Lecture on microbiological quality methods for drinking water for the faculty of Veterinary Medicine, University of Helsinki; Course in Environmental Health

1. MICROBIOLOGICAL QUALITY
OF WATER
Parameters and assessment

2. LEARNING OUTCOMES
List the microbiological water quality indicators/indexes
Explain the relationship between indicators/indexes and microbiological health
hazards
Summarize analytical methods for microbial water quality testing

3. MONITORING THE WATER QUALITY
Monitoring is a critical part in the water safety plans for both drinking and
recreational water
Microbial safety is guaranteed through knowledge of:
- The variations in quality of the source water,
- The control of the treatment process
- The integrity of the distribution or storage system

4. MONITORING THE WATER QUALITY
No a single microbial parameter is adequate to determine if all the steps in
the system are working
Goal of “zero level of pathogen” in DW and RW à implies a judgement
on the level of treatment necessary to achieve tolerable risk
Monitoring is a reactive action à any event (e.g. breakdown of the system)
can occur many hours or even days before it is detected

5. WHAT DO WE DO IF DRINKING WATER DO NOT
FULFILL THE MICROBIAL STANDARDS?
1) If many parameters are not correct
- figure out the reason and make corrections immediately (with basis on
Terveydesuojelulaki 20§)
2) If few parameters are not correct and no epidemic
- Additional sampling to confirm the result
- If no confirmation à cancel first results and new sample result stands
- If confirmationà figure out the reason and make corrections immediately

6. WHAT DO WE DO IF DRINKING WATER DO NOT
FULFILL THE MICROBIAL STANDARDS?
3) If epidemic suspicion à take actions as quick as possible
- Inform water users
- Collect multiple samples from both water and patients (search for pathogens)
- Identify an alternative water supply
- Treat drinking water: boil; use chlorine (> 2 mg Cl2/L)
- Rinse the DW network system and perform a chlorine shock (10 mg Cl2/L)

7. WHAT ARE WE MONITORING IN THE WATER?
The indicators and indexes were
developed as a measure of fecal
pollution for
- Assessing treatment process effectiveness
(Indicator)
- Diagnosing post-treatment contamination or
deterioration (Index)
Not only fecal pollution but also for
identifying aesthetic problems or the
possible overgrow of OPPPs

8. CHARACTERISTICS OF INDICATOR AND INDEX
ORGANISMS
Original assumption: same organism would serve as both index and indicator
Indicator/index should:
- Be absent in unpolluted water and present when the source of pathogens is present
- Not multiply in the environment (water)
- Be present in greater number than the pathogen
- Respond to environment stress and water treatment process similarly to the pathogen
- Be easy to isolate, identify and enumerate, and tests should be inexpensive

9. APPLICATIONS AND
LIMITATIONS
It has become clear that one microbial
organisms cannot fulfil these two roles
(indicator and index)
A range of organisms should be
considered for different purposes
There isn’t a direct relationship between
indicators and human health:
- Normal values, but presence of pathogens
(e.g. Tampere outbreak 2014)
Indicators are tools for taking decisions,
they should be coupled with supporting
information
WHO

10. WHY DO WE NOT LOOK FOR PATHOGENS?
Methods for pathogen detection are often difficult to implement, relative
expensive and generally time-consuming
We know that fecal pollution can cause disease in human à but we do not
know all possible pathogens
Examination of presence of pathogen in the water will only permit
confirmation that consumer was exposed to that specific pathogen
Methodological limitations à great care in the interpretation of the results:
- Positive: water unsafe to drink, used only in well-managed, risk-based decision-
making process
- Negative: should not be used as an excuse for complacency

11. INDICATORS & INDEXES overview

12. OVERVIEW
Heterotrophic plate counts
Total coliforms
Thermotolerant coliforms and E. coli
Clostridium perfringens
Pseudomonas aeruginosa

13. HETEROTROPHIC PLATE COUNTS
Total count (cfu/mL) of mesophilic bacterial species on tryptone-yeast extract (TH)
agar
Incubate at + 22 ° C for 3 days or/and 36 ° C for 2 days
Does not correlate with human health
Provides information on the water quality of the repeated measurements
- The effectiveness of the water treatment process
- Assessment of the condition of the distribution network
- Biofilm formation in swimming pools

14. TOTAL COLIFORMS
Defined based on the method used
Members of a genus or species within the family Enterobacteriaceae capable
of growth at 37°C and possessing β-galactosidase (able to ferment lactose)
Include Escherichia, Enterobacter, Pantoea, Citrobacter, Klebsiella, Serratia,
Rahnella, Hafnia, Erwinia, Moellerella and Morganella
Organisms in soil or vegetation and in the intestinal tract of warm-blooded
animals
Presence in water is tolerated (especially nutrient-rich water)
Used as indicator of treatment efficiency

15. THERMOTOLERANT COLIFORMS AND E. COLI
Group of total coliforms that are able to ferment lactose at 44-45 °C
Include Escherichia, Klebsiella, Enterobacter and Citrobacter
Only Escherichia coli is considered of fecal origin
Thermotollerant coliforms other than E. coli may originate from organically
enriched water such as industrial effluents or from decaying plant material and
soil
E. coli growths at 37 and 44-45°C, fermenting lactose and mannitol and
produces indole from tryptophan (there are several exceptions)
E. coli is widely preferred as an index of recent fecal contamination

16. ENTEROCOCCUS
Enterococci are found in high concentrations in human feces, usually between 104 and
106 bacteria per gram wet weight (Enterococcus faecalis and E. faecium)
Due to their ubiquity in human feces and persistence in the environment à indicators
of human fecal pollution
The use of enterococci as indicators for human fecal pollution can be problematic:
- found in animal feces in soils and on plants;
- evidence replicating in extra-enteric environments (beach sands, in water containing kelp or plankton)
Evidence for a positive association between enterococcal concentrations and swimmer
gastrointestinal illnesses

17. CLOSTRIDIUM PERFRINGENS
Good indicator of contamination from wastewater
It is conservative indicator for fecal excreta from nonherbivorous wildlife and
human-associated sewage
Presence correlated with Cryptosporidium spp. and with infection risk from
recreational activities
Spores may be detected long after a pollution event has occurred and far
from the source
Occurs at lower numbers than E.coli or enterococci - larger sampling volumes
have to be used

18. PSEUDOMONAS AERUGINOSA
It is free-living bacteria of both low nutrient or oligotrophic environments
(deionized or distilled water) and high nutrient environments such as in sewage
Found in natural waters such as lakes and rivers in concentrations of 10/100
mL to >1,000/100 mL
It is not often found in drinking water (2% of samples; 3-4 CFU/mL)
Occurrence in drinking water:
- ability to colonize biofilms in plumbing fixtures
- Deterioration in bacteriological quality
- Rose in water temperature and low rates of flow in the distribution system (DS)
- Used to assess regrowth in DS
Mena and Gerba. D.M. Whitacre (ed.), Reviews of Environmental
Contamination and Toxicology Volume 201,

19. Overview of microbial parameters and applicability

20. CULTIVATION TECHNIQUES Bacteriology

21. OVERVIEW
- Most probable number
- Presence/absence test in liquid media
- Pour plate
- Spread plate
- Membrane filtration
- Liquid enrichment with confirmation and/or isolation in solid media

22. MOST PROBABLE NUMBER (MPN)
Estimate the population density of viable
bacterium in a sample
Probable number based on observed
positive growth responses to a standard
dilution series
The sample should be diluted in a way
that higher dilutions of the sample will result
in fewer positive culture tubes in the series
The number of sample dilutions to be
prepared is generally based on the
expected population contained within the
sample

23. ASSUMPTION OF MPN
1. Organisms are randomly and
independently distributed throughout the
sample following a Poisson distribution
2. Organisms exist as single entities, not
as chains, pairs or clusters and they
do not repel one another
3. Even a single viable cell in an inoculum is
able to produce detectable growth
4. The population does not contain
viable, sub-lethally injured organisms
that are incapable of growth in the
culture medium used
P(X=x) =
!
The theoretical mean number
of point for square is 1,4 in
all three cases
In which cases do the points
follow the Poisson distribution?
1
2
3

24. MOST PROBABLE NUMBER (MPN)
Most reliable results occur when all tubes at the lower dilution are positive and
all tubes at the higher dilution are negative
When a higher number of tubes are inoculated in the series, the
confidence limits of the MPN are narrowed
MPN values are only estimates while plate counts are direct counts of living
organisms expressed in cfu/ml
MPN values are particularly useful when low concentrations of organisms
(<100/g) are encountered à Poisson distribution!!!

25. FILTRATION METHOD
From Docent Rauni Kivistö lecture YH 2014

26. DETECTION LIMITS FOR ENUMERATING BACTERIA
Method Inoculum Detection limits
(cpu/100 mL)
Reading ranges
Spread plate 0,1 mL 1000 25 – 300
Pour plate 1 mL 100 25 – 300
Membrane filtration 100 mL 1 25 – 300
MPN 5x10mL + 5x1mL +
5x0,1mL
4 4 – 100 000
MPN Quanti-Tray (Colilert) < 1 < 1 – 200.5
MPN Quanti-Tray/2000
(Colilert)
< 1 < 1 – 2419,6
From Docent Rauni Kivistö lecture YH 2014

27. Summary of the
advantages and
disadvantages of
different cultivation
techniques
Cfu count
Cfu count

28. COLIFORMS AND E. COLI
Membrane filtration 0,45 µm
Selective media
- LTTC, 36°C, 24h (SFS-EN ISO 9308-1:2001)
- mEndo Agar LES (LES Endo), 36°C, 24h (SFS
3016:2001)
- mFC, 44°C, 24h (SFS 4088:2001)
- Chromocult, 24 hours at 35 - 37°C
Species identification:
- oxidase test (all coliforms)
- Indole test (E. coli)
MPN: Colilert® (SFS-EN ISO 9308-
2:2012)
Chromocult mEndo Agar LES
Colilert
β-galactosidase coliforms - Yellow color
E. coli β-glucuronidase - Fluorescens under UV light

29. ENTEROCOCCUS
The standard method (SFS-EN ISO
7899-2: 2000)
Membrane filtration 0,45 µm
Selective medium:
- Slanetz & Bartley, 36 ° C, 44h
Verification:
- Bile-esculin-azide agar at 44 ° C,
2h
Slanetz & Bartley Bile-esculin-azide agar

30. CLOSTRIDIUM PERFRINGENS
STM No 461
Membrane filtration 0,45 µm
mCP-agar, anaerobically at 44 ° C for
24 h
In the fume hood place for 15 sec
membrane filters with straw-yellow
colonies onto cellulose pads saturated
with NH4OH.
Magenta colonies that are
approximately 1 to 2 mm in diameter
are enumerated as C. perfringens.
ISO 14189
Inoculate TSCA by the pour-plate
method (1 mL)
Incubation: 18-24 hours at 37°C or 44°C
under anaerobic conditions
Colonies producing hydrogen sulfide are
characterized by blackening due to the
reaction with sulfite and iron salt

31. PSEUDOMONAS
ISO 16266
Membrane filtration 0,45 µm, CN-agar
at 36°C±2 for 24 h
Blue-green or brown pigmentation is a
presumptive evidence of Pseudomonas
spp.
Confirmation:
- Oxidase test (positive)
- Florescence on King-agar
- Growth at 42°C±2 for 24 h
- Production of NH4 from acetamide
Pseudomonas on CN-agar Oxidase test
Pseudomonas florescence
on King-agar
Acetamide test

32. SPECIAL ANALYSES
Campylobacter (ISO 17995:2005)
Salmonella (ISO 19250)
Legionella (ISO 11731-1 and -2)
Giardia and Cryptosporidium (ISO 15553)
Enteric virus (SFS-EN 14486)
Bacteriophages (SFS-EN ISO 10705-1 and -2)