Chlorination of Water Part 7

[12] Comte. J. Pharm. Chim., 1916, =14=, 261.

[13] Ma.s.sy. J. Pharm. Chim., 1917, =15=, 209.

CHAPTER IV

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 r.e.t.a.r.ds 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 cla.s.sified 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 cla.s.sified 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 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 quant.i.ties 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 quant.i.tatively. 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 ------------------+-----+-----+-----+-----+-----+-----+-----+------