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Journal oflnfection (1980) 2, 39-51

A simple method for the study in vivo of


bacterial growth and accompanying host response

Sarah E. J. Day, Kjersti K. Vasli, Ronald J. Russell


and John P. Arbuthnott
Department of Microbiology, Trinity College, Dublin 2, Ireland

Summary
Chambers were constructed from lengths of plastic syringe barrels, having both ends scaled
with membrane filters of 0.22/~m pore diameter. When implanted intraperitoneally in mice,
the chambers were used in the study of in vivo growth of staphylococci, and the host response.
This system permits a dynamic interchange of diffusible host and staphylococcal factors,
simulating, to a certain extent, the natural state of affairs during staphylococcal infection. The
bacteria, although in close proximity to phagocytes, are protected from engulfment, and these
in vivo-grown bacteria can be recovered for subsequent examination in vitro.
The current study noted that growth of Staphylococcus aureus in chambers was slower in
vivo than in vitro. In vivo the stationary phase was more prolonged and growth attained a lesser
density than it did in vitro. An inflammatory response was observed in both control and test
chamber implantations. Test chambers, however, became thickly encapsulated with granula-
tion tissue and fibrin deposits, entrapping numerous leucocytes.

Introduction
It is possible that production of the full array of bacterial virulence factors
requires an in vivo environment. The decisive conditions in the host may
indeed be those of inflammation rather than normal physiological conditions
(Smith, 1958). A comprehensive study of bacterial virulence factors should
therefore give prominence to the growth of bacteria in vivo, observing both
the bacteria and the host response.
To allow the concurrent study of both bacteria grown in vivo and the
immune response of the host, the former should be readily recoverable.
Efficient recovery of bacteria from the host requires that they are retained
within an implanted chamber. The chamber should however be sufficiently
porous to permit dynamic interactions of bacterial and host factors such that
growth conditions simulate those of the natural infection.
Initially, intraperitoneally implanted collodion sacs were used for the
growth of bacteria in vivo. Sanarelli (1891) is believed to have been the first
to use collodion sacs in animal experiments (Harris, 1939b). To confer
resilience to distortion, a high concentration of collodion was used, and the
resultant sacs were of low permeability. Impregnation of wire gauze with
collodion (Fouard, 1909; Gates, 1921) and improved methods of preparing

0163-4453/80/010039+ 13 $01.00/0 ©1980 The British Society for the Study of Infection
40 S . E . J . Day et aL

the collodion membrane (Elford, 1931) led to increased permeability, but


also conferred heteroporosity (Harris, 1939a, b). Lam, Sweeney, Witmer
and Wise (1963) defined the porosity of their collodion sacs before use.
The first attempts to use membranes of uniform porosity were with
intraperitoneal sacs made of Visking cellophane dialysis tubing (Gladstone
and Glencross, 1960; Cybulska and Jeljaszewicz, 1969). Houser and Berry
(1961) constructed chambers from acrylic rings, sealed at both ends with
viscose dialysis tubing of pore diameter 2.4-3.0 nm, or Millipore micropore
filters of 0.45/zm pore diameter. These sacs and filter chambers permitted
the influx of nutritional factors from the peritoneal fluid and protected the
bacteria from phagocytosis, but only the 0.45/zm pore diameter chamber of
Houser and Berry was permeable to bacterial toxins and host anti-bacterial
factors. This is, however, the only type of intraperitoneal bacterial chamber,
described to date, which allows dynamic interchange of bacterial and host
factors.
Since 1972, studies of in vivo bacterial growth have concentrated on
Neisseria gonorrhoeae grown in subcutaneous chambers in laboratory ani-
mals. Implants have included plastic golf balls (Arko, 1972, 1974; Veale,
Smith, Witt and Marshall, 1975; Arko and Wong, 1977), steel springs (Arko,
1972, 1974; Arko and Wong, 1977) vinyl rings (Flynn and Waitkins, 1973),
PVC rings (Hafiz and McEntegart, 1977) and perforated plastic centrifuge
tubes (Veale, Smith, Witt and Marshall, 1975; Demarco de Hormaeche,
Thornley and Glauert, 1978). Most of these implants were left to become'
encapsulated with fibrin and granulation tissue, before inoculation. The
resultant chambers generally retained the gonococci although leucocytes
could enter.
Beyond gross pathological changes, few observations have been made on
the host response during in vivo growth of bacteria. There is a paucity of
quantitative data, and most information relates to gonococci grown in sub-
cutaneous chambers (Arko and Wong, 1977; Veale, Sen, Penn, Finch, Smith
and Witt, 1977).
Our method for in vivo growth of staphylococci is a modification of that
developed by Houser and Berry (1961) but differs in that parallel observa-
tions can be made on both the bacteria and the host response.

Materials and methods


Bacteria
Staphylococcus aureus NCTC 7121 (strain Wood 46), which produces high
levels of a-toxin, was used throughout.

Animals
Female BALB/c mice, 7-8 weeks old, were used in all experiments. Mice
were maintained under normal laboratory conditions, with free access to food
and water.
In vivo bacterial growth 41

Bacterial inoculum for chambers


The bacteria were grown for 18 hours in nutrient broth (0" 5 per cent Lab
Lemco, 0.5 per cent NaC1, one per cent Peptone Oxoid, pH 7.4) at 37°C, with
shaking at 150 cycles/min (Gallenkamp orbital incubator). They were har-
vested by centrifugation at 800 x g for 15 minutes and washed twice in
peptone water. The bacteria were resuspended at approximately 10Wml in
peptone water, dispensed as 1 ml aliquots in plastic ampoules and stored at
-70°C. When required, aliquots were thawed at room temperature and the
bacteria diluted in physiological saline, to a final concentration of 104 colony
forming units (cfu)/ml. This suspension was kept on ice prior to inoculation of
the chambers.
Construction and sterilization of chambers
Chambers were constructed from lengths of 1 ml polypropylene syringe
barrels (Becton, Dickinson and Co. Ltd., Dun Laoghaire, Ireland). Syringe
graduation marks, containing fish glue, an immunostimulant (Russell, R. J.,
personal communication) were removed by means of methanol. The barrels
were cut into 1 cm lengths, and the ends trimmed and sanded. These short
cylinders were soaked in two per cent Decon-90 (Decon Laboratories Ltd.,
Portslade, England) for 18 hours, rinsed thoroughly in distilled water and
sterilized by autoclaving at 15 lbs/in 2 for 15 minutes (i21°C). Micropore
discs, 6 mm in diameter, were punched from MF-Millipore filters of 0.22/zm
pore diameter (p.d.) (Millipore Filter Corporation, Bedford, Massachuset-
tes), using a standard paper punch. The discs were placed between sheets of
interleaving paper supplied with Millipore filters, and autoclaved at 15 lbs/in 2
for 15 minutes, between two sheets of glass to prevent distortion.
In the completed chamber (Fig. 1) micropore discs were glued on t/~ both
ends of the cylinder using U H U (Lingner and Fischer GmbH, Buhl, West
Germany), a biologically inert adhesive (Wilkinson, 1974), insoluble in
water. This adhesive was sterile as supplied and was used as aseptically as
possible.
--,t----- 7 m m

-<-5 m m -r,,.~

Length of Iml
MoCr22P~ e barrel

adhesive
Fig. 1. Chamber construction detail.
42 S . E . J . Day et aL

Procedure for filling chambers


A micropore filter disc was attached to one end of the chamber and 0- 1 ml
bacterial suspension added through the open end, which was then sealed with
a second micropore filter. Leakage from the chamber did not occur during
these procedures, due to surface tension. Before implantation, the completed
chamber was placed in sterile saline for five minutes to solidify the glue.
Control chambers were similarily prepared, containing peptone water
diluted with saline to the same extent as the stored bacterial suspension.
Implantation of peritoneal chambers
U n d e r ether anaesthesia, two chambers were inserted longitudinally in the
mouse peritoneal cavity, one to either side of a ventral incision. The wound
was closed using surgical sutures (Ethicon Ltd., Edinburgh, Scotland), stitch-
ing the peritoneum and the skin together simultaneously. The wound was
swabbed lightly with Hibiscrub (ICI Ltd., Pharmaceuticals Division, Mac-
clesfield, England) before returning the mouse to a cage lined with paper
towels.
Growth of staphylococci in vivo
Two chambers each containing 0.1 ml bacterial suspension (104 cfu/ml), were
implanted in each mouse. At intervals up to nine and a half months, mice
were killed by cervical dislocation and the chambers removed. The contents
of each chamber were washed out with a volume of saline that was nine times
that of the chamber contents. Viable bacterial counts were obtained by
spreading 0.1 ml of appropriate dilutions on agar plates and counting the
colonies after overnight incubation at 37°C. Total bacterial counts were
estimated by the Breed slide m e t h o d (Breed, 1911). Bacterial counts were
corrected for alterations in fluid volume which had taken place within the
chambers. The bacterial count at each point in time represents an average of
counts from four chambers.

Growth of staphylococci in vitro


Chambers containing 0" 1 ml bacterial suspension (104 cfu/ml) were placed in
5 ml nutrient broth, and incubated without shaking at 37°C. At intervals up
to 19 days, the contents of the chambers were washed out with volumes of
saline (nine times that of the chamber) and viable counts were obtained as
described for in vivo growth. The bacterial count at each time point repres-
ents an average of counts from a minimum of four chambers.

Kinetics of peritoneal leucocyte population


Mice were killed by cervical dislocation. A small incision was made in the
abdominal skin and the skin pulled aside, avoiding the stitches in mice
containing chambers. Saline (2 ml) was injected into the peritoneal cavity,
the a b d o m e n palpated for one minute and the saline then withdrawn. Leuco-
cytes in the peritoneal washout were counted by means of a haemocytometer,
and differential white cell counts performed on Leishman-stained smears.
In vivo bacterial growth 43

Examination of implanted chambers upon removal


The extent of encapsulation of the chamber was noted. Impression smears of
granulation tissue surrounding chambers were stained by Leishman's
method, and examined by light microscopy.
Filters were removed from chambers by soaking in ethanol. The filters
were stained with Harris haematoxylin (Wilkinson, 1974) and both surfaces
viewed by light microscopy.
For scanning electron microscopy, filters still attached to one end of the
chamber, were fixed in three per cent glutaraldehyde, and washed in six
changes of 0.05 u-phosphate buffer over a period of one hour. Dehydration
was performed in a series of ethanol solutions at concentrations of 10, 30, 50,
70, 95 and 100 per cent, the filters remaining for 10 minutes at each concent-
ration. This was followed by exposure for two periods of 10 minutes to freon
113 (DuPont). The filters were dried in a Poloron critical point dryer (Polo-
ron Equipment Ltd., Warlord, England; Model E3000). When dry, they
were mounted on double-sided tape and coated with gold to a thickness of
20 nm using a sputter-coater (Poloron, Model E5000). The two surfaces of
the filters were examined in a mini scanning electron microscope (ISI mini-
SEM, Model MSM5), at a voltage of 5 kV.

Dynamics of diffusion into chambers in vivo


Two groups of six mice were injected intraperitoneally with 1/zCi[3H] inulin
(mol. wt. 500, specific activity 200/zCi/mg, Code TRA.324, The Radiochemi-
cal Centre Amersham, England) in 0.5 ml physiological saline. One group
was injected immediately after implantation of chambers containing
staphylococci, the other group had received chambers seven days prior to
injection. Two mice from each group were killed at three, six and 24 hours
post-injection. Upon removal, chambers were washed briefly in saline and
were placed separately in vials each containing 7 ml scintillation fluid (two
parts toluene, one part Triton X-1000, PPO 4 g/l). These were counted for
two minutes per vial, using a Packard Tricarb scintillation counter.

Dynamics of diffusion out of chambers in vivo


Chambers (two per mouse) containing 0.9/zCi [3HI inulin a n d 103 cfu
staphylococci in 0.1 ml physiological saline were implanted intraperitone-
ally. Groups of four mice were killed at one half, two, four, eight and 26 hours
after implantation, the chambers were removed and radioactivity was meas,
ured as described above.
Results
Pathological changes following intraperitoneal insertion of chambers
Chambers containing Staphylococcus aureus became encapsulated with
fibrin and granulation tissue. This was first evident at two days after implanta-
tion as a thin mat adhering to the micropore filters. By six days, chambers
were totally encased; thick, cream-coloured mats covered the filters and
44 S.E.J. Day et aL

semi-transparent tissue enveloped the whole chamber. The thickness, degree


of attachment to intestines and peritoneum, and the vascularisation, of this
capsule [Plate 1(a)] increased with time. Leishman-stained impression slides
of that portion of the capsule adhering to micropore filters, at six days,
showed predominantly polymorphonuclear leucocytes on the internal sur-
face adjacent to the filter [Plate 2(a)] and both polymorphonuclear and
mononuclear cells on the external surface [Plate 2(b)]. The encapsulation
process was not evident at any stage up to 19 days in mice containing control
chambers [Plate l(b)].
Contamination of peritoneal fluid by staphylococci from the chamber was
found in four of 126 mice in which chambers were implanted.
The mesenteric and inguinal lymph nodes and Peyer's patches of test
animals appeared grossly unchanged, but splenomegaly (Plate 3) was evi-
dent at four days, progressing up to 19 days and persisting at least up to nine
and ,a half months, in mice implanted with chambers containing
staphylococci. Leishman-stained impression smears of spleens removed after
19 days revealed a high content of polymorphonuclear cells. Spleens from
mice containing control chambers appeared normal at 19 days.

Observations on filters from in vivo chambers containing staphylococci


Both light and scanning electron microscopy revealed polymorphonuclear
leucocytes adhering to the outer surface of the micropore filter [Plate 4(a)].
Clusters of staphylococci were seen attached to its inner surface
[Plate 4(b)]. Also on this surface, from 24 hours after implantation, occa-
sional deposits of electron-dense material, presumably fibrin, could be seen.
Neither bacteria nor leucocytes were seen to have penetrated the filter. Light
and transmission electron microscopy revealed only small particles of debris
emeshed within the matrix of the filter.

Comparison o f staphylococcal growth in vivo and in vitro


Both in vivo and in vitro, there was a lag of two hours before the onset of
bacterial growth (Fig. 2). During this lag, there was in vivo a slight drop in
viable count, suggesting that the environment within the chamber during this
period was less favourable for staphylococcal survival than that in vitro.
Logarithmic growth took place in vivo over approximately 10 hours, during
which the doubling time of the bacteria was 56 minutes, as opposed to 34
minutes in vitro. After 24 hours a viable count of 2.9 × 108 cfu/ml was
attained as the bacteria growing in vivo entered the stationary phase. This
number stayed relatively constant, having fallen only to 1.7 × 108 cfu/ml at
three months and 3.6 × 107 cfu/ml at nine and a half months. During this
prolonged stationary phase, the total bacterial count in vivo increased
slightly, from 8.2 × 10Vml at 24 hours to 2.5 × 109/ml at nine and a half
months. During the in vitro stationary phase the bacterial count
Plate 1. Dissection of mouse peritoneal cavity to show chambers implanted 10 days previ-
ously: (a) mouse implanted with chambers containing S. aureus NCTC 7121; (b) mouse
implanted with control chambers.
[facing page 44]
Plate 2. Leishman-stained impression slides of that portion of capsule adhering to micropore
filter of chamber implanted six days in mouse (x 1000): (a) surface of capsule adjacent to
micropore filter; (b) outer surface of capsule.
t
Plate 3. Comparison of spleen from mouse six months after implantation of chambers con
taining staphylococci (right), with normal mouse spleen (left). (Bar represents 10 mm)
Plate 4. Scanning electron micrographs of filters from chambers containing staphylococci
after 24 hours implantation in mice: (a) polymorphonuclear leucocytes attached to outer
surface (x 4900); (b) staphylococci on inner surface (x 9800).

[facing page 45]


In vivo bacterial growth 45

9 -

. .- "" ~...____:~__~" --t-../1-. ~_____


8 - ,,~"/ ".//. ~---~"-w~...~
.~ 7 ,// "'-~
.o 6
O

o~
_j 5 ,,/
41
I I I I I I /~ I /f I // I // I
4 8 12 16 20 24 6 19 3 91/z
Hours Days Days Monlhs Months
Time after chamber implantation

Fig. 2. Comparison of viable counts of$. aureus N C T C 7121 grown in chambers, m viva ( 0 )
and in vitro (©). (Bars denote standard error)

(1.2 × 109 cfu/ml) attained a level higher than that in viva, but this declined
after 24 hours and continued to do so, falling to 4.6 × 106 cfu/ml by 19 days.
This emphasizes that the in vivo chamber resembles a chemostat with a
continuous influx of nutrients and removal of toxic metabolic products,
compared With the closed in vitro system.
Peritoneal leucocyte population
Following insertion of chambers containing staphylococci, there was a slight
intraperitoneal leucopenia of less than four hours duration (Fig. 3). This was
succeeded by a rapid influx of polymorphonuclear leucocytes, elevating the

~7
o
iz T
0 I0 ~

d
.6
I I I I I I II I II I
4 8 12 16 20 24 6 19
Hours Days Days
Time after chamber implantation
Fig. 3. Peritoneal fluid leucocyte r e s p o n s e to c h a m b e r implantation: • c h a m b e r s containing
staphylococci; O control c h a m b e r s . (Bars d e n o t e s t a n d a r d error)
46 S.E.J. Day et al.

2400

2000

-~

.c
E
1600

1200
I
800

400
/
i

/
I I I I I I I I
3 6 9 12 15 18 21 24

Hours cIf4er i. p, injection of r 3HI irluiin

Fig. 4. Diffusion of intraperitonea[[3H] inulin into in vivo chambers: • [3H] inulin injected
immediately after chamber implantation; © [3HI inulin injected seven days after chamber
implantation. (Bars denote standard error)

b
24 I
._c 16
E

~lE-------t! IE
I I I I 1/ I
2 4 6 8 26
Hours ofter ¢homber implontotion

Fig. 5. Diffusion of isotonic[3H] inulin from in vivo chambers. (Bars denote standard error)
In vivo bacterial growth 47

leucocyte concentration to five times that prior to implantation of the


chamber. This response was maximal between days one and four, and its
decline was accompanied by movement of mononuclear leucocytes into the
peritoneal cavity. By 19 days, the mononuclear cell influx had passed, and the
number of intraperitoneal leucocytes had returned to that found initially.
The free peritoneal leucocyte response in mice containing control chambers
did not differ significantly from that described above.
In addition to free peritoneal leucocytes, numbers of leucocytes adhering
to the micropore filters increased greatly with time. This increase was more
rapid when the chambers contained staphylococci than on control chambers.
Numbers of leucocytes enmeshed in the capsule which formed around cham-
bers containing staphylococci, also increased with time. There was no capsule
around control chambers. These observations indicate that, although cell
counts in peritoneal fluid were similar, there was a greater peritoneal leuco-
cyte response in mice implanted with chambers containing staphylococci,
compared with those implanted with control chambers.

Permeability o f micropore filter chambers in vivo


To support bacterial multiplication in vivo, the chamber must be permeable
to growth factors present in peritoneal fluid. Also, to simulate conditions
during a staphylococcal infection, interchange of anti-bacterial host sub-
stances and staphylococcal products, across the micropore filter, should not
be restricted. It was considered probable that encapsulation of the chamber
would reduce its permeability. The permeability of the micropore filters was
assessed by measuring diffusion of [3I-1]inulin, into and out of, in vivo cham-
bers. Inulin was shown not to be metabolised by staphylococci in conven-
tional sugar utilisation tests in vitro.
There was rapid influx of a saline solution of [31-1] inulin from
the peritoneum into chambers immediately after their implantation. The
influx was significantly impeded when the chambers had been implanted
seven days prior to [31-1] inulin injection (Fig. 4), but was not halted.
Diffusion of [3H] inulin out of the chambers took place exponentially,
diffusion rate being maximal initially, decreasing with time and achieving an
almost steady state by eight hours (Fig. 5).

Discussion
The necessity to study the properties of in vivo-grown bacteria in order to
elucidate pathogenic mechanisms is emphasised by studies where in vivo-
and in vitro- grown bacteria were found to differ (Smith, 195 8; Glastone and
Glencross, 1960; Beining and Kennedy, 1963; Penn, Sen, Veale, Parsons,
Smith and Witt, 1976).
The micropore filter chamber described here provides a versatile system
for use in such studies. It is composed of physiologically inert materials,
neither syringe barrels, filters nor adhesive having immunostimulant proper±
48 S . E . J . Day et al.

ties. It is easily and rapidly constructed and filled, in contrast to collodion


sacs, which are difficult to standardise and prepare in large quantities and
which require a specia| filling device (Harris and Miller, 1941). A disadvan-
tage is that it cannot be autoclaved with filters in position due to distortion of
the filters during the process.
Chambers may be inserted with ease into the mouse peritoneal cavity, six
to eight mice being implanted in one hour. The chambers are well tolerated
by the animals, their activity being little impaired one hour after surgery.
Signs of illness are not apparent up to nine and a half months after implanta-
tion of chambers containing staphylococci. This confirms previous observa-
tions in mice implanted intraperitoneally with chambers or collodion sacs
containing staphylococci (Houser and Berry, 1961; Lam, Sweeney, Witmer
and Wise, 1963). Some earlier workers have commented less favourably.
Zinsser and Raymond (1921), using celloidin capsules containing strep-
tococci in rabbits, found that, although the animals usually survived several
months, some became emaciated.
Bacteria could be removed easily from micropore filter chambers even
after considerable time in situ in the mouse. In contrast, subcutaneous
gonococcal chambers of otherworkers eventually became consolidated with
fibrinous connective tissue and unsuitable for specimen collection; the useful
life of an implant in mice was 21 days ,(Arko, 1974).
The micropore filter chamber containing staphylococci simulates in many
aspects a natural staphylococcal infection. In the latter, the bacteria are
attacked by leucocytes and bactericidal factors from tissue fluids and leuco-
cytes. In our system leucocytes are excluded, thereby exhibiting 'frustrated
phagocytosis' (i.e. engulfment of bacteria is not possible but there is degranu-
lation of leucocytes). Leucocyte products may be secreted into the chamber;
also death of leucocytes adjacent to the micropore filters and diffusion of
necrotic products into the chamber may take place. Dynamic interaction of
staphylococcal factors (both extracellular products and intracellular factors
that are released upon cell death) with leucocytes and peritoneal fluid com-
ponents may arise. This probably results in production of leucocyte cytotax-
ins and stimulation of the immune system as in the natural situation. Presum-
ably this interaction would also cause production of staphylococcal antigens,
qualitatively and quantitatively similar to those produced in a natural infec-
tion.
The heteroporosity of collodion sacs and their eventual disintegration in
vivo (Gates, 1925) r e n d e r e d t h e m ineffective as barriers across which there
could be interaction of bacterial and host factors in a reproducible fashion.
In the micropore filter chambers, diffusion across the membrane decreased
with time after implantation, as indicated by a reduction in diffusion rate of
[3H] inulin into chambers after seven days in vivo (Fig. 4). This was probably
due to deposition of fibrin and blocking of filter pores by adherent leucocytes.
There was progressive encapsulation of micropore filter chambers containing
staphylococci. This resembled a staphylococcal abscess in that it was com-
In vivo bacterial growth 49

posed of fibrous tissue infiltrated by leucocytes. No encapsulation of control


chambers had occurred by 19 days, indicating that a diffusible staphylococcal
product was required to stimulate capsule formation either directly or indi-
rectly. Similar encapsulation phenomena have been described by many other
workers, notably Lam, Sweeney, Witmer and Wise (1963). Because the
bacteria were prevented from being engulfed by phagocytes, the infec-
tion persisted, so resembling a chronic carrier state.
In our micropore filter chambers, the growth rate of staphylococci in vivo
was lower than that in vitro. This is in agreement with observations by
Gladstone and Glencross (1960) and Houser and Berry (1961). Doubling
times in vivo and in vitro were 56 minutes and 34 minutes, respectively. In
vivo a population density of 2.9 × 10 s cfu/ml was attained by the time that
the stationary phase was reached, compared with 1.2 × 109 cfu/ml in vitro,
but with the in vivo culture there was a longer stationary phase, reflecting
continuous access to diffusible host nutrients. Transmission and scanning
electron microscopy of staphylococci did not reveal structural differences
between in vivo- and in vitro- grown organisms with the staining methods
used. In both situations the staphylococci grew in characteristic clusters•
Soon after chamber insertion (four to six hours), there was an influx of
polymorphonuclear leucocytes into the peritoneal cavity. Its peak at one to
four days was succeeded by an influx of mononuclear leucocytes. By 19 days
leucocyte levels in the peritoneal fluid had returned to normal. Similar
leucocyte responses in the peritoneal fluid were found whether the chambers
contained staphylococci or control fluid, but, in the former situation, the total
intraperitoneal insurge was greater, considerable numbers of leucocytes
becoming emeshed in the capsule. Therefore, the leucocyte response
observed in mice containing control chambers is the basic inflammatory
response resulting from trauma. In mice implanted with chambers containing
staphylococci an additional chemotactic or trapping response took place.
Veale, Sen, Penn, Finch, Smith and Witt (1977) reported a polymor-
phonuclear leucocyte influx, maximal at five hours after injection of
gonococci into previously implanted subcutaneous chambers. Using a similar
system, Arko and Wong (1977) observed a transition to mononuclear leuco-
cytes only in unsuccessful infections• In persistent infections the polymor-
phonuclear leucocytes remained predominant (90 per cent PMN). This is in
contrast to our results, but other factors may be operating in the gonococcal
system.
It is proposed that our future research should include work with chambers
of different porosities, evaluation of the immune status of animals implanted
with chambers, and studies of toxins produced in vivo. Among numerous
possibilities, this method has a potential role in the search for protective
staphylococcal antigens and for studies of leucocyte chemotaxis in vivo.
Polymorphonuclear and mononuclear leucocyte responses could be analysed
separately by use of chambers with more than one compartment and filters of
• ./ '

appropriate por e size. In addition, the method could be used to assess the
50 S . E . J . Day et aL

e f f e c t i v e n e s s o f a n t i b i o t i c s in vivo, in p a r t i c u l a r t h e i r p e n e t r a t i o n into
s t a p h y l o c o c c a l a b s c e s s e s , using fibrin e n c a p s u l a t e d c h a m b e r s as an e x p e r i -
mental model.

(We thank Mr P. Walsh for assistance with scanning electron microscopy and Miss
Pauline Christie for photography).

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