.*
I _. .. ARM2.941121.008 \ CKFMICAL CODS
Canp Detrick, Marylmd
Research and Developaent Sepprtnent
Speciil 3.eport 30. 91
Prepared by
CF~ESR. FKILLIPS Chief, Decontamination Brmcl:
SAUL KAYE .J Cecontm.inPt ion ?ranch SMALL SCALE 'r'kT3 PLEIFICATIOM WITF!
SILVX? ASIOLGXA1'3S
IiiDEX
Page I"Ti?Om-CFION
ACK#GlGDGDEi?TS
A. SILTd ASEECIG>XK?S - GEFRSL 32SC9IFTION 1 3. TH3 CAltTZX - UTILI ZING ASXLOUXE 3T?BILIZATIO!J PF3VICUS
TO FILTUTICN
1. Description of the Centcen 3 2. Special k.sboloxane Powder for Use with Canteen 6 3. ierfcrnance Tests Conducted at California Institute of Tcchnology 7 4. Performance Tests Conducted at Camp Detrick 11 C. TH3 S-FILmR - SIWlTAIOXPS FILT?ATIOl$ AND STXQILIUTION
WITR SILVD ASEOLOXXTX
1. 3escription of the S-filter 17 2. Instructions for the Yse of tt.e $-filter, Two Liter Model 24 3. Asboloxane S-filter Fowi.er 26 4. Performaice zests on the S-filter and S-filter Powder Conducted rt Zrlifornia Institute of Technology 29 5. Performpace Ti,sts on dsbolomne S-f ilter Powder Cocducted at 3~lm;,Ijctrick 33 D. SUhBA?.Y 81 .
Fkis report deals with the alsplication of the newly developed Silver
Asboloxanes to the purification of small amounts of water. Supplying the indi-
vidual soldier, or small groups of men in the field with a ready source of safe
drinking water is a constant military problem. Men cut off from sources of
-purified water must be provided with simple methods by which they can sterilize
whatever naturally occurring waters are available. Not .only must the pollution
ordinnrily found in such waters be tealt with, but also it nust be kept in mind
that in warfare the waters may be deliterately contaminated,
The present methods of treating water either with Halazone tablets for canteens or with calciun hypochlorite in Lyster bags are not altogether
satisfactory. With large amounts of organic natter or high bacterial counts,
sterilization is not achieved unless great excesses of the chlorine compound are
-used. Morover, sediment is not removed, and an objectionable taste is imparted to the water, so that even when the water treated by such methods is safe to drink, it may still be unsightly and unpalatable.
The Silver Asboloxanes, developed by Dr. Alexander Goetz at the Cali- fornia Institute of Technology as an outgrowth of theoretical studies on the oligodynamic effect of heavy metals, offer a possible alternative to these chlor- ine treatments. Two pieces of military equipment utilizing Silver Asboloxane were developed by Dr. Goetz during the war at the request of the Mexican Army.
They were a porte.ble filter pump capable of treating two liters of water a min- ute, and a canseen capable of treating one quart of water per treatment. These .. . . .__ . .. ,.- .~ -- -. ... items are of considerable interest, because in them the water is filtered as well as sterilized and no objectionable flavor is imparted.
The Asboloxanes themselves have been the subject of considerable study
in this laboratory. This report covers that information which is of interest in water purification, and particula.rly, that obtained from practical trials with
the pump and canteen. 3ata and information gathered by Dr. Goetz at the Cali- fornia Institute of Technology are included. The Asboloxanes nnd the special equipment used in this study were
furnished by 3r. Alexander Goetz and the Shellnar Corporation, who are hand-
ling the comerciel clevelonmrnt of these products. We gratefully acknowledge
their supplying us with nany products still in the experinental or develop-
mental stage and with freely supplying us with infornation concerning the
prociucts. Dr. Goete has elso supplied us with results from his own experi-
xental work Pt tha California Institute of 'i'echology and in the field in
IMexiCO. Where we have used this naterinl in the text of this report we hnve
quoted it d.irectly from his re-aorts with the necessary acknowledgements.
The experimental work at Camp 3etrick was largely the result of the
combined efforts of most of the personnel in the 3econtamination Zrrnch.
Pa-ticulrr credit is due to Dr. Xuth A. McKiniley, especinlly for the studies on bacterial enzysle system, and to the Kisses Kary F. Goffin, Dorothy M.
Portner, and Helen F. Irminger, who performed most of the bacterid ?nd
ciiemicd assays.
Tha.nks are due to Dr. 3. Warshowsky and the Chemistry Branch. 3asic
Sciences Division, nnd to 3r. J. 3. '.ateman and the Physics Zranch, Physical and Ckernical Division, for sone of the chemical itnd physical clatn,. SILm AS3OLOXANTS
A. SILIOER ASBOLOXAIJTS - G3NP,W D3SCRIFTIO1i
The term "Asboloxane" is derived from the Creek, t, oxys - C d.
It has been used 6y 3r. Goetz as a generic term for surface compounds with metals,
where the radical is represented by the acid oxidation compounds on the surface
of colloidal carbon particles. 3r. Goetz has given the following general descrip-
tion of the nature of the Silver Asboloxanes:
ltThe Silver Asboloxanes are an entirely new type of silver compound substan-
tially different from the customary chemical silver compounds such as silver
nitrate, silver.picrate, and the like, or the preparations of colloidal silver
such as argyrol, collargol, etc. In the Asboloxanes thk-silver is chemically
coupled with oxygen by the strong adsorptive bonds existing on the surface of
colloidal carbon an+. certain inert metal oxides. These physico-chemical forces which characterize chemisorption render it possible to couple silver with other
elements such as oxygen to an extent and to a degree which is non-existent in normal silver compo-mds.
"Since the forces nentioned exist only on surfaces, carrier materials of ex-
tremely fine particle size must be employed. Such substrate materials, capable of great adsorptive power and available in sub-microscopic particle sizes, 20 to
100 millimicrons, are the colloidal forms of carbcn. These colloidal carbons, while physiologically neutral on application to skin and mucous membranes, expose surface areas of as much as 10-20 acres per ounce.
"Such materials can %e made to carry large quantities of oxygen in the form of oxidation layers upon the particle surfaces and by means of newly discovered
-1- , -,. _.. .-i, , ' 1
proccsses it is possible to !h&&tb;l4n6 %kc$ &orption layers substantial
quTtitics of atomic silver. In contradistinction to normal silver compounds
of colloidal silver preparations, the silver is bo.md to and held at the surface
of the carbon particle and is only very slightly soluble.
"The advantage of such compounds lies in the combination of a highly germi-
cidal and fungicidal prowrty and an absence of toxicity and denaturation of
proteins, because the silver atom can go only very sparsely into solution and its
stability in its oxidation state on the surfaces protects it against the reducing
qualities of the proteins .It
... The germicidal effectiveness of the Asboloxanes has been explained as being
&de to their ability to catalyze the decomposition of proxides, probably by
forming an intermediary oxide of silver on the surface of the carbon. In tho
presence of the Asboloxanes, peroxides become much more destructive to ba.cteria
than are ordinary peroxide solutions. The developer of the Asboloxanes has also
given the following description of this general property:
"It has been known for a lon,: tide that silver-oxice is R very efficient
catalyst and based upon this experience a series of silver-containing catalysts
. .. 'nave been developed which have in common the proAnerty of being practically ln-
soluble, in as far as they do not send silver ions into solution, and at the
same time render available a maximum of silver pvr total mess of silver employed.
This condition can be realized by adsorbing silver on the surface of.colloida1
carbon in tHe presence of the oxygen adsorbed on the carbon so that one can as-
sume that the carbon surface exposed and acting as a catalyst consists of ad-
sorbed monomolecular layers or pclymolecular layers of an atomic mosaic of Ag
and 0. In other words the catalyst consists of a surface compound representing
an unlrnown oxidation state of silver. Colloidal carbon ippears to be particular-
ly suitable for this uurpose, partly on a.ccount of its surface per unit mass, partly because of the strong ndsorptive affinity of carF~onfor oxygen.Ib;l -n r+i--.:-,,.-. Ti,?-?
---.~ ,A_ ~~ _- t--i,TVn
' .~i. -. Two- particular ap9lications have been made'o? %e' Asboloxanes for the steril-
ization of water. In one case, a. special Asboloxane has been developed in which
sterilization of water can be achieved prior to filtration. Another, the Asbolox-
ane S-Filter Powder, is designed so that filtration and sterilization are achieved
at the same time. These special Asboloxanes have been developed respectively for
use with a specially designed military canteen and portable filter pump, cgled
the $-filter by its developer, and likewise designed for military use. These two i
pieces of apperetus, the particular Asboloxane charge designed to be used in them,
and the results of trials conducted on their performance both at California Insti-
tute of Technology and at Camp 3etrick are described in the following sections.
B. TEE CANT"%; UTIL-IZING ASEOLOXANE ST3RILIZ.4TI@N PRWIOUS TO FISTUTION
1. Description of The Canteen
The canteen developed for use with the Asboloxane treatment is an ordinary
Army Canteen with 8 specially designed filter hwd in place of the usual screw
cap. The filter head consists of a filter set into a combined cap and drinking
spout. This assembly and the filter head are illustrated in Figures 1 and 2.
The filter itself consists of a cylinder of cellulose material reinforced
by impregfiation with a thermoseaing plastic. This unit, which is replaceable. is
cemented into the metal cap. The liquid in the canteen must. pass through this filter unit to empty through the spout in the filter head. ?To vrrlve is involved.
The filter unit holds back the solid Asboloxane particles (1) as.well (18 suspenaed matter such as mud, sand, algae and,protozoa. 3acteria would for the aost wart pass through the relatively coarse filter, but as mentioned previously the Asbo- loxane charge is designed to achieve sterilization before filtration.
I--
(l! Asboloxane prticlss, RS we have observed them, have a particle size very similar to that of bacteria. "here appears to be, however, $13 pgglutination of Asbolnxane and other particles in the canteen, so that the particle size is ef-~ fectively incrnneed aft.?.r sfer4.~j zqtion. a23 th? p-r',ides irrs withb?ld..-=- -. ~ nr c,r-1 ,,! .I 9 -3 -~ w +-
-~ In ase, the canteen is filled with wa.trr and the special Asboloxame
c.-::teen charge is a.dded. The filter head is then screwed on. The canteen is
shaken for 15 to 30 seconds, rnd then allowed to stand for 15 minutes before
using. The pressure developed witnin the canteen will force a few milliliters
of water through the filter head and out through the drinking spout. This serves
to sterilize the filter cylinder and the inner surfeces of the head at the same
time as the liquid inside the canteen is being treated.
To drink from the canteen, the filter head is not removed. Rather it is
loosened about a quarter turn, and the user drinks by inverting the canteen and
sucking through the spout. Air passing along the loosened threads to the cap prevents the formation of a. vacuum within the canteen which would make it diffi-
cult to suck out the water. The Asboloxane cata.lyst and whatever solid matter
:qas originally present is held back mechanically by the filter. Not until the canteen is enpty is the filter cap removed. Then the canteen can be rinsed, re- filled with water, and the process repeated using a new charge of the Asboloxane.
The treatment, aside from its sterilization action, actually renders the water more pelatable in that a mec:?nnical filtration is included, end the odors and tastes present in the nature1 water are often destroyed by the oxidizing RC- tion. No objectionable chemical tastes or odors are added by the Asboloxane treatment.
2. Special Asboloxane Powder for Use in Canteen
A special charge, designed to sterilize water prior to filtration has been developed which can be used for treatmat of small quantities of water col- lected in containers such as canteens. portPble water tmks and the like. T'ne charge for en Army Cmteen, holding 850 cc. of water. or rpx-oximately one quart is as follows:
, -, ,. 500 mg. Urea Peroxide 1&0 mg. Sodium Carbonate - 6- Such a charge can be packaged so that it weighs no more than 2-3 grrms. The de-
velopers claim that the materiols involved, if packed to exclude moisture, have
a storage life of at least one to two years and do not have a corro;ive action
on their packaging containers. Other soluble peroxides could be used in place
of the urea peroxide. - The charge is added directly to the water. Initial shaking or stirring
is desirable, but not atsolutely necessary because the rauid catalytic destruction
of the peroxide and subsequent gas evolution provide enough agitation for a smll
quantity of liquid such as a. quart. The bactericidal action takes place as a re-
,, sult of catalytic decomposition which is usually complete within 15 minutes at whic
time only a few parts per million of peroxide renain. It is recommended that the
water stand for this full 15 minutes before drinking, although it is usually safe
before this time. 3. Performance Tests on the Cmtcen Conducted at California Institute of Technology
The following strtements are quoted from a report prepared by Dr. Goetz
at the California Institute of Technology:
"The development of the S-canteen trcatoent was largely guided by bacter-
iological laboratory tests on artificially infected waters. These tests were con-
ducted at the bncteriological laborrtory of tic Rare Metals Institute rt the Cali-
fornia Institute of Technology.
"The tests were conducted with various tpes of waters artificially inoc-
ulated with E. coli rnd mixtures of E. coli, staphylococci and streptococci. In-
hibitory agents such as organic and inorganic nitrogen in the form of peptone and
ammonia were added in certain tests in order to establish the limitations of the
action of the preparation.
"After the filling of the canteen with the test water, the chemicals were
added, and nfter the lapse of 15 and 30 minutes, water samples were withdrawn -7- -.:. ,. h c. , ,, ..
through the filter head with P vacu*m pmp under sterile conditions simulating
as closely FS possible the action of drinking through the filter head. The
sampies were distributed in the customary manner: five 10 cc smples, one 1 cc
sample md one 0.1 cc sm.ple in lactose broth, with subsequent incubation for
$9 hours at 37OC. In case gas was observed, confirmation for E. Coli was carried
out -in Erillia'nt Grem Eile troth.
Vhe following *.re typical results of such tests:
. a. "Tap water with the addition of 12O,COO/cc E. coli. Tested after 20
sinutes. Result: one confir2ing gas tube of five 10 cc tubes. , "Tap wpter with the a6dition of 62,00o/c~E. coli. Tested rfter 10 7 b. and 20 minutes. No gas.
c. "Le.ke wa.ter, very riCh in ;.lgae, with the addition of jG,OOO/cc E.
coli. Tested after 15 and 30 minutes. No gas.
d. "Well water, nixed with sewr,ge (i/2000)plus 250 parts per nillion
peptone, with the addition of 1hOo0€0/cc E. coli. Tested Ffter 15 ninutes. No
gas.
e. "Same, with 500, 1000 and 2000 prts per nillion peptcne and sane
coli inoculum. Tested after 15 minutes. No gas.
f. "Sa.me, with 500 pnrts per nillion peptone and 40 prts per nillton
NH3. Tested pfter 15 minutes. No gas.
"For the sake of comparison, the lest mentioqed substrRte with the same
inoculum WPS tested with the U.S. Amy C.3.C. (emergency over-chlorinrtion) prep
aration: after 20 minutes, confirming g?s WRS observed in all tubes down to 0.1 cc
"The same WRS found to be true with an iodine-potassiw-permngan?+te prep-
aration and with Halozoxc tablets.
"In all these tests the S-canteen wclter was found to be trsteless,color-
less and conplying with rigorous sanitary stand~rds. - - *_ 7 .- - -. e..-.. ALULD 1 A CL1 .-I 2 .L-.'>-, "ln view of the fact tha.t infccted waters tested under labor-tory condi-
'tions nay not represent the performrnce of nethod and equipment in the field, a
nmber of field tests were perforned on various types of waters in the States of
Sindaa, Guerrera, Morelos and Ptstrito Feieral (Zepublic of Mexico), in collabop
ation With the Dep. de la Salubridad Publica, In order to test the water after a
short lapse of tine, ambulant laboratory equipment was taken along to the site of
the test. Thus the conditions in the water, when collected, treated and consmed
under actwl conditions, could be studied as ciosely as possible. These tests are
enmerated RS follows:
a. "Location: Bio Zumaya, 5 north of Culiacan, Sinaloa. Temperature: . -\ km 22OC: pH: 7.5 - i3.C. Control: gas in 1 cc sanple, confirnation questionable.
Canteen water, withdrawn a-fter 17 3inutes: no gas in any smple.
b. "Location: faucet outlet in Public Health Laboratory in Culiacan.
Tem?erature: 23OC; pH: 7.5. Control: 2 confirming gas tubes in five 10 cc snmles.
Canteen water, withdrawn Rfter 18 minutes: negptive throughout.
e. "Location: Indian wter hole in river bed of Rio Siqueros nenr Pena
0 Xeca, about 40 h inland from Xazatlan. Temperature: 23.5 C; pH: 7.5. Control
showed confirming gas down to 0.1 cc. Canteen water, withdrawn after 30 minutes:
one confirming gas tube out of five 10 cc samples, other tubes negative.
d. "Location: Rio Tepecua, Chatitla, Guerrero. Tecperature: 2goC;
pH: 7.5 - 8.0. Control: confirming ga.s down to 1 cc sample. Canteen water, with-
drawn after 20 minutes: no E. coli throughout (but non-confirming gas formers in
two 10 cc smples after Lg hours incubation).
e. "Location: Tixtla, Guerrero, water ditch leading from a native adobe
swimming tank through R section used by local washe,r-wonen and subsequently unr',er
the cadaver of k burro, the latter in the beginning str2gc of p8trefa.ction. Tb
water was highly t:xkid and evil smelling. ?he sanple was taken about three meters -9-
. -. r- -T-- ~., - -
. ~~ ~~ *_--I L -.- downstrean from the cadaver. In view of the high
0 of the. chemical were taken. ?he temperature of the water was 27 , the pH S.5 (!). ' The control showed confirming $as in 0.01 cc. The canteen we-tor, withdrawn after
15 minutes showed no gas in any of the smples. It was a.lso observed that the
water had lost its putrid odor.
f., "In ord'er to test the nnture of an infected vmtcr after prolonged
storcage in the canteen under field conditions, a sanple of a stagnent wrter in a
ditch near the highway.12 km east of Acnpulcc w?.s filled into the canteen, the
chemicals added, and the water stored for 4g hours in the canteen before withdraw&
for testing. The temperature of the smple hsd been 28'C, the pH 6.5 - g.0, but
the crnteen, lying in en open truck for two days WRS exposed to temperatures ex-
ceeding 40'. The control water showed confirming g8s down to 0.1 cc. The cantee:,
;.rater was free of E. coli in all smples, but showed non-confirming gas in th-ee
10 cc samples.
g- "Location: canel of Cuautla, Morelos, at I(n 1. T'nis test was run in
the presence of Lt. Col. 3r. Granillo, as representative of the War Department and
Sr. Ing. Acosta as representative of the Dep. de la Salubridad Publica. The latter
took independent samples of the water, which was withdrawn 2 hours and 15 niniltcs
later. The tenpemture was ZO.5OC, the pE 7.5. The control wa.ter showed confirm-
ing gas down to 0.1 cc. The 'official E. coli content was estimated to be 40,000 per liter. The cpnteen wrter proved to be negative throughout in the official and
in our tests.
h. "Location: stagnant water at the highway bridge in Xochimilco, 3.F;
Temperature: 20.5OC; pH: g.0. This test alsc was run in duplicrte by t.he writer
(Dr. Goetz) and the Public Health Deprrtxent. The control wnter showed confir_ning gas down to 0.001 cc, amd the official estina-to gives a probable content of E. coli of 4 million per liter. In view of the high rlgae content nnd general turbidity of - 10 - the water,, two capsules were used. The wter wa.s withdrawn after 1 hour md 35
minutes. In the author's test, the water was conpletely negative throughout. In
the official test, two out of five 10 cc tubes showed gas after 49 hours, which di6
not confirm as E. coli. The water is therefore considered sanitary.
"The Laborntories of the Dep. de la Salubridad Publica tested the contents
of the canteen of the last test (containing two ca.psulee) Chemically, and found no
toxic substance present,
i. "In analogy to test (g) (see above) zn Prny canteen (with nornal cap)
wa.s filled at the sane location as the previous test and one capsule of the U.S.
Army C.D.C. preparation was added. Subsequently the water was withdrawn rfter 3 hours and 15 minutes. After 24 hours incubtion, the 1 cc samples showed confirm-
ing gas. It can therefore be estimated tha.t the coli content was rbout 4000 pel
liter. This water is therefore definitely unsanitary.
"The above enmeration of field tests includes tests nnde. From it,
it is obvious that in ever>- case snnit?.ry drinking water was obteined, with the possible exception of test (c). Tor this test, the local conditions for the sterile withdrawal of the water from the canteen (in a dust storm) were definitely unfavor-
able :and may have caused an error."
4. Performance Tests on the Canteen Condicted at Camp Detrick
Two types of tests were rufi at Cmp Detrick in evalwting the Asboloxane I
Canteen treptment. In the first runs, regular arny plnstic and .?eta1 canteens were used together with the special filter hend and the Asboloxrne charge. The I full charge of 750 mg. Asboloxanc catalyst, 500 mg. urea peroxide, and lg0 mg.
sodium ca.rbonate ims used. This was added to the canteen filled with rp?Poxlnatel; I ! 850 nl. of heavy suspensions of verious test organisms. The specid fiiter head I was screwed on and the contents well shaken. After the indicated tines, smples ! were syphoned out aseptically without removing the filter head, and plnte counts - 11 - ! i ...... -. ~..
I
were made in the us-dal mnner. 3ata obtained fron these trirls are presented in
Ta.ble I. Physical nensure.nents nade upon the samples renovcd from the canteen
showed that the water had become alkaline as one would expect fron the presence
of sodium carbonate in the cnarge. For example, the suspensions of E. coli in
distilled water exhibited a pH of 10.1 after being treatod in the cnnteen. The
water, however, was quite prlata.ble in spite of this change in >E, and in s?ite
of the fact that it had contained originally 35 nillion organism per milliliter.
No objectionable testes or odors T..ere noticed.
After these first prelininary tests had confirmed thnt the canteen was
indeed quite effective, a second series of trials was started in Erlc;nme.ver flasks,
using only E5 121. of kacterial suspension and one tenth the charge indicn.ted above
Using s,zall volunes of liquids in laborntory glassware, it WPS possible to control
better various fpctors such as tine of contect and to evaluate the action of the
.Asboloxane charge itself against variozs test orgmisx in conteiners nore easily
clesned and sterilized between trials. As with the trials run in the canteen, the
Asboloxme cKerge 'WRS pdded to the suspension end shaken well. The suspensions
were then filtered, this time through coarse sterile filter p?per, and plrted out
i2mediately. In this way, quick dilution into nutrient media w
eliminate my possible lethal effect caused by tte filtra.te itself r.fter the Asbo-
loxane catalyst had keen removed by filtration. The Asboloxane canteen charge vas
evaluated in this nmner aga.inst a. spectruq of eight bncteri,-l test organisas and two bacteriophages. The data obta,incd are given in Table 11.
The acbion of the Askoloxane charge is exceedingly rapid on vegetative or- ganisms. ?:lost of these orgmisms were conpletely killed 7,iithin 1/2 minute, and all such suspensions were rendered sterile within 5 minutes. -- 12 - TABLE I
PERFORMkNCE OF ASBOLOXANE CANTEEN CHARGE
(Trials Made Using Army Canteen with Special Filter Head)
Original Organisms per ml. Test Organism Suspension after Various Exposure Times
. B. globigii 532,000 36,000 ( 3 min.) 6-Hour Cultures (Veetatpre Form) 30,OOO 230 ( 3 hih.)
Ei coli 3 5 4 b0,OOO 4 (15 min.)~ 1 (24 min.1 710,000 44 ( 5 min.); 3 (15 min.); 0 (24 min.)
475,000 5 (1/2 "in.); 0 ( 5, 10, and 15 min.)
Z. typhosa 21,700,000 o ( 3 mine)
8,260,000 0 (15 min,) TABLE I1
PZRFO~NCEOF maomma CANTEEN CHARGE (Trials Made Using Glass Equipment)
Original Organisms per ml. after Various Bposure Times Test Organism Suspension 1/2 min. 5 min. 10 min. 15 min.
B..globigil (spores)# 62 000,000 - 1,560,00CQ
E. coli 257,500 2,200 0 0 0 .-- E. coli phage T-2 640,000 TNTC - 149,000 56,000 E. typhosa 7,500 0 0 0 0
0. tetragena 11,050 0 0 0 0
X. pneumoniae 1, 515 000 0 0 0 0 3. phlei Z’5,OOO 0 0 0 0
id. phlei 6,850 0 e 0 0 3. aureus 750,000 1,700 0 0 0 S. aureus phage P-209 TNTC TNTC TNTC TNTC TNTC .-. S. marcescens 245 j 000 0 0 0 0 S. marcescens 5,100 0 0 0 0
Q 2 Hours - 3,400
- :4 - Within 15 minutes, the tirx which the dcvelopers of the Asboloxnne trert- nent recomend thqt the contents be allowec! to stand before drinking, 57.5 per cent of the spores of B. globigii kLld been killed. At two hours 99.995 per cent had been killed. These spores are the nost resistant of the bacterial test organ- isms which have been studied here.
It is quite intaresting to note that the two bacteriophages tested dis- played resistirnce of the sme order of -:. itud. :'s this resistant bacterial spore.
Whether prthogenic virus orgenism would be as difficult to disinfect is an open quest ion.
Trials were also made, using 2. coli as the test agent, in which the bac- tericidal ectivity of. the v?rious ingredients of the Asboloxnne canteen charge were nenswed separately. The trials were run in exactly the sane nanner as the
Erlenp?e:mr flesk trials with the complete charge which were just described and re- gorted in Trble 11. The weights of the ingredients were the sme. .-nd in one ce.se an equal weight of nctivated carbon was substituted for the Asboloxane catalyst.
As the date which are given in Table111 show, the Asboloxane catalyst alone is a fairly effective bactericide but by no mems as active 2s it is when urea peroxide and sodim carbonate are present PS ia the case in the full charge. The urea per- oxide alone is not too effective, nor is its effect increased by the addition of sodiun carbonate slid activated carbon. No loss, but rather a gain in the EY coli count is observed in contrast when the original suspension is shaken in distilled water for 15 minutes and fiitered. This was often noted in control runs with this and othcr organisms, and probably is due to the bre&fn< up of groiips of clunped cells, preser,t in the originel suspension.
- - 15 - PERFORb.iiiNCE OF THE VARIOUS INGREDEXTS IN THE
ASBOLOXAN”, CAN TEEN CHARGE
(Trials made using Glass Equipment)
Test Organism -E. coli in all cases
Ingredients of Original Canteen Charge E. coli Organisms per ml. after various Exposure Times Present Suspension 1/2 min. 5 min. 10 min. 15 min. All. Full 258,000 2,200 0 0 0 . Charge Asboloxane 285,000 11 ,250 1,800 50 0 Catalyst mly
Urea Peroxide 258,000 TNTC TNTC TNTC 28,000 oily .. Activated Charcoal, 428,000 69,000 42,000 64,600 61, 800 Sodium Carbonate and Urea Peroxide None. Shaken in 285 ,000 - - - 840,000 Distilled Water and Filtered. .\
- 16 - I' i , & C' ;si?,ey .:.* <*.! g; I C. THX S-FILTER - SIMULTAEOUS FILT?A'?ION AN3 S?3:BILIZATION WITR SILER ASBOLOXAh@#
1. Description of the S-filter
The S-filter is a light-weight apparatus, weighing under 10 pounds; requir-
ing one .?an for operation. Water is @2+T+: into the S-filter and forced ~thpmgh ... ,
a cake of Asboloxane S-filter powder so thrt it is filtered and givep R stepiliza-
tion treatment simdltsneously. The uurified water is delivered Pt P rate of 2
liters per minute. Each charge of Asboloxane will treat about 50 liters of water.
The S-filter itself is shown in photographs in Figuree 3 thrpugh 6 and dia-
granatically in Figure 7. The photographs do not show the two hoses or rubber
tubes which are connected to the inlet and outlet valves during operation. The
inlet hose is dipped into the strear!, pond, or other water source, which is being
utilize&. Through the outlet hose, the p-ified wr.ter cen be delivered directly
to canteens, water cans, or other containers.
As shown in .Figure 5, the outer portion, or base, of the S-filter is
nothing core than a tamk. The central portion, which contains the pump, the filter,
the inlet rnd outlet connections, and the control valve, rests inside this outer
base, md is attached to it by three clamps. The Asboloxane S-filter powder charge
.is added directly to the outer tnnk. At the end of P run, the S-filter is opened
as shorn in this photograph, rnd the spent charge is discarded.
The pmp in the S-filter is e. hmd-operated piston type, with a bore of 30 m.
and a stroke of 75 mm.; delivering 53 cc. with each dorq-stroke.
The disperser, shown in Figure 6, which fits below the filter, consists
of a series of baffles rnd a. ?eta1 screen which Srenks up the flow of water and
causes it to flow evenly ebout t'.e filter. At the beginning of a run when dry
Asboloxane powder is placod below the disperser, this turbulence causes a thorough
mixing of incoming water and filter powder, and an even deposition of the filter
cake about the filter screen. - 17 -
~
I
~
~ i ! i
i I i
! i
I. FIGURE 7
c: U 0 0 W K 0
-- 22 - I... ~....
.~ ., -. . ~ The filter screen, also shown in Figure 6, is a plastic bonded celldose
cylinder. The actual filtration is ?cconplished,by the cake of filter powder,
which is formed on both sides of this cylindrical screen. !rate? passes through
the cake into the screen, and cones out through the holes bored down the center
of the screen. The total filtering surface is about 450 cm 2 . The Asboloxme
S-filter powder charge used in each cycle weighs from 35 to 40 gram. This forms
a cake approximately 2 na. thick about the filter screen.
The circulation of water through the S-filter is controlled by an operat-
ing valve which can best be seen in Figure 4. The three numbered positions for
this valve adjust the S-filter for the pre-coat cycle, filter cycle, and blow-off
nosition respectively. The flow of water through the S-filter in these three pos-.
itions is shown diagramatically in Figure 7..
Eere, the pmp, the filter cake, and the operating valve are shown, for
the sake-of simplicity, one above the other, rather than in the positions which
they actually occupy in the an?a.ratus. The check valves (1) and (2) are so ar-
ranged that the pump operates on the down stroke, syphoning water through the in-
let line and forcing it through the filtor cake in the spne operstion. Check
-I valve (j) causes positive pressure to be raintrined over the filter cake even dur-
ing the upstroke of the pump, resulting in a nore even flow rate through the filter
and preventing a sluffing off of the cake due to a drop in pressure. After passing
through the filter cake, the,water goes through the operating valve which can be
set in any one of three positions.
Position 1. (Pre-Coat Position). taen the valve is in this position,
water is re-circulated through the pump and filter cake. This accomplishes two
purposes. The Asboloxme S-filter powder is picked up and deposited as an even
cake over tbe filter screen. Secondly, all inner pnrts of the S-filter ?re steril-
ized by the ccntinuod flow through them of freshly filterGd pnd, treated. %Inter. .- - 23.- -. .. .. b Position 2. (Filter Position). In the filter or operating position,
water is dram into the $-filter through the inlet hose and passes directly
through the filter end into the outlet hose. A check valve (4) on the outlet
line is set so as to operate at a higher pressure than the other check valves.
In this way, the positive pressure on the outlet line is always higher than the
pressure of contminated water at the operating valve. Thus, any leaks or CMSS
connections, which might take place at this valve, consist of sterilized water
flowing into the untreated water and not the other way around. In addition to
this safety factor, the check valve (4) also prevents any back flow and consequent
loosening of the fi1.tcr crke, which might occur when the outlet hose was lifted
above the level of thi puup.
PositiJn 3. (?I.o,i-Off Position). When the operating valve is set at
position 3, the .wter, ,?f.;er passing the filter, is forced intc a separate small
.enilk coqressing the a?: i'lready gresent in that tank. When the front clmp on
the S-iiliis ,roc a:.nc.~ 1;. tkx ii~g::an) is released, the conpressed air forces
the wcf,er h.-w&cri.s '27.'; !.-:virg the Asboloxme cake fro3 the filter screen.
The renovad .:;-VJ ca'. :,1:;? :: p.?'.!ed out with the water, when the S-filter is
*~. opened to zh:ir:ge the isbolor.~.scharge. The water, which is forced back through
the screen, is water which has alroady been filtered and purified.
2. Instructions for the Use of the S-filter, Two Liter Model
The following instructions are those given by the developer for the use
of the Two Liter Model of the S-filter:
"Prior to use, thu ts~passembly (lid) of the filter should be removed ad
the inside inspected for cl _-i..hess; that is, absence of sand or mn.ior particles.
"The inlet .and outleb hoses should be connected with their respective
unions pnd care should be taken .that both units ere tightcncd in order to avoid
a.ir leks. - 24 -
/ I- "A sack of filter powder is opened and all of- its contents put into the tank, subsequently the lid is put on the tank and the two rear clamps are closed
while the front clamp (red) is left open. The foot pedals &e then put on the
ground. The valve hmdle is put into position .!2,!' the suction hose is inserted
into the welter supply, and the pumping is sta.rted in regular strokes (about 30-40
per minute).
"After about 30 strokes (depending upon the priming condition for the
pump), blackish wnter squirts out under tke lid of the pump which is a sign that
the punp is now filled end dispersion of the powder has begun. The front clamp
(red) is now tightened and a few nore strokes nay be given to the puxp until water
squirts out of the outslet hose.
Vhe valve is now t,urned into position '1' and pumping is continued at a
fairly vigorom rate for 50 to 40 seconds in order to complete the pre-coat cycle.
While continuing to pmp, the valve handle is put quickly into aosition '2' and
filtrt3tion begins.
'!The punping act,i,m shou1.d ke continued for the next 25 to 35 minztes;
tnat is, until. the pmp hai.slivored approxinately 50 liters of water, yielded
at 'the rate of ;!bout 2 iitsrc .:; .ninute.
"Care should be taken thet the puqping is not interrupted during this
procedure for any appreciabl-? length of tine.
"In order to remove the cuke frofi the filter screen at ti-e completion of
the filtration cycle the 'vp!ve is put in position '3' and ?-bout 3 to 5 stlpkes
exerted with the pump, at tile cc..i;detion of wi;ich the red ilrmp is quickly opened.
9y so doing the air in the j:il.t=: tank will evnd nr.3 a '>ackwash action through
tk6 screen wi1.1 r:,cult ?:?!.G?, i,.-=o~;itsthe filter cake ir. +,%e tank. Should inspec-
L....ic~ show fFri tk snk:e ;a;. not been conpletely blown off, the lid should be put
on ngain, the cil>,p ti:1?tar.r.d and t?e operation (compression and expnsion) with the valve in >osition '3' is repeated. - 25 - "The top is thfi? entirely removed aid the dis)>erserunscrcwed. If no
fresh water is availi.ble, the lptfer as well PS tke filter screen can be flushed
in t:ie urater contaii1e?. in the tmk.
This tank is subseq:ently mutied and tP.6,. dis:?ers~ris pzt back in
-,osition. Phc filter is then ready to rect.ivo anothL:!r s~??oly of filter ?owder
for t'-e nert filtmticn cycle.
"If t'7e water to 'to filtrrod. contains R considerable quantity cf sus-
-Jen?,e?. .mttCf (clay, etc.), the filter cpke will beco,ie c1ogi;ed prior to tho
coqlction of the filtration cycle of 50 liters. This is ini.icated b.i the fact '. that t:e puqing cction beco.?cs soicVint difficult and wntcr cpmts out fro?
below th lid. In this case it vi11 he necessary to blov t:*e cike off (see
above) m?. start a ncv filtr-r c:icle with 2. new su?ply of filter -0owder.
"1Jiic'.fr no coneitions should. the filter screen be tiserl as filter without
tl.e irscrtion of filter powdr?, or with a quantity which is less than that
pocked in a single bag.
"If tine operation of the jmm-$ becc-.es difficult a.i'ter about 12 to 15
cycles, representing 6co t.o 750 liters of water, the filter screen has to be
cleaned with nn acid carrier 2rovided in a sepr8t ;ly .rarked pack.
"The operation in this cns,: is the sa% rxcept that only the pre-coat
cycle position '1' is useA for .-bout 4 to 5 -tin>.tt=s,after r?ich the ?older is
blown off an4 t?-r contents of tile tpnk are noured out. It is clrsirrble that
tlle tank pn;? filter screen be rinsed after this oaerntion."
3. AsboloxPne S-f ilter Pcwder
Asbcloxanc 5-filter porider YRS developed for use iii the spr'cial S-filter,
iust described, wi-.ere water is filtered and sterilized sinultaneously. This pour-
Zer consists of uiato?a,ceous earth, togethor with an Asboloxme catalyst Rnc! an
inorganic proxick. Mcchnnicrl filtration is achievd bp fonire P cake of this .- 26 - I powder )ver B rigid filter bed just as is- acne *dth ordinary mtreatrd dintom-
ceous earth. Scch untrerted di8.tccsceocs earth filters, hcwever, if they are
of such ;>ore size os to produce eder:ua.te filtmticn rates, are by no means hac-
terial filters, even thcugh thy will retain a very largo runber of crganism
from the suspensicns passing thrcugh then. Sterility of the filtrate is achieved
by tTc dsbclcxane inccrpcrated with the diatcf2aceous enrth, which kills these
-.rganisms passing thr:.>gh the cako. Qu.,ted be1F.w is the d.escri*ti. n given by
the c?ovelr.per,
h:.?amus earth and the sterilizing pr;perties rf the -4s'~;-l.xanPs are c.qbined
in S-fi1tc.r pcwder. I - --, "lhe diatvns are cs rted with a.n extremely thin lryer r.f a therm-plaatic
material (fLr instance, E polyner r cb-p, lyner if vinyl-esters) . It is charac-
teristic uf this plastic c.at thzt it mxst be susceptible tc., water Rnd neutral
ti. fairly high alkali cr.ncentratiln. This plrstk serves Pt the sa-e tine as
vehicle fur tw, c,llbi&i ir seni-culibidal sisjicnded chezical sgcnts. One $.f
these agents is elnist water inavluble wr >xidesuch as, fkr instance, CaO.2,
Xg02 ani. tht like'. The c.ther agent is a catalyst ,.f the above mentiLned tyoe
.- such a.3 Q surface-cr,"pl:und <.,f silve? and ,.xygen adsc,rbed cn e. ILidal carbcn.
"It i3 n-,t necessary that the tw? agents (perf-'xidea.nd catalyst) ce-exist
c,n the same particle althrGgh it is 7erfectlg p,ssibl+ ti, inccrwrate bith agents
inti the srme vehicle x-iz,r :. c.-;ting t'ce dintcm. It is alsr p:;ssible t; in-
c rpr;rate each agent sqmrxtely intc. tw. be.tches i,f ve?.icle and cc.at half r,f the
dlatc.?r.s with >ne an. tLe c tk.er half r.f t:"e di,-ti.ns with the c,tPer. After drying,
the two kts of diatimaceo-Gs earth have then to be mixed with ea.ch (ther inti.mte-
ly. It is assumed that t?.? separatit,n r.f the twc acents lay prr.8.uce a filtratirn
naterial which is less easily dac<>mpr,srdwhile in stp,ragt-, in ccntmdistincti-.n
t.. the cther fLrm in which bc.th agents are in mire intimate contact, beinc sus-
pended in the sane -Gating. - p., - -.~ "This filter pcwder is then brccght int6 wattr in crder tc furrn the
filter cake in exact analc,gy tc the customry -pr;,redure f.:r the use i.f dicitt>-
macecus earth. The filter cake, hilt frm filter pGrrder3 su ci.atnd consists
thus cf particles which have the structura.1 sha?e ;f dintons. It exposes, however,
t:. the pilssing f'iltrate a. surface which can adsc!rb, by virtue .f th.e water s~s-
ceptibility c)f the plastic, P sufficient amount .-#fwater in order tfJ start the
catalytic decLmpositi\:n of the p(-roxide, due to the clcse juxtapcsition of the
two a,.ents in the cuoting. The silver-bearing catolyst can be designed to yield
e csrtain (minute) quantity uf silver-ion if this is desirable in order tc ac-
celerate the killing 8ctiL.n and fc,r producing a certain retention bf gerniciw
agent in the water.
"As the acti,-n .f the filter pc,i#der is rim ti the deccn,uusiticin c.f the
peroxi6.e end the catalyst, it is o-~vi*-.usthat the activity cf such a cake is
linited. The weight rati6 betsreen the water to be treated and the quantity of
pi,wder necessary for its treatnent, even in hegvily infected strtes, is favorable
indeed. Frr instance, 5-7 gall(;ns of wrter can be filtered thr,;ugh a cake, the
dry wight ;.f -1hich is 112 cz. cr kss.
1 "Powders Lf this type can be stored, are relatively temperature insen-
zitive (boli,w l5IlLF.) but have tt.ba kept dry.
"In edditivn tc thr sterilizing rction, f'ilter mw(?ers of this tpe have,
,,f c\.urse, the same retentiin qualities as untreated di?tcnacrous earth so thnt
the filtration acconplish?s .iechanical retentirn t,f solids PS well RS the ge-Ti-
cidal actim. The layer of retained s. lids dvea nut inpair the acticn 9z.f the
cake 'cecadse <;f the fact that the retair:ad s.,lids remain un the. tt>g of the cake
and ib not penetrate intz the latter."
- 28 - k. Perfdrmance Tests on the S-filter and S-filter- P:.wder Conducted at California Institute i.f Technology
The following is quoted directly frcm a repcrt cf Dr. Goetz a# the
California Institute of Technclogs:
"The development of the S-filter powder and t!e a,-plication of the
latter to the S-filter was largely guided by bacteriological 1aboratGry tests
on artificially infected waters. These tests were conducted at the bacterio-
logical laboratory of the Rare Metals Institute at the Zalifornia Institute of
Tech.:ology.
"The tests were conducted with varicus types of waters artificiplly
inoculated with E. coli and mixtures of E. ccli with staphylococci and strep-
tococci. 1nhibitGI-y agents such as organic and inorganic nitmgen in the form
of peptone and am!!onia were add.ed in certain test.6 in order to establish the
1.initations of the action of the areperation. The technique of these tests was
as foll~ws:
"After prepering 20 liters of test water with chemical and bacterial
additions, filtration was startad, and, in generd, a sample taken from the
first, seventh and fifteenth liter filtered. These sanples were divided in two
?qual portions, one of which was plated imediately (i.e.,within one minute) while the other portion was allowed to stand for 15 minutes before it was brought into contact with the nutrients, Each of these samples (i.e., 6 altogether) was divided into five 10 cc, one 1 cc and one 0.1 cc samples, inserted into lactose
50th and subsequently incubated for 4s hours at 37'c. In case gas was obsemed,
confirnation for E. coli was carried out in Srilliant Green Bile broth.
"The laboratory tests gave always sanitary writer (i.e., no confirming gas in any tu>e) for coli concentra,isns up to aproximately ~oo,OOO;/CC (or
2C0,000,000/liter). This is similarly true for suspensions of stre2tococci and staphylococci. At higher concentratiors of X:coli, occasiond gas tubcs .) . - 29 - 7 in the 10 cc samples occurred, 3ut it is assumed that such concentrations do
not occur in nature.
"As aqueous subptrates, well water with sewage (1:2@00), lake water
rich in protozoa and algae. well water with the a,ddition of up to 500 parts
per nillion peptone 'as representative of organic nitrogen and up to 25P parts
2er million anrnoaia as representative of inorganic nitrogen, were used. Further-
nore, as a representative of heavy organic contamination, up to 25,O)C parts per nillion urine was used. To each of these substrates, artificial inocula
of E. coli up to 1~~~:;,030/ccwere treated successfully and resulted in an efflu-
ent fulfilling the bacteriological sanitary standards, either imediately or
at least after 15-20 minutes elapsed time. "In view of the fact that infected Praters tested under laboratory coa-
ditions -!ay not represent the performnce of nethod and equipment in the field,
a number of field tests were performed on varying ty?es of infected waters in the States of Sinaloa, Guerrera, Morelos and Distrito Federal in collebora.tion with the Dep. de la Salubridad Publica. In order to test the water after a short lapse of tine, rmtulant la5oratory equiFnent was taken dong to the site of tiie tests, in most cases. The results reported refer to an incubation tine of 40-48 hours. Thus the condition in the water when filtered and consumed under actual conditions would be studied ES clossly as possible. These tests are enumerated as follows:
e.. "Location: Bio Rumaya, 5 ko. north of Culiacan, Sinaloa. Temper- ature: 22OC. pK: 7.5 - 8.C. Control: gas in 1 cc sawle, confirmation ques- tionaUe, growt3 till 0.OO;Ol cc. Filtrate: sanple after third liter, plated after 12 minutes: no gas in any sa.nple, growth in three 10 cc tubes only.
-:. "Location: Bio Culiacan, 3 b west of cdi8.Can at the pint of confluence with the sewage canal (Canal Almada). Temperature: 25OC. pH: 7.5 -
8.0. Control: con irning gas in 1 cc sam?le, growth till O.OC01 CC. Filtrate: - 30 - s-e after sixth and fifteenth liter, tested after 15 an6 20 minutes respec- tirely: no gas in any sanple,growth in four 10 cc saxples only.
c. IILocation: Rio Cmazula near pu?lic laundries of Culiacan, Sinaloa.
Tenperature: 24.5'C. 'H: g.0 - g.5. Coctrol: confirming gas in 0.01cc, no growth in 0.001 cc and lesser dilutions. Filtrate: san:de after fifth liter,
tested after 20 minutes: no gas in any saqle, no g:-owth in any'simple.
d. Uocation: faucet in Put.lic Health La3oratcry in Cdiacan. Te.qer-
ature: 23OC. pH: 7.5. Control: confirning gas in two of five 10 cc tubes-
Filtrate: sample after fifth liter, tested imediately: no gas in any sanple,
growth in two 10 cc sarnples.
e. "Location: sewer canal (Canal >.hpciaj of Cuiacan. Temperature:
28'C. fl; g - S.5. (Water turbid and odsrous). iio control t-ken. Fil-
trate: sample after fifth liter, tested after 15 minutes: confirming gas in one
of five 10 cc samples, no growth in 1 and 0.1 cc..
f. "Location: stagnant water Sody in an arroyo near Eanchita "La Divisa," appoximately g kilo.2eters east of Culiacan. (F.& of mosquito larvae, algae and with a very mddy bottog). Temperature: 24OC. pH: 8.0. Control: no coli >ut growth till 0.001 cc. Filtrate: smple after sixth liter, tested after 20 miiiutes no coli and no growth in any sample.
g. "Location: Fiio Te7ecua near Xatitla, Guerrera. Tenperature: 29OC. pH: 7.5 - g.0. Control: confirming gas in 1 cc, growth till C.0001 cc. Filtrate: sapple after third liter, tested after 15 minutes: no gas in any sample. growth in one 10 cc s-le only.
h. "Location: stagnant pool at tke entrance of Tixtla, Guerrera. The water WBS extremely turbid from algae growth, so that t??e filter clogged after three liters. Tezperature: 27OC. pH: g.5 (1). Control: confirming gas till
G. '1 cc, growth till 0.' .:-lcc. Filtrate: sanple after two liTers, tested after
20 jT,inutes: confirming gas in one of fiirn cc t.nhna, pn crn,.r+h in 1 "?A. n.1 CC. - 31 - -. -. i. "Location: Xi0 Sahana, 8 km east of Acaoulco, Guerrera. Stagnant
pool in river ked, clear water. Temperature: 36.5'~. (1). pH: 8 - 8.5. Call-
trol: confirning gas in 0.1 cc, growth till 0.001 cc. Filtrate: sample after
seventh liter, tested after 15 minutes: no gas in any sanple. no growth in 1 cc.
j. "Loc&ion: Rio Sabana, same location as (i.) but flowing river water. Temxrature: 35OC. pE: 8.0. Control: gas in 0.01 cc., growth till
0.001 cc. Filtrate: sam?le after tenth liter, tested after 15 ninutes: no E.
coli. Two s-confirning 10 cc sayles out of five, no growth in 1 and 0.1 cc.
k. "Location: Canal de Cuautla, tdorelos, at Xiloxter 1. Clear water.
... Temperatme: 2O.5OC. pH: 7 - 7.5. Control: confirming gas in 0.1 cc., growth till 0.601 cc. Filtrate: sample after fifth liter, tested after two hours and
15 dinutes: no gas in an>- sanple, no growth in 1 and 0.1 cc. 1. "Location: Arroyu near Yautepec, Xorelos., at Rn 3G. Fast flowing,
veq? mddy water. Ten:gerature: 25OC. pH: 7.5 - g. Control: confirming gas in
0.01 cc-. growth till 0.0001 cc. Filtrate: sample after third liter, tested
zfter 1 hour and 10 minutes: 110 ges in any smgle, no growth in any sample.
rn. "Location: Xochimilco, D.F. Stagnant canal under street bridge.
Temperature: 22OC. pR: g.0. Control: confirming gas in O.f.'O1 cc. Yiltrate: * -sanple after third liter, tested after 2 hours and 40 minutes: no &as in any siuaple, growth in one 10 cc tube only.
n. "Location: Xochimilco, D.F. Sane location as (2.1 but two packages
of filter powder used, in view of extremely tur'2id water. Filtrate: smple
after third liter, tested after 2 hours and. 35 sinutes: no gas or growth in a.nI
smple.
0. "Location: Xochinilco, D.F. E13)1arc8dero, San Cristobal. Temper-
ature: 2O.'joC. pH: 7.0. Control: confirning gas in 0.001 cc. Filtrate: sample
after second liter, tested s.fter 2 hours and 20 minutes: no gas in any sample, growth in four 10 cc. snnplez cnly. - 32 - - canteen trials, a larger series of tests was' run with the Asbolcxane Fowder
in laboratory equipment, where its perfrrmance could be studied separately
from the mechanical functioning of the S-filter.pump, and where various factors
influencing its action could 'oe better st'idied. The Camp &trick trials largely
centered about investigatirg tine performance cf the S-powder when extremely
heavy suspensions of various txJeS of crganisms were used. The data from the
California Institute of Technology, already quoted, had shcwn What the S-filter
and the Asboloxane S-powder could do towards handling the amounts of Contamin-
ation ordinarily found in naturally occurring waters. The tests at this instal-
lation were designed more to indicate what t:,e upper limits of performance were,
and how broadly effective the apparatus was against all types of agents. For
this reason, tests wex not confined to the common water-borne organisms us:ially
found in natural wpters, nor were they usually run with concentraticns as low
as those which might be expected to occur naturally.
a. Trials Utilizing both the S-filter and Asboloxme S-pcwder
The first preliminary trials with the S-filter were run with 20-
liter suspei:sions of B. globigii spores snd of E. coli Cells. Samples were
taken for bacterial counts after approximately 2, 10 and 18 liters respectively
of the 20-liter samples had passed through the filter.
The $-filter was operated according to the instructions already
viote6, given by the Cesigner. in -.c~ cos3 b grams of tke Asbolojtane, S-filter powder were used in the pre-coat cycle so that a filter crke apgroximately 2 m.
thick is formed. Through this cake the rrater was pumped, filtered, and sterilized at the rate of about 2 liters a minute.
The E. globigii spore suswnsions were red.uced in count f-&om an origiml concentration of 200, .:OO,&?O orgapisms/ml. to 16, 26, and 12 organisms/ nl. respectively in the three maples of the filtrate. E. coli was reduced in count from 35,~.C.COO to. 13, 13- and 1 rganiss/ml. . - zb - These filtrates were pooled and then run back through the S-filter using 2 fresh charge of Asboloxane S-powder. The counts irere about t*e same as in tke first passage.
A set of experimerts was then run utilizing in each case 50 liters of very heavy suspensions of typical bacteria. Samples of the filtrate were collected after 5, 15, 30, and 50 liters respectively heU passed through the filter. In several of the eq3eriments no collections were made after 30 liters. The data collectet are given in 'Fable IV. TXTC indicates that the colonies were too numerous to count at the dilutions used. In such cases it can be as- sued that tile filtrate concained over 5W organisma/ml.
It is evident that the S-filter can effectively remove very high concentrations of various tpes of organisms, an6 do this at the relatively repid rate of 2 liters a minute. It is also evident that it is possible to ex- ceed its sterilizing capacity especially when counts rise to the order of magni- tude of 106 organisms/ml. This is apprent usaally in increased counts toward the end of the 5G-liter filtrations, or in at least me of these tests, in high counts throughout the entire course of the filtration.
~ Some evidence was obtpined which indicated that, with' StaFh. aureus at least, bacteriostasis was occurring in certain cases. Additional colonies ap;jeared on a few platas, which had not beeCdestroyed after the usual reading at the end of 48 &ours incubation. Five trials islere set up, all with Staph. aureus as the test agent, in which counts were .-de of viable organisms in the filtrate after the usual $9 hours and again after the same plates or broth tubes had been incubated for 9 hours. Xmy :?lates recorded as sterile at 48 hours stowed some grONth at 96 hours, as indicated in the data in Table k' from two of these trials.
- 35 - TABLE IY
EXPiRIKhNTS WITH
THE S-FILTKR AND ASBOLOXANE S-FILTER POWDER
Original Con tamin ation Bacterial Count per cc. in Successive Filtrate Samples Test Organism Count per ml. -SL -15L -30 L -50 L
S. marcescens 670,000 1 0 2 0
~ S. albus 750,000 1 0 0 0
B. globigii (Veg,) 1,760,000 7 4 TNTC
B. globigii (Veg.) 1,000,000 8 2 2 62
B. globigii (Spore) 970,000 1 7 11 B. globigii (Spore) 16,000,000 263 294 TNTC TNTC E. coli 15,000,000 10 13 TNTC TNTC
S. aureus 145jOOOj OOO Tarn: TNTC TNTC TNTC
- 36 - TABLE V
DIFFERENTLAL 48 AND 96 HOUR COUNTS ON FILTRATES I OBTAINED USING ASBOLOXANE S-FILTER POWDER IN TdE S-FILTER
(40 grams Asboloxane used in all tests) - Orieinal Volume Filtered Ratio Number Plate Count Broth Count Contaibtion Before Sample Taken Organisms to or anisms ml. wpN/ml. Mg. Asbo. & 48 hr 96 hr
Staph. aureus 5 liters 48 T 0 31 0 5 385,0W/ml 15 144 T 0 730 0.2 8 20 193 T 0 2950 0.2 8 30 288 T 0 1040 .0.4 18
Staph. aureus 5 17 T 0 4 0 5 136, COO/ml. 15 51 T 0 38 0 1602 20 68 T 0 30 102 ,T 0 13l5 0.2 2
-.37 - Since Staph. aureus is sensitive to high pi; values, and the Asbolox- me filtrates are alkaline, it was thought possitle that this bacteriostasis or lag vas a p4 effsct, and not due to the As'coloxane. To evnluate this factor, two sterile broth solutions were ade alkaline, one by passing the zolution through an Asboloxane cake; the other b:- ad6.ing Ea09 until its pH value matched that of the Asboloxane fil'trato. A third lot of broth kegt at pH 6.8 served as ccntrol. These broths were inoculated nith Stash. aureus;giving the theoretical counts inaicated belcw, and after the indicated times, 1 ml. portions were re- moved ?ad plated with nutrient agar in petri dishes. These plates were ree.d both after 24 hours a.nd after 56 hours of incubation at 37OC. The da'a. appear in
Table VI.
The data shov that contact with alkali can clefinit:ly so affect
Staph. aureus organisms that colonies develop :mch more slowly than usual from those orgnnisms which survive the trcatment. The time lag in the d-evelopgent of Stan?. aureus colonies caul?. thus 'ce accounted for by the alkalinity of the
Asbololinne filtrate. Of greater interest, however, is tho evidence that the
Asboloxane filtrates poseess a definite anti-bacterial activity. There is a much greater killing action in the Asboloxane filtrate than can be accounted for by alkali alone. Rot only are viable organism killed in passage through thiAsbo- loxane %filter cake, but Also a lethal groperty is iqarted to the filtrate which, as shown in these tests, is effective agaiilst crganisms added hater to the filtrate. This residual anti-bacterial property of the Asboloxane filtrates has been called its "antibiotic potential," and will be further demonstrated in experiments done with laboratory equipment, rather than utilizing the S-filter pu.q. GFKT OF pH IN ASBOLOXANS FILTRATE
ON LAG PHASE OF STAPH. AUREUS
Plate Counts Inoculum of Treatment Given Broth- -Staph. aureus/ml. Contact time
Filtered thru Asbolexane 31,500 5 0 12 PH 10.5 30 0 7 60 0 0
Adj 4 with NaOH 31,500 5 0 TNTC pH 10.5 30 0 652 60 0 4
No treatmefit 31,500 5 TNTC I pH 6.8 30 TNTC -- 60 TNTC -
. .. ,. - 39 -. ,.; ...-. b. Trials with Asboloxane S-filter Powder on a Sintered Glass Filter I I Bed i I . I While the trials with the Asboloxane S-filter powder were being 1 conducted in the S-filter designed fcr dlitary use, laboratory scale trials ii were inaugurated. Working on a s.mll scale ra.ther than with the 50 liters of
bacterial suspension and 40 grams of Asboloxane %filter powder required for
each trial in the S-filter, it was possible to step uio the tempo of the work.
BL-sides, closer control was possible with the laboratory trials, when it was
desired to vary certain factcrs in the filtration process and to use high con- - centrations of various pure culture suspensions, which necessitated sterilizing
tke total equinment between runs. The first of these laboratory trials was run
with very simple glass equipment using vacuum suction flasks and Suochner ty:oe
fmnals of coarse sintered gla.ss, which served as the filter be6. against which
cakes of Asboloxane S-filter powder were formed. The sintersd glass alone re- g tained very few organisms, 0,s evidenced by a drop from 1.30 x 10 organisms Der ml. to 1.03 x 10l3 organisms per ml., when a 3. globigii spore suspension was
pass& through it.
The recommended charge of Asboloxane powder in the %filter WRS
one gam per 15 cm2 of filtering surface. A correspo 'ding char.ye for the 3uech-
ner fuliiel, which had a sintered glass bed approximately 30 cm2 in area WRS two
grams. The filtering cake so obtained was about 2 mm. thick. Utilizing the
service vacuum lines, ho?rever, it was not possible to filter bacterial suspen-
sions through these cakes at the rate of 150 ml./minute which corresponded to
the rate achieved in the S-filter pump. Tho 300 ml. suspensions used in most
' cases required from 3 to 4 minutes to filter.
5he ceke WDS formed 3y mlxing the dry Asboloxane S-powder with a
small amount of sterile water and drawing by vacuum prectically all of this - 40 - - .. .. - ~. wator into a storile scction flask. T'-e bncterial susnension was then odded
and the vacuum turned on fully. -4fter all the sus9ension had. passed through
the filter, the filtrate was usually allowed to stand for at lee.st 15 minutes
in tha vecuum flask, then romoved and ploted out with the usual dilution tech-
nique into nutrient agar.
Pelpresentative data obtained from th.e use of the Asboloxane 5-filter
powder in this glass laboratorp equipment are given iil Tables VI1 through XII,
presented in chronological order, as the tests were run in the laboretory.
Variations in the techniques described above are either npparent from the head-
ings, or explainoddan foot notes.
The dat:, obtained with the isboloxane S-filter powder on the sin-
tered gloss filter beds showed, as hPd the trials in the S-filter itself, that
the treatment was effective against widely different types of bacteria present
in very lar;,e numbers. Suspensions up to the neighborhood of 106 organisms per
ml., varying of course with type of orgmis::? and other factors, could be effec-
tively sterilized by a single passage through a cake of the thickness reccmmended
for use in the military S-filter. A5 might be expected, reppassage through an
Asboloxaile cake of this thickness or single passage through crkes of greater
thickness causrd increased reduction ir Guspensions so high in count that nor-
ally a.iJprecia.-le numbers of viable orgo.nisms would he found in the filtrate.
Parallel filtrations through Astoloxanc S-f ilter powdt-r and diatomeceous earth
showed thst somethin: more than mechanical retention of crgrnisms wits oper?.ting
here, although large percentages of, orgnnisms from heavy suspensions are trapped
on the filter cake.
When viable org?nisms do appear in the flltrato, tkeir count drops
as the filtrate stands. This is another indication of tho "antibiotic potential'' - 41 - .. . ,. .. ~ .. TABLE VI1 ... ..-
ASBOLOUNE S-FILTER POWDER ON SINTEED GLASS FILTER BED (&gram Cake)
Repassage --of E. globigii Suspension Through
The Same Filter Cake
Treatment B. glob;@ spores/ml.
Original Suspensioli 160 000 000
1st Passage thru cake 3 000 000
2nd Passage thru cake 76 - - . . .. -. NOTE: The E, globigii suspension made by resuspending dried spores contained many lumps or clusters of spores and non-viable organic material. ASBOLDXANE S-FILTXR POWDm ON SINTERED GIASS FILTZR BED
Single Passage of 8. globigii Suspension Through Cakes of Varying Thickness
Treatment B. globigii spores/ml.
Original Suspension 10,000,000 Passed thru 2 gram cake 66,000 Duplicate 37,000
Passed thru 4 gram cake 0 lhplicate 3 Passed tnru 6 gram cake 5
NOTE: B. globigii suspension made by resuspending dried spores. TAB= IX
ASEOLOXEtNE S-FILTa PCNDER ON SINTDZD GUSS FILTER BED
Comparative Trials with ksboloxace S-Filter Powder
and Diatomaceous Earth .
Bacterial Counts/ml. - Treatment Plated Immediately Plated after 24 hours Original Suspension 10,000, ooo Thru 2 grams Asboloxane 45,000 8,500
Thru 4 grams Asboloxane 2 3
Thru 6 grams Asboloxane 2 3 (All above colonies E. globigii)
Thru 2 grams Diatomaceous Earth L1,OM) 1,300,000 (21,000 B.g.)
Thru 4 grams Diatomaceous Earth 6,700 TNTC in dilutions (29 B.g.) plated
Thru 6 grams Diatomaceous Earth 120 Over 5,000 (5 E.g.1
'> NOTE: The same crude resuspension of dried E. globigii spores was used. olly B.g. colonies appeared in the filtrate which had passed through the Asboloxana. Many small white colonies xere found in the fil- trate which had passed through diatomaceous earth. These were counted differentially. Apparently, a contaminant was present in . the B. globigii suspension which was killed by the Asboloxane but which passed through the diatomaceous earth more easily than did the E. globigii spores, and showed up when the large excess of E. globigii spores vrsre removed from the suspension. Since the filtrates had not been discarded, duplicate plates were made from them the next day. Then interestinzly enough, the ksboloxane filtrates still contained only E. globigii spores in lesser number than when first filtered, while the filtrates from the untreated diatomaceous earth showed greatly increased counts of the contaminating organism.
- 44 - , , . --. TABLE X
ASBOLOXANE S-FILTER POVDER ON SINTERED GUSS FILTER BED
(L-gram Cake Used)
Action Against Various Organisms and Drop of Count -in Filtrate U?on Standinc
ilriginal Contamination Time Lapse After Fil- tration Be- Test -_ Organism Count per fore Plats Bacterial Count per ml. Staph, albus 560,000 0 - 5 min. 26 15 min. 8 30 min. 2
Staph. aureus 1,100,000 0 - 5 min. TNTC 15 min. TNTC 30 min. 386
Staph. aureus 930,000 0-5min. 238 15 min. 155 30 min. 70
Staph, aureus ' 620,000 0 - 5 min. 207 15 min. 137 30 min. 11
Staph. aureus 338,000 0 - 5 min. 0 15 min. 0 30 min, 0
E. coli 288,000* 0 1 5 min. 1 15 min. 0 30 min. 0
E. coli 272,000 0 - 5 min. 0 15 min. 0 30 min. 0
B. fLz:jji.i 960,000 0 - 5 min. 4 15 min. 3 30 min. 5
5. globigii 623,000 0 - 5 min. '0 (veg. 1 15 nin. 0 30 m% 0 - 45 -- TABLE XI
ASBOLOXANE S-FILTER POl’WJER ON SINTEFZD GUSS
FILTZR BED
Action Against E. coli Bacteriophage
Original Contamination Filtrate Count per ml. Test Organism Count per ml, 4 Gram Cake 6 Gram Cake
T3 Phage 18, ooo,000 below 100 below 100 T3 Phage 6,800,000 300 belo-nr 100
NOTE: Phage counts are made by plating out dilutions in agar heavily seeded with E. coli and counting the clear plaques where the phage is destroying the host cell. When the filtrate through the Asboloxane was added to the seeded agar directly and in a 1:lO dilution, the 2. coli growth was so inhibited that it was not possible to determine whether or not phage was present. At dilutions of 1:lOO or greater the E. coli cells developed, and in one of these plates, three clear plaques were found indi- cating a count of 300 ml. for that filtrate. The other filtrates could have had any count, ranging from 0 to 100 phage particles per ml.
TABLE XI1
ASBOLOXANE S-FILTER POWDER ON SINTXED GLASS
FILTZR BED
(4-gram Cake Used)
Effect of Successive Filtrations and of Temperature
Upon Action Against E. coli
Original Bacterial Count per ml. for Successive Suspension Temp. Gne Liter Lots of the Filtrate Count per ml. OC .- 1st 2nd 3rd 4th -5th 67,200 3O - 100 150 100 - 22O 0 0 0 10 135
45; 500 2O 0 0 0 10 TNTC 37O 0 0 0 10 20
48, ooo 2O 20 0 0 50 50 37 O 0 0 0 0 0
42,120 20 0 0 0 100 0
126,000 3O 0 0 250 19 50 1400 220 20 200 200 200 400 37 O 0 0 650 250 200 86,800 0 1 5 7 56; 373: 0 4 6 13 13
+* Counts made using 5 tube broth series, Other counts are direct plate counts.
- 47 - imprtcd to tbsolution ugn passage through the Asboloxane S'aowder. %'hen
large volumes of susptnsionwere passed through a single cake, exanination of
successive lot6 of the filtrate showed larger nunbers of organisms appearing towards the end of the filtration, indicating the chemical a.ction of the powder became spent as more end more liquid passe& through it. One point of distinct
Sractical importance was brought out in the trials conducted in the cold (2-3OC) I, and warm (37'C) rooms. The temperature coefficient seems to be quite smell, as the performance at the lower temuerPturo compares very favcrnbly vith the ster- ilization obtained at 22 end %.t 37 degrees.
There appeared. however, definite limitations in the use of the sihiple 3uechner sintered glass fun.:els nnd vacuun flasks for USB in laboratory trie.16. Particularly, it became evident the.t it would be a.dvisable to have an apparatus where successive samples could be takon of the filtrate for bncterio- logical md chemicd exeminetion without interrupting the course of the f iltra- tion, and to hrve the filter cake -.iore protected, since it @ften-b6ctmedis- turbed as more suspension was added to the sintered glass funnels, or as the funnels were removed to other sterile suction flasks, when one became filled.
Accordingly, the laboratory apparatus described in the next section was designed, and subsequent laboratory evaluations of the Asboloxane S-filter powder were conducted in it.
c. Laboratory Pressure Filter Apparatzs
A pressure-filtration system was set up to simulate the action of the S-filter on a laboratory scale. A closed reservoir w8s used tc. c8ntain the water to be filtered. Compressed air forced the aus_oension out through an open- ing at the botttm of this reservoir end through a Seitz filter unit, from which the valve and diephragm were remcved. The effluent from the Seitz filter led - 48 - to a calibrated glass chamber in which measured portions of the filtrate
could be held and removed for chemical and bacteriological determinations.
One gram of the Asbolcxane S-filter powder was weighed out, and
filter cakes were made by filtering a water suspension on a Euechner funnel.
The cskes and filter papers were placed unon the metal screen of the Seitz
filtor, which was then clamped tight. The water used in these pressure fil-
trations was autoclaved distilled wilter, unless otherwhe noted, to which might :f be adted any type of bacterial or chemical agent, depending upon the purpose
of the test. The tine which it took for each successive 200 nl. portion of
1,faterto pass through the filter cake was noted. ?hen the nm was over, tk&e
thickness of the filter cake was determined by measurements with a Randall and
Stickney thickness gauge.
Chemical tests performed on the filtrate included Winkler's deter-
mination of dissolved oxygen, and later, th.e cupric dithizonate method for sil-
ver. A discussion of these factors will be presented later.
Eacteriological assays were done on every 200 ml. portion of fil-
trate. Triplicate plates were poured of "direct" and "1:100'1 samples of fil-
trate,. using Nutrient agar or Friedlein's synthetic agar or both (1). Plates
were incubated at 37OC for ag hours.
In some cases the suspensions being filtered were made by adding
the broth in which they were grown directly to the water. In most ca.ses, how-
ever, where comparative data win-+? desired, bactr>rial sus-oensions were centri-
fuged. the precipitated organisms wcre taken up in distilled water. centrifuged again, and re-suspended before being added to the test water.
(1) Plating was begun within one minute of collection of tho sample to avoid the self-sterilizing action or antibiotic potential of the filtrates themselves - kc - In all tests, counts were made of the bactcxia in the test water
both before and after a run, to determine original concentration and fllsc the
normal die-off, due to standing in the reservoir during the time portions were
being treat Ed.
mcal3ata: in Table XI11 is a standard laboratory sheet, such
as is obtained from a single filtration experinent of this type. From this
table we can see the method of deriving the time of contact of any particle
with the actual filter cake. Tho time for 200 ml. to pass through the filter
cake is recorded, and transformed to rate of flow in ml/sec. This rete is
divided by the area of the filter cake, to give the velocity with which a mass
of water penetrate-, the cake. From this figure, we may obtain the tine of con-
tact of any particle with the cake (Tc) by dividing it into the thickncss of
the cake. It will be noted that this time of contact increases as the filtra-
tion pioceeds wder any given pressure of air in the reservoir. This may be
accoilnted for by mechanical compression of the cake during filtration, or by
the stoppage of pores due to a.ccmulation of microscopic debris. Correlation
of this tine of contact with chenical or bacteriological assay of the filtrates has not as yet been fruitful, but we havc continued to gather these data ageinst the tiso when the latter determinations become more standardized.
In this table WE can also note the typical characteristios of Asbo- loxane filtration. The fixt filtrates are essentially sterile, and then a point arrives w!:an small numbers of organisms begin to cone through the filter.
In no case does the number of organisms ever i.ec.ome exceedingly great, dw, probably, to.mechanica1, rether than chemical effects from this point forvrard.
The volune of fi1tra.to which contained no more t.han one or two vipble bacteria per nl. has been reported as ttvolume sterilized," and 1s used PS the basis of
Coqarison. . .I TABLE XI11 ASBOLOXANE S-FILTER PO9t3ER JJ LABOWTORY PRESSURE FILTER APPARATUS Typical Pata Sheet
Test 26 Asboloxace S-Filter Powder 1.0 gm
Vol. Water 3500 ml. Cake Thickness 0.070 cm Inoculum 20 ml coli F Broth Plated F Agar Incubated 48 hrs. Vol. of Samples 200 ml Control: 220,00c org/d at start of test, 18?,OOC org/ml at end of test.
Titer Sample Time Rate Velocity Tc ml N XgO2 Bac teria/ml No. sec ml/sec cm/sec sec thio/100/ml 100 ml aO, Filtrate
1 123 1.63 0.129 0.54 7.57 1.71 0.70 0 2 138 1.45 .115 ,61 - - - 1 3 135 1.48 .118 59 - - " 1 4 140 1.43 .113 .62 7.57 1.71 .70 0 5 143 1.40 ,111 -63 - - - 0 6 149 1.34 ,lob 66 - - 0
7 152 1.32. .lo5 -67 6.45 .44 0 8 158 1.27 .lo1 .69 - - 2 9 163 1.23 .098 -71 - - 5 10 165 1.21 .096 -73 5.72 e28 5 11 168 1.19 094 .74 - - 8 12 172 1.16 .092 .76 - 10 13 180 1.11 ,088 .79 5.20 .16 6 14 181 1.10 .om .80 - - 9 15 185 1.08 .086 -81 - - 9 Control - - - - 4.47 - -
- 51 - Thickness of Cake and Contact-_ Time: From runs of this type in the laboratory pressure filtcr aLqaratus, conditions closely approximating
action in the S-filter could be set up and variables, such as thickness of
the filter cake, could be studied under closely controlled conditions. In
the S-filter. 36 to 40 grams of S-filter powder are spread on approximately
450 square Centimeters of surface, or roughly one gram to 11-12.5 cm 2 of screen
surface. The Seitz filter use& in thi: laboratory pressure filtration apparatus
had an inside area of 12.6 centimeters, so the use of one gram of powder very
closely approximated conditions in the pump. A flow from the S-filter of two
liters per minute is equivalent to 50 ml/min/gram %filter powder, or .e4 nil/
second pcr gram. In the tests run, the flow rate could be controlled by vary-
ing thc pressure on the reservoir. In most runs, the ilverage was milintained
very close to .9 ml/sec/g. Variations in flow rate from 2.0 to .5 ml/sec/g.,
however, were not found to be the primary muse of varimtion in sterilizing
effectiveness. This factor, nevertheless, needs further study. The weight
or thickness of the cake, and hence contact time, does have a decided effect
upon sterilizing effectiveness, however, as the relatively few data collected
in Tablc XIV indicate.
Tests -4gainst Various Types of Organisms: Using the methods de-
scribed previously, a series of tests against various organisms in water sus- pension was conducted. In a11 cases the organisms were grown in nutrient broth,
centrifuged, end added to distilled water. One gm of powder was used in all
tests. In testing the T2 coli bacteriophage, it was necessmy to suspend the phage particles in Friedlein's synthetic broth rather thar? in water. Table
XV presents R summary of results obtained in this series of tests.
- 52 - TAELE XIV
ASBGLOXANE 3-FILTER PO~DLRIN IABOFATORY PRESSUR3 FILTER AZTkRkTUS
Conparison of Filter Effectiveness with Cake Thickness
Grams Cake thick- Average Volume of E. coli Suspension Run No. S-Powder cess - cm. Tc - sec. Filtered Sterilized
28 0.25 .021 -25 3000 10
.a 0.50 .a27 e29 2600 50
29 1.0 .070 .64 3000 2000
31 1.0 .r112 -69 300@ 1600
20 2.0 ,114 2.49 2600 2600
21 2.0 .173 3.73 2400 2400
- 53 - TABLE XV
ASBOLODNE S-FILTER PCWDER IN JAaOAkTOFE PRESSW FILTER A 'PAGTUS
Sterilizing Action Against Various Types of Crganisms
V0l.X Test Orig. Conc. Sterile Contaminant No. org./ml. Zffluent
E. coli - 37 400,000 2800 S. aureus 38 250,000 2400
S. aureus 70 2,800,000 2800
B. globigii (veg.) 39 50,000 2600
E. globigii (spore) 40 45,000 2800
B. globigii (spore) 47 200,000 2800
B. globigii jspcre) 43 13,500,000 0
M. phlei 42 45@,000 2800 I,!. I,!. phlei 44 1,250,000 2800 K. pneumoniae L5 570,000 2800
E. typhosa 68 118,000 1400
E. tyFhosa 78 450,000 2800
E. typhosa 79 3,000,000 2300
S. marcescens 73 650,000 2000
8. marcescens 71 2,500,000 1600
G. tetragena 72 86,000 2800
E. coli T2 Phage 76 4,000,000 0
* 2800 ml. was the total amount filtered in all cases.
- 611 - In evaluating these aata it is iaporttid r;u rsiL=abar tkat WL hrc
r?ealing with a system which stands to tho S-filter in tho ratio 1:40. There-
fore, each 200 ml. of filtrate collected in this test is equivrlent to eight
liters of filtrate from the S-filter. The rat-d safe cqacity of the pump is
50 or 60 liters, so thet 1250 or 1500 ml. of sterile effluent from the labora-
tory mo8.el mry be considered a sterile run in the pump. On the other hand,
these tcsts were carefully conducted in the absence of organic matter, so they
represent conditions which are rather idoal.
Storage of Asboloxane Powder: Stompered ;.nd open vials were filled
with one gram samples of Asboloxane S-filter powder and exposed to different
conditions of temperature by being stored RE follcws: in a dry ice chest (-600~):
in a cold room (5O); in a laboratory (25'); in m incubator (37OC); and in a
wator bath (45'). Water containing waslled E. coli was filtered through one
grm c'&es of these powders efter six months of storrse at the various conditions.
aesults are reported in Table XVI.
Effect of Various idaterials added to the Suspension: In an erirly
attempt to rule out silver ion P.S the antibacterial ngent, a. solution of sodium
chloride (1.25 per cent) was used as suspending nedium for 3. coli. Three
liters of this solution werc filtered through one gram of Asboloxane - all the filtrates were storiie. Leter work, of course, indicated that the silver present
is much below the solubility product of silver chloride, and so this experiment
did not at all rule out the possibility that Ionic silver was the active agent.
In a.nother test,, cysteine hydrochloride was rd.ded to distilled water
in a coccentration sufficient to give a titer of zero when the solution was as- sayed for dissolved oxygen by Winkler's alkpline mangenous hydroxide method.. It was of interest to see whether the strong reducirg pmpcrties of such a solution - 55 - TABLE XVI
ASBOLOXhNE $-FILTER PCVJER IN LABChTCBX PUSUELE FILTZR Ar’PAMTUS
Surveillance of Stored Powders After Six Months Storage
Temperature Original c oncer,tration, VolmeK Vial OC E. coli/d water Sterile Effluent
Stoppered -60 470,030 1700 men 500,000 800 Stoppered 5 1,600,000 i800 wen 6,000 2800 Open 600,000 2800
Stoppered 25 1,000,000 2800 men 500,000 2800 Stop3ered 37 250,000 2000 t@en L60, OCO 2300
Stoppered 45 560,000 2800 open 550,000 2600 x 2600 ml was the total volume filtered in all cases,
NDTZ: It is possible that samples stored in the dry ice chest were affected by the large amount of C02 in the atmosphere. The effect of very low temperatures upon storage should certainly be studied further.
- 56 - interfered with the death of E. coli by filtration through Asboloxene, In
this test a few contaminmts appeared, but essentielly all of three liters of
E. coli suspension were sterilized by pnssage through one grum of Asboloxsae.
It is interesting that in the test the value of ,302 for the first filtrrte
was almost that of a normal water filtration, but this value dropped exceed-
ingly repidly, until there YRS very little determinable oxygen after one liter
had passed tke filter. The reducing conditions of the water did not aljparently
affect the effectiveness of the sterilization.
A detrimental effect on sterilizing cqxicity is noted, however, when nutrient broth is Rdded to the water. Normally Friedlein's synthetic nedim was used tG grow the E. coli, and 20 ml. of this culture medium was added to 3530 ml. of wrter. However, when nutrient broth W~SPdded, fer less water was sterilized. To show this point, and to rule out the possibility that bacterin. gmwn in differcnt mEdin might have different resistances, or that the prosucts of their growth were interfering with the action of Asboloxnne, the tests described in TRble XVII were performed. Tiro gams of filter powder were used in ench test.
It appears obvious that it is the nutrient broth, and not factors of rosistmce or of metabolic end-products, which gives rise to the observed differences in effectiveness of the Asboloxnne filtration process.
The same nntP.gonism of nutrient broth for some bectericidal factor cont8ined in the filtrpte is evident when dilutions are na8.o in nutrient and in Friedlein's agar. The Friedlein's plates me likely to be ell sterile, while nutrient agar plates my show growth, due to the reversal of bacterio- stasis by the components of the nutrient broth. It is thus important that test ncthods be standardized as to media used, nnd RS to time of incubation. if com>e.rnble- results are to be obtained. - 57 - 7--, ;;- ~..,i. ,.. ,.. 9 ..~, _-.-.~ ^x - w A UJ TABLE XFII
ASBOL0XANE S-FILTLR FOXDER IM LABOUTCRY FRESSURE
FILTLR APTARATUS
Effect of Nutrient Broth OR Asboloxane Filtrations
Additional Original Volume of water E. coli Material added canc. Filtered Sterilized Run No, grow. in Treatment to water E. coli,/d ml. ml.
19 20 ml. N* Added t.o 3500 None 600,000 2400 800 ml. water ++ 21 20 ml. F 11 None 215,000 2400 2400
23 20 ml. F II 20 ml. N 270,000 3200 800 (sterile)
24 20 ml. N Centrifuged, None 285,000 2000 1800 twice washed, added to 3500 ml. water
Q N = Nutrient broth F = Friedleints synthetic medium Antibiotic Potentiel: Kention ha.s been nade in previous sections
that the Asboloxsne filtrptes pppeared to be bacteriostatic. Dr. Goetz had re-
ported this interesting phenolenon end called it "Antibiotic Potential." By
passing various suspmsions through Asboloxane S-filter powder cnkes in the
labor?tory pressure filter Ppparatus, and re-inoculating portions of this fil-
trate with different anounts and types of bacteria, the existence of this mti- biotic potential has definitely been established. E. coli, E. typhose, S.
aurous, S. marcescens and G. tetragenn have all been observed to die off more
rapidly in Asboloxane filtrates then they do in the sNne water before filtra-
tion. This killing action is not too rapid, and when relptively heavy inocula
are adted to the filtrete and aliquots exmined for viable bpcteria at periodic
intervals up to 3 hours, the killing is often not yet complete. -*v .e ,' Comparison of the effectiveness of this Rntibiotic potential can be nade however
by comprripg .under different circwstpnces the velocity constant or death rate,
k, which is often used for corparing vnrious b?.ctericidal effects. This con-
stant, k, cones from the expression for ,- first-order reaction, log XOB = kt, where No is the original bpcterid concentration, N is the concentration after
any contact time, t, and k is the velocity constant or death rntc. The bacteria
die off in a 1oge.rithnic order iii the Asboloxane filtrR.tt!s as in so nrny CPSOS
and thus obey the pbove expression. When the logRrithxI of the concentration
(Log W) is plotted sgainst t, x strright line cm be drawn through the points annd the velocity constmt k can be calculated by determining the slope of that line. It is customarily mid that k is a numericd c'onstant ha.ving nc exact meaning, but tha.t it becoues krger in value a.s the death rate becomes faster.
ActuFlly k has the dinensional units of time -', and it is nunerically the re- ciprocal of the time required to kill 50 per cent of the organisms in any given - 59 - suspension, since in this special case Log becomes 1 and k T1 in the equetion given above.
A typical exanple of the fdl in concentrution of E. coli when eddod in two different mounts to an Asboloxn,ne filtrate is given in Table
XVIII. The bacteriostatic action is hard to conpnre in these two cases be- cause of the difference in original concentration. ?he death rates however
(.078 ns against .G56) tell us that the bactcrip aro dying off faster in the lower inoculr. where 90 per cent of the orgpnisms die in just over 10 ninutes
on nn nveroge as agsinst just under 13 ninutes.
Usingthese denth rete figures,it w2s possible to compere the ef-
fect of various fnctors such as orgmic or other material present in the water
before it wns filtered, and of the number of contnninnting organisms ndded to
the filtrete RS is done in Ta.ble XIX.
Our datp., to date, nade b:r conpnring these death rates, have in-
dicated thpt the nntibictic potentiel is greptest in the first portions of
suspensions passed through Asboloxme S-filter powder than in later portions,
although it is present even towards the end of the filtrntion. Also as is
evident in Table XIX referred to above, the dcath rxts falls off as the number
of orgnisns added to the filtrate is increhsed.
The litervture is filled with similar observadions, pnd silver, as
a sterilizing agent, shows this relationship to P msrked d.egree (1). This
factor must be borne in mind in conpnring the results of different investiga-
tions.
Another conclusion which is rea.dily nppnrent from these date is
that pure wn.ter, passing through the Asboloxane filter cnko, acquires considerabl.
(1) "Abstrscts of Articles on Oligodynnnic Sterilization" - The.. Engineer 3oard. Project WS 766, 1 May 1247 -60- TABLE XVIII
DEATH RATE OF E. COLI IN FILTSTi FROM ASBOLOXIWE S-FILTER FGWD'iR
Bac teria/ml. Time of 0.5 ml. 0.005 ml. Contact E. coli inocula E. coli inocula
0 min. 5,220,000 160,000
15 min. 700,000 4,400
30 min. 7,800 246
60 min. 800 6
120 min. 35 0
~ ~~ k (death rate) .078 .096
- 61 - ?
TABLE XM
DUTH PATS CF E. COLI IN VARIOUS
ASBOLCWZ FILTRATES
Filtrate from water Organisms added per criginally containing ml. filt.rate -k* 3 ml. mtrient broth - 1.5 x LO56 030 3. coli 1.8 x 10 ,036 2.1 x 16. .048
E. coli only 2.1 x 106 .Ob2 3.5 105 .om 5.7 104 .120 6 Nothing 3.6 x 105 093 3.4 x lo4 ,126 2.4 x 10 ,138 * k deat.h rate of added orzanisms. The greater the number, the faster is the rate of death of the added bacteria.
- 62 - inore ontibiotic potent ia,l than docs water which originally contained washed
3. coli orgacisns. The differencc is still gre?ter when the wter originally contained broth as well as well as 3. coli. These rc?sults pro not in accordmce with tke findings of Dr. Goctz, who reported that the entibiotic potential of
Asboloxane filtrates wos greater if viable orgnnisms hrd been present in the water before it w*s filtered. Our findings, confirned in nany experinents utilizing both E. coli ard S. aureus to re-inoculate the filtrate, show thnt any orgeiiic natter present in the weter as it is being filtered, whether viable or not, results in less antibiotic potentid in the filtrate. Although many at)te:.gts have been made to elucidete the exact nature of this antibiotic potentiel, it is still IIJ~unexplained factor.
Its iqortrnce, however, na,y be considernble, for wrter sterilized with the Asboloxrne 3-filter pump idill rebin self-sterilizing pro?erties for a considerrble length of tize. Thus the usud precnutions for rseptic hnndlirg of drinking supplies may possibly be dispensed with to a certain extent.
d. Various Chemical and 3iological Properties of the Filtrates from
Asboloxane S-Filter Powder
In this section are reported various tests conducted at Canp Detrick on Asboloxane S-filter powder and the filtrates obtaineA from it. These tests are largely exploratory or fact-finding in nature, and describe some of the char4es which occur both in the S-filter powder itself, the liquids which are passed through it, and the organisms which have been exposed to Asboloxane ac- tion. Certainly no definitive explanation can be given at t%istime as to how or why microorganisms are killed b)- Silver Asboloxanes, or by any other silver complexes. ?he tests do give, hovever, a little insight into a few of the chemical and biologicel forces at action in Asboloxnne sterilization.
- 63 - !
-Silver ?elationships: Chemical analysis for sil-er was mnd.e on
Asboloxane S-filter powder. Also, spectrographic analyses were mde of the
porcder and of the dried residue from an Asboloxane filtrate. The data SO ob-
tained are in good agreement with thio data for the composition of the powder,
f~xnishedlater by Dr. Goetz, save for the fact that no zinc was reported in
the spectrographic data. The figures are given together in Table XX. Routine tests for silver were performed on .r:any of tha filtrates
in the laboratory pressure filtration tests with the Asboloxane S-filter powder.
The method used was the cupric dithizonate method (1) which detects as little
as 5./liter. TpyIJical bta ObtailJed are presented in Table ?XI. Silver c?oes
a?pear in the filtrates, but in nuantitips far below the solubility of silver
chloride. Spectr0::raphic data also have shown that, when Asbolo>-anc S-filter povder is deposited on hardened agar plotes, small amounts of silver diffuse oilt into the agar.
It apears that silver is still present towards thn c-'nd of the fil- trations, where viable organism often begin to show cp in the filtrates. More- over, ionic silver at a concentration several times PS great as the silver con- centrations usually found in the filtrate, is but slowly toxic to 3. coli RS measured in experiments conducted iz this 1a.boratory and shown in Table XXII.
For thcse reasons, attempts thus far to correlats the data on silver in the filtratLs with killinpi action of Silvcr Asboloxane have been unsuccessful.
It is intcrcsting to note in t:,is connection that very little silver
(5)lliter or less) a;>pears in the water in canteen trials where the special As- boloxanc Canteen chargc is added to the rrater,and is in contact with it for 15 rinutes bcfore filtering.
(1) E. 3. Sandell, "Colorimetric Determinntion of Trnces of b!etals" - Inter- Science Publishers Inc., New York, N.Y. (1944) p. 399. - E4 - TABLE XX
COWEITION OF ~LSBOLOUKE5-FILTER POhDZR
Chemical Test for Silver
Asboloxane S-Filter Powder Ap7rcx. 0.5% silver
Spectrographic Data (Combined for both Asboloxane %Filter Poxder and residue from Filtrate)
Xajor Elements Ca, Si, Xg Ti+:* Yinor and Trace Elements Al, Fe, Na, Ag, Cu':', y Reportad only for Filtrate Residue Mt Reported only for bsboloxane S-powder
Composition of Asboloxane S-filter Powder as Furnished by Dr. Goetz
% by Ingredient Dry ;;eight
Diatomaceous Earth 76.4 Ca02 . nH20 + Ca(GH)2 13.2 ZnO (U.S.?.) 1.9 Colloidal Carbon (Black) 2.9
Ag (as kg20) (U.S.P.) 0.5
hsboloxane 5.3
Polyvinyl Compds. (dry) 2 .? 0.4 Detergent (wet) - Total 100.0 % IISBOLOXMiZ S-FILTLR POWDER IN LKBORATORY
PREs3Um FILTER B??AFM”l’S
Urrount of Silver Appearing’in Filtrate ,’ (Filtrstes dige.sted with H2S0,+)
Liquid Being Filtered Volume Distilled passed T~F water. Distilied Tap throuch Water washed coli Tap water + water t Filter Cake Distilled (Average (Average xater + nutrient nutrient (d) Water of 2) Gf 2) washed coli broth broth Silver in Filtrate in Y/Liter
200
400 5 50 10 25 100 150 600 65
800
1000 5 60 17 80 70 90 1200 17
1400 30 75 , L5 45 1600
1802 10 50 17 65 35 25 2000
22m 20 L5 15 60 20 30 2fb00
2600 20 12 50 10 20
2800 30
Control 0 3 5 0 0 3 NOTL: For Comparison - Solubility P.gC1 in Water - 1,50Op/L or 1,130 dLof Silver Sol-ability Agar in Vater - l30\//L or 75 V/L of Silver
- A6 - .~ 3ACT~ICIEALACTION OF AgN03 AGAINST Z. C@LI
(Concentration of Silver 104 Y/Liter)
Contact Clrganisms/ml at Various pH Values Times PH 3.42 PH 6.35 pH 9.85
0 rnin. 190,000 30,500 14,100
5 min. 10,700 10,800 375
10 min. 4, 800 3,100 101
15 min, 1,850 920 35 30 mix. 260 172 2
60 nin, 4 28 0
120 min. 0 2 0
120 control” 2,900 25,600 11,100
‘e In contact with Buffer only. No AgN03. Experimental Coizditions:
1 ml of 0.00005 N 1 ml of 8. coli Suspension Mded to 50 ml Vhrious Buffers. kliquots removed for viable comt at indicated tinles. Tests mn at room temperature. ri7-r.. Cxvgen_L Rel*yio&hips: Tie Asbplg-xdd-ilter powder contains both a silver catalyst and an insoluble peroxide (Ca02). In the course of filtration the peroxide is consumed, and the filter powder loses effective- ness. It is therefore interesting to try to trace what is happening to the peroxi6.e originally present in the powder when it comes in contact with water.
One possibility is that the silver could catalyze the decolnposition of the calcium peroxide to oxygen and calcium hydroxide according to the fol- lowing equation:
Ca02-gE20 ->Ca(GH)2 + &)2 + 7H20 (216.2) (74.2) (16) (126)
Actually some gas is given off when the powder becomes vet. 3- periments were conducted in the Vrrburg respirometer to collect end analyze this gas. As the data in Table XXIII indicate, the gas is oxygen since it is adsorbed by pyrogallol and not by elhli. In 30 minutes shilking with water,
0.32 milliliters or 0.42 milligrnns of oiTgen ere given off by one gram of As- boloxane S-filt er poi-idcr.
Some simple calcula,tions cm show the: this ?mount of gaseous oxy- gen is not enough to account for all of the peroxide originally present in the filter powder or extrscted by the wter. For emniple tbc equivdent weight of calcium peroxide, according to the equation above, is 0.42 x 216/1,6 = 5.69 mg. of calcium peroxide per gram of Asboloxme. Thus the oxygen liberated -ccount? for only pbout 0.6 Ter cent CaG2 in the originol Asboloxane composition.
In anothw experimpnt ? smple of Asboloxmo S-filter uowder wos carcfally .weighed and then extracted with 100 ml. of water. The extmcted pcwLcr WEIS dried to a constant weight in R v~cuumdesiccator for two d~nys, and tho filtrate was dried frozen under high vacuum in a lyophilizer. The drying - 68 - (iiilliliters of Gas per gram of Powder after 30 minutes)
Collected in the presence of: Water- 20% KOH Pyrogallol .25 -42 0
.43 .27 0
.37 .22 0
.29 - - - Average .34 -30 0 ~ processes were such that no decomposition due to heat should teke place. The
not loss in weight wn8 0.017 grems per grem of Asboloxane S-filter powder. If
one assumes that this non-recovernble weight is due to oxygen and. woter, formed according to the equation above, then 0.017 x 216/142 or .026 grms of Cdr
must be decomposing per grm of filter powder. This figure is about five times
as high es that obtnined in the Varburg experiment.
If analyses are run on the Asboloxane filtretes, hovever, oxygen
compo-unds Ere found in the filtrate. The method used in this lnboratory to
aetermine these compounds vas that of Wfnlrler (1) for total dissolved oxygen
in water. Peroxides are also determined by this method, so that the values ob-
tsined for ''Total Oxygenll in the filtrates represent the total of oxygen gas
and soluble proxy type compounds. When the control values, representing the
amount cf dissolved oxygen prescnt in the water 5efore filtration, .?re subtracted, one obteins values for -102, or for oxygen and peroxy compounds nddpd to the water in the course of filtra.tion. The values for total oxygen md for ;.02 as obtained in this way when 2gOO ml. of distilled or tap water nre filtered throuph gne &ran of Asboloxnne are given in Figures 6 and 9.
The values for 202 must largely represent peroxides of some sort, sirce the water usually is nearly saturated with oxygen before passage through the filter cake, and any oxygen 1iber;rted as such would mainly be lost to the atmosphere before the samples could be titrnted. Dr. Goetz ~PSrmorted to US that he has used acid permangmate solutions to titrpte peroxides in Asboloxme filtrates. When this method is used, curves very similar but slightly lower than the do2 curves are obtained, as is also shown in Figures 8 and 9 where data are presented for both methods used on the same filtmte. For comparative
~uq:cscs, a1.1. -~:l~iesir, these figures are given PS hydrogen peroxide, although it --is __doubtful that this is the actual compound formed. (1) Mathad ei-ren in most analytical texts such as Treabell-Hall 'lknalytical ChamiStPY.n Manganous hydroxir?e is oxidized by oxygen to manganeze dioxiao, which i5 than d.zterdr,e:?. iotcmtIics:1.::. - 70 - hAU5 FIGURENO. %
From thesc graphs, by swing up thc nrw under the ,302 curves,
one cun calculate the amount of pcroxy type compound added to the water during
filtration. This amounts to 0.02h grans of iI202, or its equivalent in oxygen or other peroxides, for the distilled wter curve, and to 0.0210 grams of H202
in the figure for the tap water filtration. ?he former figuro is equivalent
to that which would be formed by the 8.ecomposition of 0.170 grams of calcim
peroxide per gram of filter powder. ?he latter is ecpivdent to 0.134 grams
of the calcium .peroxide pcr grm of filter powder.
When these figuros are cocrpnred with thoso appearing in Te.ble XBI for the per cent cmposition of As'coloxane S-filter :Jowder (13.2 pcr cent cs-lciun poroxide as stated by the manufpcturer), it is apparent th.at dlof the cdciun peroxilc must be decomposed during\ the c0.urs.e of filtration. The values for ,,: 0: obtained by the h'inklcr nethod indice.te a little more calcium peroxide than Ch;t.t
stated. The values for peroxides as determined by the pcrmanganate nethod would
indicate a little less calcium peroxide in the powdpr than this sta.ted amount.
Almost all of this calcium peroxifie (nbout 13 uer cent) which is decomposing appears as peroxy compounds in the filtrflte, since thnt which decomposes to form gasso& oxygen is very little (about 0.6 per cent in the Warburg test), and thnt which goes to form compounds which are lost when the products are dried in the absencc of heat is not vcry ge-t (about 2.5 2er cent in the loss of weight test).
The low figure in the loss of weight test indicates that the SOhbblQ peroxy coapound in the fi1tra.t.e is not hydrogen peroxide. ?here nre other good reasons for believing thrt it is some other compound or co.3pounds. The comm qualitative tests for hydrogcn peroxide ?re negative for the Asboloxene filtrates,
(Table XXIV) . Moreover, when hydrogcn peroxide and sodium carbonnte are odded to distilled wa.ter in mounts large enough to produce R -total oxj.gcn value Sust - 73 - - TLSTS FOR IiYilROWi PEROXIDE IN FILTRkTLS
FROM ASBOLOXdfZ S-FILTL’R POWDB
Sensitivity Result, if Reagent KdLiter Peroxide present Observed
CO(NO~)~(alkaline) - Black ppt. Nothing
FeC13 + K2Fe ( CW)6 0.02 Green 4- blue ppt. Hothing K2Cr0 etner (acid) c.1 Blue ether layer Nothing 4 -+ KI+ starch (acid) 0.05 Blue color immediately Blue, slight, slow
- 74 - under that ottgined for the 8sboloxane~~~~~~~.tes,the kiiling'e.ct~a h .. is con- siderably grtater than thpt of the filtr>.tes, ns shown in Table m. Expressed in another way, the mtibiotic potential of the Asboloxme filtrates is less than that of hydrogen wr0xifi.e solutions of equivalent or nlizhtly lower strength.
The exact composition of the p,roxy substance or substances inparted to the fil- trate by the Asbolmnne S-filter powder is not evident from the work done in this leboratory to dfite.
The earlier work done here iadica-ted thrt there m~ybe some correl- ation between the amount of these conpounds appenring in the filtrrte, find the sterilizing action obtained. In :&le XXVI appear data obtrined fron earlier runs giving the :!I03- values found in the lpst portion of the filtrate which cmc tFzou&h sterile. In the errlier ~crtionsof these filtrates the C2 values were higher, and the fil:rfi.tes pll sterile. ,In the later portions,
vie.ble organisms began to ~7 pear in the filtrates Rnd the D2 values were lower. It war; hoped that it night be possible to USE these data to ade.pt a chaicel test for evnlueting the effectiveness of Asboloxane filtrptions by sinply 'checking the filtrate to see if the d 02, or peroxy titer, WRB above a certain level. This value differs, however, according to tne aEount of OrgWiC netmi81 present, as is shorn in Table XXYI reforred to above. Moreover, it is possible to achieve sterility even ishen no d 02 is evident in the filtrate
8,s in tke run described on ppge 55 in which cysteine hydrochloride wns present.
Tor naturnlly occurring waters it still mry ?e possible to set uu such a control test for sterility.
sffect on Zpctcrirl Enzpme Systems: The effect of the Silver As- boloxeiies on vnrious ba.cteri;..l enzyme 6yste.w h~sbeen studied, since it is by attnck on these sensitive components of the cell that most disinfectants work.
- 75 - TABLE XXV
AND HYDROGEN PEROXIDE SOLUTION
(ksboloxane Filtrate - 1200 ml thru 1 gram asbcloxane S-Filter Powder) (Teroxide Solution = 0.47 ml 3% H2C2 +- 5 g Wa2C03 to 1 Liter water)
Heavy E. coli Suspension Orga?isms/ml After Added To 1 min. 5 min. 10 nin. 30 min. 60 min.
Distilled Water 4.5 51 4.4 M 3.9 M 4,g hi 4.4 M ksbolcxane Filtrate 4.3 M 0.8 M 3.6 N 1.2 M 58 T Peroxide Solution 15 9 8 8 7
Total O2 and peroxides ksboloxane Filtrate 0.3030 gi100 mi (Expressed as H202) Peroxide Solution 0.3026 g/lGG ml
Light E. coli Suspension Crganisrnsjml After Added to 1 min. 5 min. IC min. 30 min. I Distilled Water 12,300 10,700 8,850 6,753 Asboloxane Filtrate 7,500 7,300 5,650 3,350
Peroxide Solution 67 r) 0 0
Total 0 and peroxides Asbolcxane Filtrate 0.0029 dl00 ml (Exprzssed as H2C2) Peroxide Solution 0,0026 dl00 ml
- 76 - -.-- kSBOLOXAN3 S-FILTXt IF Llli?OF&TOHY PFLSSURZ FILTSR !-WARATUS
Lowest Value of i>O2 Accompanying 4terile Filtrates of E. coli Suspensions
Frisdlein Broth Suspension Nutrient Broth Suspension Run No. 602 (minimal) Run No. ~02(minimal) - (mg. 02/’100 ml.) (mg. 02/100 ml.)
14 .26 15 . .74 21 .37 17 .7 5
24 a30 19 .58
25 t 29 20 * 59
26 * 41 23 .80
29 .24 27 .7 5 31 a32 28 .54 -- - Average .31 +- .04 Average .68 +- .09 - A technioue of treating organism to varying dosages of Asboloxanc S-filter
powder WPS evolved in which pour platcs of hardened nutrisnt Rgar was prepared
rrnd the surface uniformly seeded with a. suspension of the organism chosen for
study. In the center of the plate about half a grem of Asboloxanr: S-filter powdor VPS pleced,and the plate incubted. The bacteria are thus subjected to
succcssively diminishing dosrges the frrther away thy arc fron the solid powder
in the Liiddle of the plate. Che.yica1 tests hove shown that silver, peroxides, and alkali all diffuse out from the S-filter powder into the ner.rby &gar.
??.e orgmisms farthest away, neer the ecge of the Petri dish, grow norndly. Usually those in the area next to Asboloxane S-filter powder fRil to grow e.t nll, vnd P cle-r zone of i&ii?ition in which no colonies develop en- circles the filter poi,rder. These zones of no growth, and. of normrl prorrth, do not 'rerge iziperceptibly, RS one Digkt iwgine. Insted, with S. Pureus st lea&, the zone of inhibition sto2s obru;jtly vnd thvre is P norrow band just around it wherc the growth of the colonies is ?.ore vigorous thm in the rreR of nornal grcmth behind it. This is rnot.:er exand? of contact with sub-lethnl concen- tretions of F toxic substrnce cnusizg a stinula.tion i3 growth, as has been noted in so Wny fields of biology. The tests swrrizeti in Table lixvII Were performed on orgabisns which survived P. sub-lethrl dosnge of Asboloxa.ne S-filter powder.
These organiscs were collected fron the zoze of enhanced growth or stirnuletion just next to the lethal zone on the plate. ?he ectivitifs of the enzyne systms in these prrticulrr organisms me conp?rrec? with those of nor.m.1 unexposed or- ga.nis.?ls.
At the present stP.tc of the investigation, no theory cen be postu- lated as to the rxchp.nim of thc Askoloxone mtion bnsod on these enzyme studies.
It is evident, however, that with S. cureus nt least. n wid6 range of enzyne system arc rffected RS the orgp.nis.?i attenpts to overcone the Asboloxme rction. - 7R - TABLE XXVII
EFTECT OF SUE GTIUL DOSAGS OF ASBOLOUNE S-FILTER POWDER UPCN ZNZYXk OF STAPH. AUREUS kctivity in Enzyme How Measured hsboloxane Treated Organisms
Coagulase Clotting of human Increased blood plasma
Catalase Ability to decom- , Increased* pose H202
Gelatin Liquefaction Stab puncture in No effect Gelatin tube
Hemolysin Action against defi- Decreased* brinated rabbit blood cells
Succinic Ehydro genase Ability to oxidize Decreased substrate - dye indicator
+$ Thioglycollic acid does not inhibit this action. Its ability to produce CPtdRse, nnd hence destroy peroxides, is enhanced md
so is its maduction of corgulnse. On the other hmd, tho proriuction of pro-
teolytic enzymes rrhich cRn liquefy gelrtin are not affected. The succinic de-
hydrogenP.se, which is used by the organism for metnbolisn in the Pbsence of
oxygen, is produced in lessened mount; ?.gain indicating tht the orgpnisn is
adapting itself to nn envirorinent of increasing oxygen supply.
Sulfhydryl conpounds, which me prok-blg contained in many of the
enzynes, often hrve an effect on the Bsboloxflne action. Yith S. aureus for
exanple, no zone of inhibition rpwrrs about the Asboloxane poder in the r%ove
described technique, if :li~rcaptoethrnol or cysteine h-ydrochloride is present in
the Pger. The orgcniszs grow u:Y to the very edge of thc AsboloxFno deposit.
Thioglycollic ncid, however, does not hrve this power of ovcrconing the Asbo-
loxrile action agPinst S. nureus.
When E. coli is seeded on cutrient agar pla.tes, the zone of inhib-
ition ebout the Asbolomne S-filter powdar which one observes with S. aureus
does not apmrr. On Friedlein's coqlftely synthetic agm, however, the zone
of total inhibition mc' zone of incremed growth do apperr. Ey ndding the various conyonents of nutrient agar to the Fricdloin's .?gar, it can be shown that t?e beef extract, en&, to P. lesser degree, the yeast in tke forrner ennble
E. coli to overcome the Asboloxme toxic nction. ::?e sulfhyckyl conpound, thio- glycollic ?cia, likewise neutrnliacs this action. Cystcine hydrochloride arid nercnptoethmol do not rct as neutralizers, plthough the57 both contain the saw reactive SI group.
These exFrinrnts with various neutrnliring agents point out ouite strongly the difference in enzyrie systais of different bpctorial soecies, md
Imphasize how difficult it is to bnse R tbeory PS to *iec5rnism of nction of a aisinfectent on tests with only one orgnnisn. - 80 - D. summy
The Silver Aslloloxanes have been described. Two pieces of apparatus, a canteen with a. special filter head, end the S-filter, designed es militc.ry items to be used in conjunction with the Silver Asboloxe~nesto sterilize water on a small scale, have keen described. The eqeriments of Dr. Goetz in which these pieces of equipment were given field trials in Mexico, to determine their ef- fectiveness in removing water-borne organisms from surface waters, are quoted in full.
Results of laboratcry trials conducted at Camp Detrick on these items are given in stme detail. These trials have in general centered aromd their per- formance in the presence cf very high concentrations of organisms chosen to represent various typical grcups of bacteria. Chemicnl tests and biological tests w1:ioh shed some light on the mechanism of action of the Asboloxanes are reported in lesser detail.
Zriefly, the findings from the Camp 3etrick trials may be summarized as follows:
The use of the Asbolorane pcwders, in a canteen or in the S-filter cnn pro- vide a o_uiok, convenient, reliable method of obtaining small scde supplies of sterile drinking weter. The canteen po-der is intended for the treatment of individual supplies of water, while the pump may be used to deliver about 100 liters of sterile water per hour.
Experiments at Camp 3etrick have indicated that both types of Asboloxane powder, when used in the prescribed method and apparatus, can sterilize mter ous2msions of bactvrie. of concentrations as high as 3 million viable organ- isms per ml. Organisms shown to be susceptible to the action of the Asboloxanes hclude Xscherichia coli, Staphylococcus nureus, F!L:bsi,-.lla pnewnoniae, Eberthell?
- gl - tnkosa, Mycobacterium phlei, Serratia marcesceno, Gafflva tetragena, and
Bacillus globigii. Tests against bacteriophage hwe indicated that these
organisms are more resistant to the Asboloxanes.
Our most intensive work has been done on the 5-filter and povder. This aFpears to be remarkably effective in removing high concentrations of bacteria from water. It appenrs that a considerable proportion of the becteria are removad mechanicaliy, tat this filtration effect is not the significant part of the action of the S-filter. The Asboloxane powders actually kill bacteria
(or renC.er them incapable of growth) 7,rhile the watw containing t,he.n is in con- tact with the filter cake.
Water treated by the mparatus under consideration is c1ea.r and perfectly potable. It has, however, a rat.1ic.r high pH (9-10) and a considerable degree of temporary hrdness (Ca7*). The efficiency of the S-filter can be improved by usin, more powder, or by repeated treatment of the water, thou&h these pro- cedures would not seem to be necessary under oriinary circmstances, since viable oreanisms agpear in the filtrates only when initia.1 concentrations are about 106 orgenisms/ml. The filter powder is still effective sfter storage for six months in sealed containers at temperatures between 0 0 and 45OC.
The nresence of nutrient broth and, presumably, of other protein materials has a detrimental effect on the sterilizing capacity of S-filter powder. This interfering nction of nutrient broth extenns a.lso to a diminution of the self- sterilizing properties or anti?iotic potential of Asboloxane filtrates.
This self-sterilizing capacity, which is apparently of long duration, may be of considerable practical importance in preventin,: the recontaminstion of treated water. Its intenFity appears to dti:;end upon the concentration of con- rminati.?? qry?ts..!s pddoc? to the trcat.cn wrfcr, an3 upr,r. the a'?oiint ?f organic. aatter orieinal.ly ;re-,ent - 82 - In an attempt to deternine the ective agont resnonsiblu for the bacteri-
cidal action of S-filter powder, a great number of filtrates obtained under
various conditions wre examined. %IO observations as to the chemical compos- ition of th filtrates have been .=de; nnmely, that silver and some form of
active oxygen compound are present. The role of these materials has not yet
been clearly delineated. Some of the data obtained in experiments d.one to do-
termine the effect of ionic sil-rer, and of hydrogen per0xid.e. nre orpsented.
' It may be possible by using the oxygen-peroxy ccntent of filtrptes to devise a
ra2id spot-test system which will indicate whether such filtrfitcs are likely
to be bacteriologically safe to drink.
Som interesting resultg have been qtrtained from investigations of the en-
zyme systems of bacteria which have barely escaped bnctcriostasis by Asboloxane
S-filter powder on solid r.edia.. It e,j:pt-ars that the ability of S. aureus to
produce catrlese (vhich destroys Fro?i:tes) is grestly enhanced while the suc-
cinic dehydrogonaso system is dccreaspd in mount. ?hose results might be ex-
Fectod in such a case rhcn organisms rre growing cn a substrate rather rich in
oxygen. It is w-pDrent that the enzymr systems are being Rffected by Asboloxane
T,o.rdcrs, but gcncr~lizationscannot be made on the basis of the limited number
of tests we have performet.
- 83 -