Professional Documents
Culture Documents
09 01 29 - PG - Bacteriological Examination of Water
09 01 29 - PG - Bacteriological Examination of Water
Nationalestraat, 155
B – 2000 Antwerpen
MODULE 2
CLINICAL & BIOMEDICAL SCIENCES OF TROPICAL DISEASES
Practical notes
__________________________
WATER ANALYSIS
What to do:
In groups of 2 persons:
What to remember:
Practical 1I
What to do:
In groups of 2 persons:
Individually:
What to remember:
WATER SAMPLING................................................................................................6
TURBIDITY MEASURMENT:..............................................................................13
Target values:.......................................................................................................15
The most important factor to take into account is that, in most communities, the principal risk
to human health derives from faecal contamination. In some countries there may also be
hazards associated with specific chemical contaminants such as fluoride or arsenic, but the
levels of these substances are unlikely to change significantly with time. Thus, if a full range
of chemical analyses is undertaken on new water sources and repeated thereafter at fairly
long intervals, chemical contaminants are unlikely to present an unrecognized hazard. In
contrast, the potential for faecal contamination in untreated or inadequately treated
community supplies is always present. The minimum level of analysis should therefore
include testing for indicators of faecal pollution (thermotolerant (faecal) coliforms), turbidity,
and chlorine residual and pH (if the water is disinfected with chlorine).
It is ideal to look for individual specific pathogen but it is not practical since they are few in
numbers than the non-pathogenic organisms and methods to detect them are costly in time
and money. Therefore indicators of human/animal pollution e.g. coliforms are used. Faecal
streptococci are regularly present in the faeces in varying numbers but their number is fewer
than Escherichia coli and they probably die and disappear at the same rate. The presence of
faecal streptococci along with coliforms in absence of Escherichia coli is also confirmatory of
faecal pollution.
Coliform organisms (total coliforms): Coliform organisms have long been recognized as a
suitable microbial indicator of drinking-water quality, largely because they are easy to detect
and enumerate in water. The term “coliform organisms” refers to Gram-negative, rod-shaped
bacteria capable of growth in the presence of bile salts or other surface-active agents with
similar growth-inhibiting properties and able to ferment lactose at 35–37°C with the production
of acid, gas, and aldehyde within 24–48 hours. They are also oxidase-negative and non-
spore-forming and display b-galactosidase activity. Traditionally, coliform bacteria were
regarded as belonging to the genera Escherichia, Citrobacter, Enterobacter, and Klebsiella.
However, as defined by modern taxonomical methods, the group is heterogeneous. It
includes lactosefermenting bacteria, such as Enterobacter cloacae and Citrobacter freundii,
which can be found in both faeces and the environment (nutrient-rich waters, soil, decaying
plant material) as well as in drinking-water containing relatively high concentrations of
nutrients, as well as species that are rarely, if ever, found in faeces and may multiply in
relatively good-quality drinking-water, e.g. Serratia fonticola, Rabnella aquatilis, and
Buttiauxella agrestis. The existence both of non-faecal bacteria that fit the definitions of
coliform bacteria and of lactose-negative coliform bacteria limits the applicability of this group
as an indicator of faecal pollution. Coliform bacteria should not be detectable in treated water
supplies and, if found, suggest inadequate treatment, post treatment contamination, or
excessive nutrients. The coliform test can therefore be used as an indicator both of treatment
efficiency and of the integrity of the distribution system. Although coliform organisms may not
always be directly related to the presence of faecal contamination or pathogens in drinking-
water, the coliform test is still useful for monitoring the microbial quality of treated piped water
supplies. If there is any doubt, especially when coliform organisms are found in the absence
of thermotolerant coliforms and E. coli, identification to the species level or analyses for other
indicator organisms may be undertaken to investigate the nature of the contamination.
Sanitary inspections will also be needed.
Faecal streptococci: Faecal streptococci are those streptococci generally present in the
faeces of humans and animals. All possess the Lancefield group D antigen. Taxonomically,
they belong to the genera Enterococcus and Streptococcus. The taxonomy of Enterococci
has recently undergone important changes, and detailed knowledge of the ecology of many of
the new species is lacking; the genus Enterococcus now includes all streptococci that share
certain biochemical properties and have a wide tolerance of adverse growth conditions—E.
avium, E. casseliflavus, E. cecorum, E. durans, E. faecalis, E. faecium, E. gallinarum, E.
WATER SAMPLING
Several type of bottle may be used for sampling, but glass bottles are best. These should
have securely fitting stoppers with non toxic liners. The bottles should hold at least 200 ml of
water and should be sterilized.
When water that contains or may contain even traces of chlorine is sampled, the chlorine
must be inactivated. If it is not, microbes may be killed during transit and an erroneous result
will be obtained. The bottles in which the samples are placed should therefore contain sodium
thiosulfate to neutralize any chlorine present. The sodium thiosulfate should be added to the
bottles before they are sterilized.
For 200 ml samples, five drops of aqueous sodium thiosulfate solution [100 g/litter (w/v)]
should be added to each clean sample bottle. The stopper is loosely adapted to the bottle
and aluminium foil cover is tied to the neck of the bottle to prevent dust from entering. The
bottle is the sterilized [in hot-air oven for 1 hour at 160°C or for 40 minutes at 170°C; or in an
autoclave at 121°C for 20 minutes.
Although recommendations vary, the time between sample collection and analysis should, in
general, not exceed 6 hours, and 24 hours is considered the absolute maximum. . It is
assumed that the samples are immediately placed in a lightproof insulated box containing
melting ice or ice-packs with water to ensure rapid cooling. If ice is not available, the
transportation time must not exceed 2 hours. It is imperative that samples are kept in the dark
and that cooling is rapid. If these conditions are not met, the samples should be discarded.
The cool box used to carry samples should be cleaned and disinfected after each use to
avoid contaminating the surfaces of the bottles and the sampler’s hands.
Water sources can be divided into three basic types for the purpose of sampling.
1. Attach a stone of suitable size to the sampling bottle with a piece of string.
2. Tie a 20 meter length of clean string on the bottle and to a stick.
3. Open the bottle as described above and lower into the well.
4. Immerse the bottle completely in water without touching the sides of the well
and lower it down to the bottom of the well.
5. Pull it out when the bottle is filled.
6. Discard a little water to provide airspace.
7. Stopper and label the bottle.
Where the quality of the water is totally unknown, it may be advisable to test two or more
volumes in order to ensure that the number of colonies on the membrane is in the
optimal range for counting (20–80 colonies per membrane).
The multiple-tube method is applicable to all kinds of water: it can be used with clear,
coloured, or turbid water containing sewage or sewage sludge, or mud and soil particles,
provided that the bacteria are homogeneously distributed in the prepared test samples.
Double strength broth: Dissolve 71.2 g of lauryl sulphate broth in 1 litre distilled water.
After solubilisation, dispense 10 ml into each test tube containing inverted Durhan-tubes.
Sterilise by autoclaving at 121°C for 15 minutes. Cool down slowly to prevent bubbles in
Durhan-tubes.
Single strength broth: Dissolve 35.6 g of lauryl sulphate broth in 1 litre distilled water.
After solubilisation, dispense 10 ml into each test tube containing inverted Durhan-tubes.
Sterilise by autoclaving at 121°C for 15 minutes. Cool down slowly to prevent bubbles in
Durhan-tubes.
Total Coliforms:
Arrange three rows of 3 (5) tubes each in a test-tube rack. The tubes in the first row hold
10 ml of double strength lauryl sulphate broth, while the tubes in the second and the third
rows contain 10 ml of single strength lauryl sulphate broth.
With a sterile pipette (syringe) add 10 ml of the sample to each of the 3 (5) tubes of the
first row.
With a sterile pipette (syringe) add 1 ml of the sample to each of the 3 (5) tubes of the
second row.
With a sterile pipette (syringe) add 0.1 ml of the sample to each of the 3 (5) tubes of the
third row. Add also in each tube 0.9 ml of sterile distilled water.
3. The media receiving one or more of the indicator bacteria show growth (turbidity)
and a gas production which is absent in those receiving an inoculum of water
without indicator bacteria. Presence of grow and gas indicates positive reaction
whereas absence of either or both these features denotes a negative reaction.
4. The presumptive positives are read and remaining negative bottles are re-
incubated for another 24 hours. Any further positives are added to the previous
figures. The probable numbers of coliforms are read from the probability tables of
McCrady (Tables in annex).
5. From the number and distribution of positive and negative reactions, count of the
most probable number (MPN) of indicator organisms in the sample may be
estimated by reference to statistical tables. The test gives presumptive coliforms
count as the reaction observed may occasionally be due to the presence of some
organisms other than coliforms.
For every tube showing fermentation (primary fermentation, presumptive coliforms), you
may inoculate one new tube of Lauryl sulphate broth, from the tube showing primary
fermentation, and incubated this tube at 44°C respectively. If there is fermentation in the
tube incubated at 44°C after 8 to 24 hours, perform indole test by adding Kovac’s
reagent. A positive indole test in a broth tube showing gas production at 44°C indicates
the presence of E. coli.
From the number and distribution of positive and negative reactions at 44°C, count of the
most probable number (MPN) of indicator organisms in the sample may be estimated by
reference to statistical tables. The test gives E. coli count
Arrange three rows of 3 (5) tubes each in a test-tube rack. The tubes in the first row hold
10 ml of double strength lauryl sulphate broth, while the tubes in the second and the third
rows contain 10 ml of single strength lauryl sulphate broth.
With a sterile pipette (syringe) add 10 ml of the sample to each of the 3 (5) tubes of the
first row.
With a sterile pipette (syringe) add 1 ml of the sample to each of the 3 (5) tubes of the
second row.
With a sterile pipette (syringe) add 0.1 ml of the sample to each of the 3 (5) tubes of the
third row. Add also in each tube 0.9 ml of sterile distilled water.
3. The media receiving one or more of the indicator bacteria show growth (turbidity)
and a gas production which is absent in those receiving an inoculum of water
without indicator bacteria. Presence of grow and gas indicates positive reaction
whereas absence of either or both these features denotes a negative reaction.
4. The presumptive positives are read, (perform indole test by adding 0.5 ml of
Kovac’s reagent: a positive indole test in a broth tube showing gas production at
44°C indicates the presence of E. coli) and remaining negative bottles are re-
incubated for another 24 hours. Any further positives are tested with Kovacs and
added to the previous figures. The probable numbers of E coli are read from the
probability tables of McCrady (Tables in annex).
5. From the number and distribution of positive and negative reactions, count of the
most probable number (MPN) of indicator organisms in the sample may be
estimated by reference to statistical tables. The test gives E. coli count.
Turbidity may change during sample transit and storage, and should therefore be measured
on site at the time of sampling.
Add water slowly to the turbidity tube, taking care not to form bubbles. Fill until the mark at the
bottom of the tube just disappears.
Read the turbidity from the scale marked on the side of the tube. The value is that
corresponding to the line nearest to the level of the water in the tube. The scale is not linear,
and extrapolation of values between the lines is therefore not recommended.
The method recommended for the determination of chlorine residual in drinking water
employs N,N-diethyl-p-phenylenediamine, more commonly referred to as DPD. Methods
employing orthotolidine and starch–potassium iodide were formerly also recommended.
The first of these reagents is a recognized carcinogen and the method is not reliable. The
method based on the use of starch–potassiumiodide is not specific for free chlorine, but
measures directly the total of free and combined chlorine; it is not recommended except
in countries where DPD cannot be obtained or prepared. In this Annex, therefore, only
the DPD method is considered.
In the laboratory, colorimetry or spectrophotometry may both be used for the
determination of chlorine by means of DPD. However, it is common practice and highly
recommended for field measurements using simple color match comparators to be done
on site. The color is generated following the addition of DPD to the water sample and is
matched against standard colored discs or tubes. The method can be used by staff
without extensive specialized training. The reagent may be solid (e.g. individually
wrapped tablets) or in the form of a solution; the former is more stable. If the solution is
used, it should be stored in a brown bottle and discarded as soon as it starts to become
discolored.
It is important to measure pH at the same time as chlorine residual since the efficacy of
disinfection with chlorine is highly pH-dependent: where the pH exceeds 8.0, disinfection is less
effective. To check that the pH is in the optimal range for disinfection with chlorine (less than
8.0), simple tests may be conducted in the field using comparators such as that used for chlorine
residual. With some chlorine comparators, it is possible to measure pH and chlorine residual
simultaneously. Alternatively, portable pH electrodes and meters are available. If these are used in
the laboratory, they must be calibrated against fresh pH standards at least daily; for field use, they
should be calibrated immediately before each test. Results may be inaccurate if the water has a
low buffering capacity.
The objective of zero E. coli per 100 ml of water is the goal for all water supplies and should
be the target even in emergencies; however, it may be difficult to achieve in the immediate
post-disaster period. This highlights the need for appropriate disinfection. An indication of a
certain level of faecal indicator bacteria alone is not a reliable guide to microbial water safety.
Some faecal pathogens, including many viruses and protozoan cysts and oocysts, may be
more resistant to treatment (e.g., by chlorine) than common faecal indicator bacteria. More
generally, if a sanitary survey suggests the risk of faecal contamination, then even a very low
level of faecal contamination may be considered to present a risk, especially during an
outbreak of a potentially waterborne disease, such as cholera.
Typical sample volumes and numbers of tubes for the multiple-tube method
Another reason for maintaining a predominance of hypochlorous acid during treatment has
to do with the fact that pathogen surfaces carry a natural negative electrical charge. These
surfaces are more readily penetrated by the uncharged, electrically neutral hypochlorous
acid than the negatively charged hypochlorite ion. Moving through slime coatings, cell walls
and resistant shells of waterborne microorganisms, hypochlorous acid effectively destroys
these pathogens. Water is made microbiologically safe as pathogens either die or are
rendered incapable of reproducing.
A typical bacterium has a negatively charged slime coating on its exterior cell wall, which is
effectively penetrated by electrically neutral hypochlorous acid, favored by lower pH’s.
(Reprinted from The Chlorination/Chloramination Handbook by permission. Copyright ©
1996, American Water Works Association.)
CAS#Chemical NamePercentEINECS/ELINCS
10102-17-7Sodium Thiosulfate, Pentahydrate100 unlisted
EMERGENCY OVERVIEW
Appearance: colourless to white solid. Caution! May cause eye and
skin
irritation. Hygroscopic (absorbs moisture from the air). The
toxicological properties of this material have not been fully
investigated. May cause respiratory and digestive tract irritation.
Target Organs: No data found.
Potential Health Effects
Eye: May cause eye irritation.
Skin: Prolonged and/or repeated contact may cause irritation and/or
dermatitis.
Ingestion: Ingestion of large amounts may cause gastrointestinal
irritation. The toxicological properties of this substance have not
been fully investigated.
Inhalation: May cause respiratory tract irritation. Low hazard for
usual industrial handling. The toxicological properties of this
substance have not been fully investigated.
Chronic: No information found.
RTECS#:
CAS# 10102-17-7: WE6660000
LD50/LC50: Not available.
No information available.
US DOTIATARID/ADRIMOCanada TDG
Shipping Name: No information available.
Hazard Class:
UN Number:
Packing Group:
US FEDERAL
TSCA
CAS# 10102-17-7 is not on the TSCA Inventory because it is a hydrate.
It is considered to be listed if the CAS number for the anhydrous
form is on the inventory (40CFR720.3(u)(2)).
Health & Safety Reporting List
None of the chemicals are on the Health & Safety Reporting List.
Chemical Test Rules
None of the chemicals in this product are under a Chemical Test Rule.
Section 12b
None of the chemicals are listed under TSCA Section 12b.
TSCA Significant New Use Rule
None of the chemicals in this material have a SNUR under TSCA.
SARA
European/International Regulations
European Labeling in Accordance with EC Directives
Hazard Symbols:
Not available.
Risk Phrases:
Safety Phrases:
S 37 Wear suitable gloves.
S 45 In case of accident or if you feel unwell, seek
medical advice immediately (show the label where
possible).
S 28A After contact with skin, wash immediately with
plenty of water.