen. at intervals during incubation, to obtain equal
Protection of guinea pigs was lower with sonically volumes of cells for preparation of cell extracts
treated than with untreated filtrate antigen. The and of culture filtrates from a single source.
protective effect of addition of serum during Assay results of the cell extracts are presented
sonic treatment of cell suspensions was investi- in Table 3; it can be seen that maximal intra-
gated, since Strange and Belton (1954) found cellular antigen was present at about 23 hr of
that normal horse serum stabilized their fil- incubation. This was similar to the results first
trate antigen. Normal horse serum (5%, v/v) obtained with the sonically treated cell extracts
added to a 23-hr cell suspension, or to culture (Table 2). Direct comparison of antigen in the
filtrate antigen prior to sonic treatment, had no
stabilizing effect. Accordingly, less drastic TABLE 3. Protective antigen in extracts of cells
methods of cell disruption were investigated. from a complete medium
Effect of freezing and thawing on release of intra-
Antigen assay
cellular antigen. Cells from 4 liters of 23-hr culture Growth time Survival Avg day ComplementDiffusion
were subjected to three cycles of freezing and slow ratio* of death fixationt titer
thawing. After centrifugation to remove cellular
debris, the extracts were sterilized by filtration hr
through either membrane or ultrafine sintered- 20 2/4 7.0 100 1:2
glass filters. The extracts showed no evidence of 23 2/4 5.0 120 1:6
anticomplementary activity; they had comple- 26 1/4 5.0 100 1:4
ment-fixation titers of approximately 120. Agar 29 1/8 4.8 75 1:2
plate diffusion titers were the same as those of the Control
filtrate 7/8 5.0 160 1:8
sonically treated cell extracts. Increasing the
number of freeze-thaw cycles did not release more *
Ratio of the number of animals surviving
protective antigen. This method of extracting over the total number challenged.
intracellular material was used throughout the t Expressed as 50% units per ml.VOL. 85, 1963 CONTROL OF CELLULAR PERMEABILITY 241
extracts and in the culture filtrates revealed that TABLE 4. Protective antigen in extracts and culture
intracellular antigen reached a maximum, and filtrates front bicarbonate-free cultures
then began to disappear from the internal en- Antigen assay
vironment concurrently with the accumulation Antigen type Growth Corn- Gel
of antigen in the culture filtrate (Fig. 1). time Survival Avg day piement diffusion
ratio* of death fixationt titer
Protective antigen in extracts from a bicarbonate-
free medium. Preliminary studies on extracts of hr
cells obtained from a bicarbonate-free medium Filtrate 23 0/8 3.5 0 Neg
indicated the presence of protective antigen in Extract 0/7 3.5 6 Trace
these extracts. To examine this more fully, large Filtrate 34 0/7 3.9 0 Neg
volumes of cells were grown in the bicarbonate- Extract 3/7 6.2 80 1:4
free medium, and were sampled at intervals Filtrate 41 0/16 3.5 0 Neg
during growth to obtain both cells and culture Extract 1/15 4.5 24 1:2
filtrates. Extracts of these cells showed that pro-
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*
Ratio of the number of animals surviving
tective antigen was present in a measurable over the total number challenged.
amount within the cells, and that a peak of intra- t Expressed as 50'S units per ml.
cellular antigen was reached after about 34 hr of
incubation. No protective antigen could be TABLE 5. Protective antigen in extracts and culture
demonstrated in the corresponding culture fil- filtrates from pH-adjusted bicarbonate-
trates from the bicarbonate-free medium (Table free cultures
4). The difference in time of occurrence of maxi-
Antigen assay
mal internal antigen in bicarbonate-free cell
Antigen type Growth Gel
extracts and in control cell extracts corresponded time Survival Complement diffusion
to the lag in initiation of growth between these
ratio* fixationt titer
two types of cultures, as described in a previous hr
report (Puziss and Wright, 1959). Cells that were
grown anaerobically in a bicarbonate-free medium Filtrate 40 17.5 Neg
Extract 4/8 80 1:4
in the presence of 0.04 M tris buffer (pH 8.4) Filtrate 50 1/8 10 Neg
showed normal growth. Only a trace of pro- Extract 2/8 40 1:2
tective antigen, however, was detected in this
* Ratio of the number of animals surviving
culture filtrate, as shown by a complement-fixa-
tion titer of 5 and a guinea pig survival ratio of over the total number challenged; no animal
1/8. data for the 40-hr filtrate available.
t Expressed as 50% units per ml.
140
It was observed that anaerobic bicarbonate-
120
free cultures, in the absence of any other buffer,
CELL became more acid after the 40-hr incubation
CULTURE
period than did control cultures, despite the
-100 FILTRATES initially high pH in the medium. This may have
resulted from the absence of the buffering effect
of bicarbonate. The greater acidity in the medium
*, 80
was one possible reason for the low level of pro-
E tective antigen in extracts of cells grown longer
860 than 34 hr in the bicarbonate-free medium. To
determine the effect of the low pH on the in-
ternal protective antigen, bicarbonate-free cul-
40
tures containing a pH indicator (m-cresol purple)
I_
were adjusted at 3- to 4-hr intervals during incu-
I
0 20 30 40 50 60 65
Time (hours)
bation by periodic addition of sterile 1 N NaOH
FIG. 1. Intracellular and filtrcate antigen ac- to maintain the pH at the initial level of pH 7.6
cumulation in a complete medium. to 7.8. Extracts prepared from cells grown in the242 PUZISS AND HOWARD J. BACTERIOL.
pH-adjusted bicarbonate-free medium for 40 hr reported that cell-free extracts of the organism
had considerable protective antigen activity. are free from any toxic activity (Gladstone, 1946;
Protective antigen was still evident in the cell King and Stein, 1950).
extracts even after 50 hr of incubation of culture. The extracts described in this study contained
Despite the alkaline pH level maintained in the significant concentrations of the protective anti-
culture vessel, however, only a trace of antigen gen; this is in contrast to the findings of Keppie,
was detected in the corresponding bicarbonate- Smith, and Harris-Smith (1953) that no antigen
free culture filtrates (Table 5). was present in the internal contents of cells, and
that antigen was, therefore, essentially extra-
DISCUSSION cellular. Results of the studies reported here show
It is evident from these studies that bi- that protective antigen of B. anthracis is probably
carbonate had no effect on antigen elaboration, of internal origin and is not a surface type of
if it was added to a suspension of matured cells cellular antigen. As demonstrated in this study,
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grown in the absence of bicarbonate ion. High antigen is apparently formed fairly early within
bicarbonate concentrations, as well as addition the cells, when grown anaerobically in the com-
of a surface-active agent, and the presence of plete medium. Peak protective antigen concen-
additional glucose, did not act upon these cells to tration within the cells occurs before peak accu-
remove or "strip" any protective antigen from mulation in the culture medium is found. As the
the cell surface. Bicarbonate was necessary, ap- cells approach maturity and exhaustion of nu-
parently, during the early growth of the cell for trients, antigen apparently passes out of the cells
any appreciable antigen accumulation in the cul- and into the ambient medium, as though it were
ture medium. The earlier the cells were exposed a metabolic by-product; it is not, however, a
to the bicarbonate, the more antigen accumu- product dependent on cellular autolysis. In cells
lated. grown in a bicarbonate-free medium, internal
Several methods of disrupting the cell structure antigen reaches a maximum later than in cells
and extracting the internal cell contents were ex- from a complete medium; this corresponds closely
plored. The results with lysozyme treatment to the lag in growth initiation of cells grown
showed the ineffectiveness of this method. As anaerobically in the absence of bicarbonate
Gladstone and Johnston (1955) reported, not all (Puziss and Wright, 1959).
B. anthracis strains are susceptible to lysozyme The salient observation reported in this paper
action; it is also possible, as they indicated, that is that of significant antigen formation within
the bicarbonate and CO2 concentrations present cells grown in the absence of bicarbonate, but
during growth were too low for synthesis of the with no significant antigen accumulation in their
lysozyme substrate in the cell walls. Freezing corresponding culture filtrate. Culture of cells
and thawing of cells suspended in buffer was the in a bicarbonate-free medium, maintained at a
method chosen for obtaining intracellular antigen constantly alkaline pH, supported the observa-
with minimal destruction. The cell-free extracts tion of high antigen levels within the cell and
could be lyophilized, in a manner similar to cul- very low levels in the culture medium outside the
ture filtrates, without appreciable loss in antigen cell. Strange and Thorne (1958) reported that
activity. It is difficult to understand why the some antigen elaboration occurred in a bicar-
frozen-thawed extracts of Smith, Keppie, and bonate-free medium buffered at an alkaline pH
Stanley (1953) should not have contained at least with tris buffer. This was confirmed in our anaero-
some protective antigen, since this is the least bic cultures grown in the presence of tris buffer,
rigorous extraction method. Cell disruption by where the concentration of antigen in the culture
shaking with "ballotini" (Smith et al., 1953; filtrate was very low in comparison with control
Gladstone, 1946), in the absence of a suitable culture filtrates. The small accumulation of pro-
buffering system, might readily result in destruc- tective antigen can be attributed to the efficient
tion of the labile antigen liberated, particularly retention of metabolic CO2 by the tris buffer in a
at pH levels below 8.0. In the study of Smith et bicarbonate-free medium. In this study, an alka-
al. (1953), as well as in our study, no antigen was line pH was maintained by intermittent addition
detected in the washings from the cell suspen- of small amounts of NaOH to the bicarbonate-
sions. In addition to these authors, others have free cultures. The data obtained from study of theVOr,. 85, 1963 CONTROL OF CELLULAR PERMEABILITY 243
cell extracts strongly suggest that bicarbonate Bacillus anthracis. II. Some biological prop-
in the medium affects cellular permeability, erties of bacterial products. Brit. J. Exptl.
allowing passage of the internally formed antigen Pathol. 34:486-496.
across the cell membrane. In the absence of bi- KING, H. H., AND J. H. STEIN. 1950. The non-
carbonate, antigen apparently is unable to pass toxicity of Bacillus anthracis cell material.
across the cell membrane and accumulates within J. Gen. Microbiol. 4:48-52.
MCGANN, V. G., R. L. STEARMAN, AND G.
the cell. Bicarbonate-free anaerobic cultures G. WRIGHT. 1961. Studies on immunity in
incubated for 3 weeks retain normal turbidity anthrax. VIII. Relationship of complement-
and very little autolysis. Cultures grown in a fixing activity to protective activity of cul-
complete medium, however, usually show the be- ture filtrates. J. Immunol. 86:458-464.
ginnings of autolysis after about 72 hr of incuba- McILwAIN, H. 1948. Preparation of cell-free
tion; nearly complete clearing of the medium is bacterial extracts with powdered alumina.
seen by 3 weeks. J. Gen. Microbiol. 2:288-291.
The mechanism by which bicarbonate could Puziss, M., AND G. G. WRIGHT. 1954. Studies on
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affect cellular permeability is not understood; immunity in anthrax. IV. Factors influencing
perhaps some alteration of the integrity of the elaboration of the protective antigen of
Bacillius anthracis in chemically defined
cellular membrane occurs. Similar effects on media. J. Bacteriol. 68:474-482.
cellular permeability by other inorganic ions Puziss, M., AND G. G. WRIGHT. 1959. Studies on
have been observed with mammalian cells. For immunity in anthrax. VII. Carbohydrate
example, Dubes and Klingler (1961) increased metabolism of Bacillus anthracis in relation to
monkey kidney cell permeability to polio virus elaboration of protective antigen. J. Bac-
by calcium ion depletion. Adams and Burgess teriol. 78:137-145.
(1960) decreased catalase migration from mouse- SMITH, H., J. KEPPIE, AND J. L. STANLEY. 1953.
liver granules by addition of calcium or The chemical basis of the virulence of Bacillus
magnesium ions. Bicarbonate ion concentration, anthracis. I. Properties of bacteria grown
in vivo and preparation of extracts. Brit. J.
therefore, may indeed affect permeability of some Exptl. Pathol. 34:477-485.
species of bacterial cells. STRANGE, R. E., AND F. C. BELTON. 1954. Studies
ACKNOWLEDGMENTS on a protective antigen produced in vitro
from Bacillus anthracis: Purification and
The authors are indebted to Ralph G. Kanode chemistry of the antigen. Brit. J. Exptl.
for technical assistance. Pathol. 35:153-165.
STRANGE, R. E., AND C. B. THORNE. 1958. Further
LITERATURE CITED purification studies on the protective antigen
ADAMS, D. H., AND E. A. BURGESS. 1960. Some of Bacillus anthracis produced in vitro. J.
factors affecting the permeability of large- Bacteriol. 76:192-202.
granule membranes to mouse-liver catalase THORNE, C. B., AND F. C. BELTON. 1957. An agar-
in vitro. Biochem. J. 77:247-252. diffusion method for titrating Bacillus an-
DUBES, G. R., AND E. A. KLINGLER. 1961. Facili- thracis immunizing antigen and its applica-
tation of infection of monkey cells with tion to a study of antigen production. J.
poliovirus "ribonucleic acid." Science 133:99- Gen. Microbiol. 17:505-516.
100. WRIGHT, G. G., AND M. PUZISS. 1957. Elaboration
GLADSTONE, G. P. 1946. Immunity to anthrax: of protective antigen of Bacillus anthracis
Protective antigen present in cell-free culture under anaerobic conditions. Nature 179:916-
filtrates. Brit. J. Exptl. Pathol. 27:394-418. 917.
GLADSTONE, G. P., AND H. H. JOHNSTON. 1955. WRIGHT, G. G., M. PUZISS, AND W. B. NEELY.
The effect of cultural conditions on the sus- 1962. Studies on immunity in anthrax. IX.
ceptibility of Bacillus anthracis to lysozyme. Effect of variations in cultural conditions
Brit. J. Exptl. Pathol. 36:363-372. on elaboration of protective antigen by
KEPPIE, J., H. SMITH, AND P. W. HARRIS-SMITH. strains of Bacillus anthracis. J. Bacteriol.
1953. The chemical basis of the virulence of 83:515-522.You can also read
