BACTERIA SURVIVING CHLORINATION
A disinfectant is usually described as a substance capable of destroying
bacteria and other micro-organisms, and an antiseptic as one that
restrains or retards their growth or reproduction. This distinction is
entirely arbitrary as the ability of a substance to kill organisms or
merely inhibit their growth depends upon the concentration employed.
Chlorine and hypochlorites, even in minute doses, exert a toxic effect
that is sufficient to produce death in organisms but when still smaller
concentrations are employed the toxic effect is transient and the
reproductive faculty may be entirely regained.
The enumeration of bacteria by means of solid media depends upon the
ability of the organism to reproduce at such a rate as to produce a
visible colony within the period of incubation and any substance that
prevents the growth of a visible colony is classified as a disinfectant;
if on further incubation the bacterial count approximates that of the
untreated sample the added substance has acted mainly as an antiseptic.
In practice no substance acts entirely as an antiseptic as the organisms
present have varying degrees of resistance and the less viable ones are
killed by doses that are only antiseptic to the more resistant ones.
An example of an antiseptic effect followed by a mild disinfectant
action, caused by small doses of bleach is shown in Table XV. In this
experiment the water designated as control was from the same source as
the treated water. In order to make the bacterial count in this water
approximately the same as in the treated water, the original count was
reduced by diluting the sample with water from the same source,
sterilised by boiling, and afterwards reaerated with sterile air.
TABLE XV.[A]--ANTISEPTIC EFFECT OF CHLORINE
Sample treated with 0.1 part per million of available chlorine.
-------------+---------------------------------------+------------------
| | RATIO OF
PLATED. | INCUBATION PERIOD, DAYS. | BACTERIAL
| | COUNTS.
--------+----+-------+-------+-------+-------+-------+-----+-----+------
Time. |Day.| 2 | 3 | 4 | 5 | 6 | 2:4 | 2:5 | 2:6
| | | | | | |Days.|Days.|Days.
--------+----+-------+-------+-------+-------+-------+-----+-----+------
11 a.m.| 1 | 520| 940| 1,350| 2,360| 2,780|1:2.6|1:4.5|1:5.3
12 noon| 1 | 390| 770| 1,080| 2,040| 2,320| 2.8| 5.2| 5.8
2 p.m.| 1 | 187| 260| 690| 1,840| 2,080| 3.7| 9.9| 16.4
4 p.m.| 1 | 91| 130| 280| 760| 840| 3.1| 8.3| 9.2
10 a.m.| 2 | 42| 120| 670| 920| ...| 15.9| 22. | ...
10 a.m.| 3 | 320| 1,210| 3,500| ...| ...| 10.9| ...| ...
10 a.m.| 4 | 8,700| 14,200| 26,000| ...| ...| 2.9| ...| ...
--------+----+-------+-------+-------+-------+-------+-----+-----+------
CONTROL. NO CHLORINE ADDED
-------------+---------------------------------------+------------------
| | RATIO OF
PLATED. | INCUBATION PERIOD, DAYS. | BACTERIAL
| | COUNTS.
--------+----+-------+-------+-------+-------+-------+-----+-----+------
Time. |Day.| 2 | 3 | 4 | 5 | 6 | 2:4 | 2:5 | 2:6
| | | | | | |Days.|Days.|Days.
--------+----+-------+-------+-------+-------+-------+-----+-----+------
11 a.m.| 1 | 121| 184| 285| liquid| ...|1:2.4|1:...| ...
12 noon| 1 | 115| 171| 223| 380| 392| 1.9|1:3.2|1:3.2
2 p.m.| 1 | 109| 152| 221| 362| 375| 2.0| 3.3| 3.4
4 p.m.| 1 | 121| 175| 251| 410| 415| 2.1| 3.4| 3.4
10 a.m.| 2 | 6,200| 8,500| 8,800| 8,900| liquid| 1.4| 1.4| ...
10 a.m.| 3 |425,000|650,000|670,000| liquid| ...| 1.5| ...| ...
--------+----+-------+-------+-------+-------+-------+-----+-----+------
ORIGINAL SAMPLE. UNTREATED AND UNDILUTED
--------+----+-------+-------+-------+-------+-------+-----+-----+------
11 a.m.| 1 | 915| 1,410| 1,630| 2,150| 3,200|1:2.2|1:2.8|1:3.5
--------+----+-------+-------+-------+-------+-------+-----+-----+------
[A] Results are bacteria per c.cm.
Table XVI shows the effect of a concentration of 1.0 p.p.m. of chlorine;
the hypochlorite at this concentration acted almost entirely as a
germicide or disinfectant.
TABLE XVI.[A]--EFFECT OF CHLORINE AS A DISINFECTANT
AVAILABLE CHLORINE 1.0 P.P.M.
---------------+------------------------------
PLATED. | INCUBATION PERIOD, DAYS.
---------+-----+-----+-----+-----+-----+------
Time. |Day. | 2 | 3 | 4 | 5 | 6
---------+-----+-----+-----+-----+-----+------
11 a.m. | 1 | 2| 5| 7| 8| 10
12 noon | 1 | 1| 1| 2| 2| 4
2 p.m. | 1 | 0| 0| 0| 2| 2
4 p.m. | 1 | 1| 2| 2| 6| 6
10 a.m. | 2 | 0| 0| 0| 1| ..
10 a.m. | 3 | 0| 0| 0| ..| ..
10 a.m. | 4 | 5| 13| 16| ..| ..
10 a.m. | 5 | 79| 166| ..| ..| ..
Untreated| | | | | |
water | .. | 915|1,410|1,680|2,150|3,200
---------+-----+-----+-----+-----+-----+------
[A] Results are bacteria per c.cm.
Table XV shows a recovery of the anabolic functions after treatment with
0.1 p.p.m. of chlorine but since this was obtained by plating on such a
suitable medium as nutrient gelatine, it is probable that reproduction
in water having a low organic content would be still further diminished.
This is indicated by the results obtained.
There is no evidence of any marked difference in the resistance of
ordinary water bacteria to chlorine and these are the first to be
affected by the added germicide. The common intestinal organisms are
also very susceptible to destruction by chlorine and there is
considerable evidence that _B. Coli_ is slightly more susceptible than
many of the vegetative forms usually found in water. The specific
organisms causing the water-borne diseases, typhoid fever and cholera,
are, on the average, not more resistant than _B. coli_.
The spore-forming bacteria usually found in water are those of the
subtilis group, derived largely from soil washings, and _B. enteritidis
sporogenes_, from sewage and manure. The spores of these organisms are
very resistant and survive all ordinary concentrations. Wesbrook et
al.[1] found that 3 p.p.m. of available chlorine had little effect on a
spore-forming bacillus isolated from the Mississippi water and the
author has obtained similar results with _B. subtilis_.
Thomas,[2] during the chlorination of the Bethlehem, Pa., supply, found
four organisms that survived a concentration of 2 p.p.m. of available
chlorine: _Bact. aerophilum_, _B. cuticularis_, and _B. subtilis_, all
spore formers and _M. agilis_.
In practice no attempt is made, except in special cases, to destroy the
spore-bearing organisms as they have no sanitary significance and the
concentration of chlorine required for their destruction would cause
complaints as to tastes and odours if the excess of chlorine were not
removed. Such doses are unnecessary and result in waste of material. It
is found that, when the dose is sufficient to eliminate the _B. coli_
group from 25-50 c.cms. of water, the majority of the residual bacteria
are of the spore-bearing type. Smeeton[3] has investigated the bacteria
surviving in the Croton supply of New York City after treatment with 0.5
p.p.m. of available chlorine as bleach. Table XVII gives the results
obtained.
The organisms of the _B. subtilis_ group outnumbered all the others, 66
(62.8 per cent) belonging to this group alone. This group contained _B.
subtilis_--Cohn (36 strains), _B. tumescens_--Chester (15 strains) _B.
ruminatus_--Chester (13 strains), and _B. simplex_--Chester 1904, (2
strains). Three of the four coccus forms were classified as _M. luteus_.
No intestinal forms were found.
Clark and De Gage[4] in 1910 directed attention to the fact that the
bacterial counts, made at 37 deg. C. on chlorinated samples, were often much
greater than the counts obtained at room temperature. "This phenomenon
of reversed ratios between counts at the two temperatures," they stated,
"has been occasionally observed with natural water, but a study of the
record of many thousands of samples shows that the percentage of such
samples is very small, not over 3-5 per cent.... On the other hand 20-25
per cent. of samples treated with calcium hypochlorite show higher
counts at body temperature than at room temperature." Clark and De Gage
were unable to state the true significance of this phenomenon but were
of the opinion that it was not due to larger percentages of
spore-forming bacteria in the treated samples. Other observers, on the
contrary, have invariably found the spore-formers to be more resistant
to chlorine and thermophylic in type.
TABLE XVII.--ORGANISMS SURVIVING TREATMENT NEW YORK
(SMEETON)
---------------+---------------+-------------+-------------+-------------
| Morphology | Spore | Gelatine | Reaction in
| | Formation |Liquefaction | Litmus Milk
+--------+------+------+------+------+------+------+------
|Bacilli.|Cocci.| Pos. | Neg. | Pos. | Neg. | Pos. | Neg.
---------------+--------+------+------+------+------+------+------+------
No. of strains | 100 | 5 | 89 | 16 | 68 | 37 | 98 | 7
Per cent. | 95.2 | 4.7 | 84.7 | 15.2 | 64.7 | 35.2 | 93.3 | 6.6
===============+========+======+======+======+======+======+======+======
| Indol | Acid | Reduction | Inhibition
| Production | Production | of | by Gentian
| | in Glucose | Nitrates | Violet
+--------+------+------+------+------+------+------+------
| Pos. | Neg. | Pos. | Neg. | Pos. | Neg. | Pos. | Neg.
---------------+--------+------+------+------+------+------+------+------
No. of strains | 75 | 30 | 61 | 44 | 40 | 65 | 98 | 7
Per cent. | 71.4 | 28.5 | 58 | 41.9 | 38 | 61.9 | 93.3 | 6.6
---------------+--------+------+------+------+------+------+------+------
The removal of intestinal forms is, of course, merely a relative one and
when large quantities of treated water are tested their presence can be
detected.
The author, in 1915, made a number of experiments to ascertain whether
the _B. coli_ found after chlorination were more resistant to chlorine
than the original culture. The strains surviving treatment with
comparatively large doses were fished into lactose broth and subjected
to a second treatment, the process being repeated several times. The
velocity of the germicidal reaction with the strains varied somewhat,
but not always in the same direction, and the variations were not
greater than were found in control experiments on the original culture.
No evidence was obtained that the surviving strains were in any way more
resistant to chlorine than the original strain; in considering the
results it should be borne in mind that the surviving strains were
cultivated twice on media free from chlorine before again being
subjected to chlorination.
A number of the strains that survived several treatments were cultivated
in lactose broth and the acidity determined quantitatively. All the
cultures produced less acid than the original culture, and the average
was materially less than the original. These results point to a
diminution of the bio-chemical activity by action of the chlorine.
A point of perhaps more scientific interest than practical utility is
the relative proportion of the various types of _B. coli_ found before
and after treatment with chlorine. The author, in 1914, commenced the
differentiation of the types by means of dulcite and saccharose and
obtained the results shown in Table XVIII. These figures are calculated
from several hundreds of strains.
Although there is a slight difference in the relative proportions of the
types found at Ottawa and Baltimore, both sets of results show
definitely that there is no difference in the resistance of the various
types to chlorination.
=Aftergrowths.= In Tables XIII (p. 44) and XV (p. 51), it will be
noticed that, after the preliminary germicidal action has subsided, a
second phase occurs in which there is a rapid growth of organisms. This
is usually known as aftergrowth. When the contact period between
chlorination and consumption is short, the reaction does not proceed
beyond the first phase, but when the treated water is stored in service
reservoirs the second phase may ensue. At one purification plant, where
the service reservoirs are of large capacity, the aftergrowths amounted
to 20,000 bacteria per c.cm. although the water left the purification
plant with a bacterial count usually lower than 50 per c.cm.
TABLE XVIII.--TYPES OF _B. COLI_ SURVIVING CHLORINATION
------------------+------------------------------------------------
| PERCENTAGE OF ORGANISMS.
+-----------+-----------+-----------+------------
| B. coli | B. coli | B. lactis | B. acidi
| communis | communior | aerogenes | lactici
+-----+-----+-----+-----+-----+-----+-----+------
| |Chlo-| |Chlo-| |Chlo-| |Chlo-
|Raw. | ri- |Raw. | ri- |Raw. | ri- |Raw. | ri-
| |nated| |nated| |nated| |nated
------------------+-----+-----+-----+-----+-----+-----+-----+------
Ottawa, 1914 | 5 | 4 | 40 | 48 | 44 | 36 | 11 | 12
Ottawa, 1915 | 8 | 8 | 50 | 46 | 34 | 31 | 8 | 15
Baltimore, 1913[A]| 11 | 14 | 33 | 25 | 35 | 31 | 21 | 30
------------------+-----+-----+-----+-----+-----+-----+-----+------
[A] Thomas and Sandman.[5]
Regarding the nature of this aftergrowth, there has been a considerable
difference of opinion: some regard it as the result of the
multiplication of a resistant minority of practically all the species of
organisms present in the untreated water; others, that it is partially
due to the organisms being merely "slugged" or "doped," i.e. are in a
state of suspended animation, and afterwards resume their anabolic
functions; whilst others believe that with the correct dosage of
chlorine, only spore-forming organisms escape destruction and that the
aftergrowth is the result of these cells again becoming vegetative.
The aftergrowths obtained under the usual working conditions vary
according to the dosage of chlorine employed, and none of the above
hypotheses alone provides an adequate explanation. When the dosage is
small, a small number of active organisms, in addition to the spore
bearers, will escape destruction, and others will suffer a reduction of
reproductive capacity. The flora of the aftergrowth in this case will
only differ from the original flora by the elimination of a majority of
the organisms that are most susceptible to the action of chlorine and
the weaker members of other species of greater average resistance. As
the dose is increased these factors become relatively less important
until a stage is reached when only the most resistant cells, the spores,
remain. The resultant aftergrowth must necessarily be almost entirely
composed of spore-bearing organisms. A small number of the most
resistant members of non-sporulating organisms may also be present but
they will, in the majority of instances, form a very small minority.
This is the condition that usually obtains in practice and it is
necessary to consider whether the aftergrowth may have any sanitary
significance.
Concerning the secondary development of _B. coli_, the usual index of
pollution, there is but little information. H. E. Jordon[6] reported
that, of 201 samples, 21 gave a positive _B. coli_ reaction immediately
after treatment, 39 after standing for twenty-four hours, and 42 after
forty-eight hours. These increases were confined to the warm months, the
cold months actually showing a decrease. The following figures, taken
from the author's routine tests for 1913 and 1914, show a similar
tendency, but an analysis of the results by months did not show that
this was confined to the warm season. The sequence of the results from
left to right, in the following Table, is in the same order as the
contact period. Approximately 290 samples were taken at each sampling
point.
At station No. 2 the germicidal action was still proceeding but at No.
5, representing an outlying section of the city, the increase in the _B.
coli_ content is very apparent.
During 1915 and 1916 the author endeavoured to duplicate these results
under laboratory conditions and entirely failed. These experiments,
which were made with the same materials as were in use at the city
chlorination plant, but in glass containers, were usually only carried
to a forty-eight hours contact, as this was the extreme limit for the
city mains; one, however, was prolonged to five days. Many experiments
were made under varying conditions, with similar results. Typical
examples are given in Tables VI, VIII and IX on pages 33 and 37.
TABLE XIX.--AFTERGROWTHS OF B. COLI
PERCENTAGE OF SAMPLES SHOWING B. COLI IN 10 C.CMS.
-----+----------------------------------
| SAMPLING POINT NO.
+------+------+------+------+------
| 1 | 2 | 3 | 4 | 5
+------+------+------+------+------
1913 | 15.2 | 14.4 | 16.3 | 16.8 | 26.8
1914 | 7.0 | 5.7 | 6.0 | .... | 11.6
-----+------+------+------+------+------
In every case there was persistent diminution in the number of _B. coli_
with increase of contact period. Determination of the bacterial count on
nutrient agar showed that, in several experiments, the aftergrowth had
commenced, and in some instances there was evidence that the second
cycle was partially complete i.e. the number had reached a maximum and
then commenced to decline. The time required for the completion of the
two cycles, comprising the first reduction caused by the chlorine, the
increase or aftergrowth, and the final reduction due to lack of suitable
food material, is dependent upon several factors of which the dosage and
temperature are the most important. With a small dosage the germicidal
period is short and the second phase is quickly reached; with large
doses, the second phase is not reached in forty-eight hours; the higher
the temperature the quicker is the action and the development of the
aftergrowth. These statements refer only to the bacteria capable of
development on nutrient agar. The _B. coli_ group behaved differently
and persistently diminished in every case. If _B. typhosus_ acts in a
similar manner to _B. coli_, the laboratory experiments show that
aftergrowths are of no sanitary significance and can safely be ignored,
but as the results obtained in practice are contradictory to the
laboratory ones, the matter must be regarded as _sub judice_ until more
definite evidence is available.
It is common knowledge that samples of water from "dead ends" of
distribution mains show high counts and much larger quantities of _B.
coli_ than the water delivered to the mains. This is another phase of
aftergrowth problem that often causes complaints and can only be
eliminated by "blowing off" the mains frequently or by providing
circulation by connecting up the "dead ends." One extreme case of this
description might be cited. A small service was taken off the main at
the extreme edge of the city to supply a Musketry School two miles away
and was only in use for a few months in the summer season. This service
pipe delivered water containing _B. coli_ in a considerable percentage
of the 10 c.cm. samples and in a few instances in 1 c.cm., although the
water delivered to the city mains never exceeded 2 _B. coli_ per 100
c.cms. and averaged about one-tenth that quantity. No epidemiological
records of the effect of this water are available because it was put
through a Forbes steriliser before consumption.
In some instances the rate of development of the organisms after
chlorination is greater than in the same water stored under similar
conditions. This is especially noticeable in the presence of organic
matter and has been ascribed to the action of the chlorine on the
organic matter with the production of other compounds that are available
as food material for the organisms.
Houston, during the treatment of prefiltered water Lincoln in 1905,
found that although the removal of _B. coli_ and other organisms growing
at 37 deg. C. was satisfactory, there was almost invariably an increase in
the bacteria growing on gelatine at 20 deg. C. This was ascribed to the
action mentioned above and the chemical results supported this view,
more organic matter being found in the filter effluents than in the
prefiltered water. Rideal's experiments with sewage at Guildford
indicate that a similar action may occur in contact beds. The addition
of bleach to the prefiltered water at Yonkers also resulted in an
increased count and in these instances the aftergrowths are due to a
disturbance of the equilibrium by the action of the chlorine on the
zooglea and other organic matter invariably found in ripe filters.
Similar results can be produced by the addition of chlorinated water to
small experimental sand filters. This is shown by the results in Tables
XX and XXI.
TABLE XX.--AFTERGROWTHS IN SAND
--------------+---------------+---------------+----------------
| Bacteria Per | |
Available | Gram of |Typical B. coli| FREE CHLORINE
Chlorine in | Sand After |After 24 Hours.| AFTER 24 HRS.
Water p.p.m. +-------+-------+---+---+---+---+-------+--------
| | |100| 10| 1 |0.1|Without| After
| 3 Hrs.|24 Hrs.|Gr.|Gr.|Gr.|Gr.| Acidification.
--------------+-------+-------+---+---+---+---+-------+--------
Nil | 12,000| 21,000| + | + | + | - | - | -
3.0 | 80|114,000| - | - | - | - | - | -
5.0 | 50|150,000| - | - | - | - | - | -
7.0 | 25|214,000| - | - | - | - | - | -
10.0 | 26|500,000| - | - | - | - | - | -
--------------+-------+-------+---+---+---+---+-------+--------
TABLE XXI.--AFTERGROWTHS IN SAND
--------------+---------------------------------
| BACTERIA PER GRAM OF SAND AFTER
Available in +----------+----------+-----------
Water p.p.m. | 3 Hours. |24 Hours. |48 Hours.
--------------+----------+----------+-----------
Nil | 70,000 | ..... | .....
0.1 | 7,200 | 20,400 | 12,800
0.3 | 5,240 | 6,400 | 11,200
0.5 | 5,120 | 4,700 | 10,800
1.0 | 1,100 | 8,800 | 20,400
--------------+----------+----------+-----------
It is observable that the effect of small doses was comparatively small
and transient; large doses of bleach reduced the bacteria very
materially but the reduction was not maintained and the subsequent
increase was abnormally rapid.
BIBLIOGRAPHY
[1] Wesbrook, Whittaker and Mohler. J. Amer. Pub. Health Assoc., 1911,
1, 123.
[2] Thomas. Jour. Ind. and Eng. Chem., 1914, 6, 548.
[3] Smeeton. Jour. of Bact., 1917, 2, 358.
[4] Clark and De Gage. Rpt. Mass. B. of H., 1910, p. 319.
[5] Thomas and Sandman. J. Ind. and Eng. Chem., 1914, 6, 638.
[6] Jordan, H. E. Eng. Record, 1915, May 17.