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A number of visits were paid to the outfall works at Barking and Crossness during the
construction of the filter bed. These visits will be continued in the future from time to time.
It is proposed, in the first place, to describe some of the more important methods used in the
bacteriological examination of sewage. These need not be repeated in future reports, which will
be mainly concerned with the consideration of the results arrived at during the progress of the
investigation.
After a description of these methods a summary of the results shown in Table I. (pp. 8-10) will
be given, and these results will be briefly discussed. The enquiry is not as yet sufficiently advanced to
make it advisable to enter more fully into details.
In the Table which follows the above summary certain experiments are included which were
undertaken under my direction by Mr. E. Brooke Pike, the chemist in charge at the Northern Outfall
works. These experiments were carried out with great care and yielded valuable results.
A number of micro-photographs illustrating the work accompany the report. These have been
specially taken by Dr. Norman.
B.—DESCRIPTION OF SOME OF THE METHODS USED IN THE BACTERIOLOGICAL
EXAMINATION OF SEWAGE.
1. Collection of samples.
The samples were invariably collected in sterile bottles. The bottles were glass-stoppered, and
they were completely filled with the sewage. In every case the cultivations were made on the same
day as the samples were collected, and as soon after collection as possible.
2. Dilution of sewage.
The microbes in sewage are so numerous that it is necessary to dilute the sewage with a large
quantity of sterile water, in order to render their enumeration and study possible. The two most
convenient dilutions are 1 in 10 and 1 in 10,000.
3. Total number of bacteria.
In cultivating the germs in sewage with the view of estimating their number, gelatine plates
should be used. No doubt the rapid liquefaction of the gelatine, which is produced by the liquefying
bacteria in sewage, is a decided disadvantage; but if the sewage has been suitably diluted, and if the
observations are made day by day, it is not difficult to obtain plates with colonies sufficiently advanced
in their growth and sufficiently numerous to allow of their being accurately counted, even when
rapidly liquefying species are present. When agar-agar is employed as a nutrient medium for the
germs, the numbers counted are too low, and the differentiation of the species of micro-organisms
present is more difficult. Further the liquefaction of the gelatine is a positive advantage, inasmuch as
it gives some indication of the number of bacteria present in the sewage, which are likely to cause
liquefaction of the suspended organic matter. From 0.1 to *1.0 c.e. of a liquid, consisting of crude
sewage diluted with 10,000 times its volume of sterile water, is used in these cultivations. As soon
as the gelatine film, which has been spread upon the plates has become solid, the plates should be
turned upside down and kept in this position, if possible, until the colonies are ready for being counted.
Contamination of the films with germs from the air is thus prevented, and difficulties arising from the
condensation of water on the lid of the capsule are obviated. The plates are best "incubated" at
a definite temperature, which is usually 20° C. If, on the afternoon of one day, it is found that the
colonies are nearly, but not quite sufficiently advanced in their growth for being counted, it is advisable
to remove the plates to a cool place, as otherwise before the next morning the progressive liquefaction
of the gelatine may altogether prevent the colonies from being accurately counted.
4. Spores of bacteria.
In estimating the number of spores of bacteria in sewage the best method is to add 1 c.c. of
diluted sewage (1:10) to 10 c.c. of melted gelatine in a test tube, heat the mixture to 80° C. for 10
minutes, and then pour it into a Petri's capsule.
5. Liquefying bacteria.
The number of liquefying bacteria present in the sewage may be judged to some extent by
careful observation of the ordinary gelatine plates. But in these plates many of the colonies are
necessarily imbedded in the nutrient medium, and in this case they may not liquefy the gelatine at all,
or only liquefy it when they reach the surface as their growth proceeds, or they may display their
liquefying power so late that the plate has become overcrowded. It is therefore usually advisable to
supplement the ordinary plate cultivations by making "surface" plate cultivations. It is perhaps best
for this purpose to use 0.1 c.c. of diluted sewage (1: 10,000). The gelatine is first allowed to become
solid in a Petri's capsule, and the diluted sewage is then added and spread over the entire surface with
a sterile platinum " spreader " or with a sterilised camel's-hair brush
* See No. 2 (Plate I.), which shows a gelatine plate cultivation containing 1 c.c. of diluted Crossness
crude sewage (1 : 10,000).