Transcript Presentation - Standardsx

NEAGU LILIANA
Microbiologist for Romanian National Institute of Public Health
Dr.A. Leonte 1-3, Sector 5, Bucharest, Romania
[email protected]
European Drinking Water Directive 98/83 EC
European Drinking Water Directive 98/83 EC
E. Coli and coliforms
EN ISO 9308-1 or
EN ISO 9308-2
Intestinal enterococci
EN ISO 7899-2
Total count
EN ISO 6222
Pseudomonas aeruginosa
EN ISO 16266
bottled water only
Clostridium perfringens
EN ISO 14189
surface water only
Water quality – Sampling for
microbiological analysis
EN ISO 19458
General guide to the enumeration
of microorganisms by culture
EN ISO 8199
Limits for drinking water
Limits for bottled water
* According to this directive all tests (excluded the
detection of total count) must be made by
membrane filter method.
* The membrane filter must have a pore size of
o,45µm sufficient to retain the bacteria.
* Sampling shall be carried out in accordance with
ISO 19458.
Detection and enumeration of E.coli and coliform
bacteria – according to EN ISO 7899-1
E. coli
Coliform bacteria
Membrane filtration unit
Disinfect by flaming
Put sterile membrane filter on filtration unit
Filtrate 100 ml sample
Put membrane filter on Chromogenic
Coliform Agar (CCA)
Incubation at (36±2)ºC for (21±3)h.
Escherichia coli
Coliform bacteria
- Count all colonies giving a positive β-D-galactosidase and
β-D-glucuronidase reaction (dark –blue to violet) as E. coli
- Count all colonies giving a positive β-D-galactosidase reaction (pink
to red) as presumptive coliform bacteria that are not E.coli
Identification coliform bacteria
The confirm the presumptive coliform bacteria that are
not E.coli.
Preparing subculture onto a non selective agar at
(36±2)ºC for (21±3)h:
- oxidase test (commercial tests can be used).
Detection and enumeration of E.coli and
coliform bacteria – according to EN ISO 7899-1
Expression of results
Calculate the numbers of E.coli and coliform bacteria present
in 100 ml of the sample in accordance with EN ISO 8199.
The count of coliform bacteria is the sum all oxidase
negative pink to red colonies plus all dark –blue to violet
colonies.
E. coli are all dark-blue to violet colonies
Detection and enumeration of E.coli and coliform
bacteria – according to EN ISO 7899-1
Calculation of results
Since each colony is assumed to have arisen from on microorganism or from a single aggregate of micro-organism, the results
is expressed as the number of colony-forming units(cfu)or colonyforming particles(cfp) in a specified reference volume of the
sample by Equation:
Detection and enumeration of E.coli and coliform bacteria –
according to EN ISO 7899-1
Where:
Cs = estimate of the number of cfu or cfp in the reference volume
Vs
Z = is the sum of colonies counted on plates or on membranes
derived from dilutions d1, d2, …di, or derived from separate volume
of the test portion (sample or dilution)
Vs = is the reference volume
Vtot = is the calculated total volume of original sample included in
the plates enumerated.
Vtot = (n1V1d1) + (n2V2d2) ….+(niVidi), where:
n1, n2, ni = is the number of plates counted for dilution d1, d2,… di
V1, V2, Vi = is the test volume use with dilution d1, d2,… di
d1, d2,di =is the dilution used for the test volume V1,V2,Vi (d=1 for
an undilute sample, d= 0,1, for a ten-fold dilution, etc)
Case after identification or confirmation
After identification or confirmation, calculate for
each of the plates the confirmed results as the
proportion of presumptive colonies complying with
the identification or confirmation criteria, usig
Equation:
Where:
x = is the estimated number of confirmed colonies per
plate;
k = is the number of colonies complying with
identification or confirmation criteria among the
inoculated colonies n;
n = is the number of presumptive positive colonies
inoculated from a plate for confirmation;
z= is the total number of presumptive positive colonies
counted on the plate
Detection and enumeration of intestinal enterococci –
EN ISO 7899-2
microbiological parameters
- gram-positive, catalase-negative, facultative anaerobic cocci that
grow in pairs (diplococci) or in short chains, and possess
Lancefield’s Group D antigen;
- bacteria which are able to reduce 2,3,5-triphenyltetrazolium
chloride to formazan and to hydrolyse aesculin at 44ºC;
- the main species are:
- Enterococcus faecalis
- Enterococcus faecium
- Enterococcus durans
- Enterococcus hirae
Considered indicators of fecal pollution, can be found in water,
soil, where they can survive for longer than coliforms.
Detection and enumeration of intestinal enterococci – EN
ISO 7899-2
membrane filtration technique
- 100 ml water sample
- filter on Slanetz and Bartley medium
- incubation at (36±2)ºC for (44±4)h
CONFIRMATION AND ENUMERATION
After incubation, consider all raised colonies which show a red, maroon or
pink colour, either in the centre or throughout the colony, as typical.
If there are typical colonies, transfer the membrane and the colonies, with
sterile forceps without inverting it, onto a plate of bile-aesculin-azide agar
which has been preheated to 44°C
Incubate at 44°C ± 0,5ºC for 2h.
Regard all typical colonies showing a tan black colour in the surrounding
medium as giving a positive reaction and count them as intestinal enterococci.
Detection and enumeration of intestinal enterococci –
EN ISO 7899-2
Expression of results
Calculate the results in accordance with ISO 8199.
Procedure – preparation and inoculation
Preparation of the sample
Place a volume of the test sample (or it dilution) not
exceeding 2ml in the Petri dish.
Procedure – preparation and inoculation
Add 15 ml to 20 ml of the molten medium, allow to cool and
maintain it at (45±1)ºC in the Petri dish.
Time between addition of the test sample (or it dilution) and
addition of the molten medium shall not exceed 15min.
The inoculated doublets for incubation at each temperature.
Procedure – preparation and inoculation
Mix carefully by gentle rotation.
Leave the Petri dish on a flat surface aprox. 10 minutes
until the gel completely solidifies.
Procedure – incubation and examination
Invert the plates and incubate one set at (36±2)ºC
for (44±4)h;incubate the other set at (22±2)ºC for
(68±4)h.
Examine the plates as soon as they are removed from
the incubators.
Examination of the plates
>300 colonies
Countig of colonies
Count all colonies (from the surface and interior of
the culture medium).
A counter of colonies with a magnifying glass helps
with counting small colonies grown in culture
medium.
Calculate the results in accordance with ISO 8199.
”Water is the essence of life”