Microorganisms (The Coliform Group Bacteria) by XQ68htCW


   Objectives:
   Check water distribution system in the
    university and determine contaminated
    point in the system to be treated.

   Suggest appropriate treatment to eliminate
    the possible contamination

 Limitation of the Study:
 To investigate drinking water distribution
  system in Islamic University by detection of
  primary indicator pathogenic bacteria of
  220 sample and perform three chemical
  tests including dissolved oxygen, nitrate and
  chloride on each sample and PH
  measurements as physical parameter
Literature review
Introduction of water contaminations
 Microbial contamination
 There are four main types of micro
  organism that can contaminate drinking
 Bacteria
 Virus
 Protozoa
 Fungi
Bacterial Contamination
 • Water borne Disease
   can caused by
   different type of

 • Most of bacterial
   pathogen may found
   in distribution system
   illustrate in this table.
        organism             Major disease
Salmonella typhi          Typhoid fever
Salmonella paratyphi      Paratyphoid fever
Salmonella typhimurium    gastroenteritis
Other salmonella sp.      Gastroenteritis(salmonell
Shigella                  Bacillary dysentery
Vibro cholerae            cholera
Enterovirulent E.coli     gastroenteritis
Yersinia enterocolitica   gastroenteritis
Campylobacter jejuni      gastroenteritis
Legionella pneumophila    Legionnaires disease,
                          Pontiac fever
Virus Contamination
Water may also play a role in the transmission of
 virus with different mode of action such as:
 Hepatitis A and B virus, Rotavirus and others.
   Others include:
 Poliovirus : poliomyelitis
 Enteric adenoviruses: gastroenteritis
 Hepatitis E : liver inflammation
Protozoa Contamination:
 Giardia lamblia, cryptosporidium
 parvum and Entamoeba histolytica are
 the major intestinal protozoal
 pathogens that contaminate drinking
Primary Indication of Contamination
    Total coliform bacteria
    The organisms most commonly used as
    primary bacterial indicators of faecal
    pollution are the coliform group which .
 Member of Family Enterobacteriace.
 Non spore forming bacteria
 Can ferment lactose at 35-37ºC
Total coliform bacteria
   The total coliform bacteria group
    includes both faecal and environmental
    species which include:
   Escherichia coli.
   Citrobacter.
   Klebsiella.
   Enterobacter.
Bacterial fecal indicator should be:
  •   Abundant in faeces and sewage.
  •   Absent or at least very small in
  •   number from all other sources.
  •   Capable of isolation and
      identification easly.
Total coliform bacteria

   These Organisms can survive and grow in water
    distribution system, they can be used as:

 Indictor of treatment effectiveness.

    To assess the cleanliness and integrity of distribution
    system and the potential presence of biofilms.
Faecal coliform
   E. coli is the predominant coliform in
    faeces and the only member of the coliform
    group exclusively associated with faeces.
   Other organisms can also be used as
    indicator of faecal pollution such as:
   Faecal streptococci.
   Clostridium perferingens.
Biofilm in Distribution System
 Many different microbes have demonstrated
  the ability to survive in the distribution system
  with some possessing the ability to grow and
  produce biofilms.
 Water distribution system biofilm is a complex
  mixture of microbes organic and inorganic
  material accumulated amidst a microbially
  produced organic polymer matrix attached to
  the inner surface of the distribution
Steps of biofilm development:
   Trace organic material deposits on
    water/solid interface forming conditioning
    layer which allow initial attachment of
    material cell.
   Planktonic (free floating) bacteria approach
    the pipe wall and become entrained with in
    the boundary layer where flow velocity falls
    to zero result in reversible adsorption.
   Some of reversibly adsorbed cells may
    permanently adhere the cell to the surface
    and become irreversibly adsorbed.
   Biofilm bacteria excrete extra cellular
    polymeric substance (sticky polymers)
    which :

 Hold the biofilm together.
 Act as nutrients for bacterial growth.
 Protect bacteria from biocides.
Chemical contamination
chemical contaminant of drinking water are often
considered a lower priority than microbial contaminants.

Major chemical contaminant:
   Nitrate:
   Excessive concentration of nitrate in
    drinking water can be hazardous of health,
    especially for infants.
   Methmoglobinemia result from high
    concentration of nitrate.
   Chloride in drinking water is generally not
    harmful to human beings until high
    concentration are reached, although it may be
    harmful to some people suffering from heart or
    kidney disease

   Other health effect chemical contaminants:
   Fluoride
   Sodium
   Arsenic
   Lead
Literature review
    Treatment of water contamination.

                       Sanitization Method
                           Of bacteria

 Oxidizing biocides                   Non oxidizing biocides

- Chlorine                                     Quaternay-
- Chlorine dioxide    Physical Treatment       ammonium -
- Ozone
                                               - Anionic and
                        --- Heat
                                               nonionic surface
                        - Mechanical removal   active agent.

This is microbiologically and
chemically study to detect the quality
of water distribution system.
Distribution of samples:
  Type of sample       Number of sample before   Number of sample after
                            treatment                 treatment

 Municipality source             4                         -
    Well source                  2                         -

    Central filter               2                         2
  Reservoir (mun)                37                        7
  Reservoir (filter)             20                        6
   Kitchen (mun)                 9                         -
   Kitchen (filter)              23                        7
    Refrigerator                 30                        9
        bath                     39                        -
        Lab                      12                        -
       Other                     10                        0
        Total                   188                       31
Sampling collection
 Collection of sample is vary depend on the type of the source of
 water .
 Sampling from Taps
 - Flaming the tap by 70% ethanol saturated cotton swab.
 - Water was allowed to run for at least 2-3 minutes in order
 to                       flush

 refrigerator for sample collection

Reservoirs Sample Collection

- Sterilization of outer surface of bottle by
70%           ethanol

- Dipping of sterilized bottle inside reservoirs
          using long forceps.

Well Sample Collection
 - Collection from well was performed after
   sterilization of nearest tap water before water
   passes into reservoirs.

Central Filter Sample Collection
 - Sterilization of plastic tap filter with 70%
  - Water were allowed to run for 5
 minutes          before the sample was taken.
Microbiological analysis
 Media and Reagent
  - Nutrient Agar

Total plate count analysis

 - 100µl of sample were spread
 on       NA by L- shaped glass
 - Incubation at 37C for 24 hr
   before counts was done.
 - Colonies were counted
 as           CFU/100ml.
Total Coliform
Membrane Filtration Method:

 -Filtration of 100
 ml            water sample
 on             membrane

 -Picked up filter paper
 on specified media.

 - Incubation at 37C for
   for 24hr

          - Pink colony counted as
          presumptive        total coliform.

          - Green metallic colony counted as
            presumptive E.coli.
Chemical and Physical Analysis
 Nitrate Analysis
 Ultraviolet spectrophotometric method used
 for nitrate determination.
Chloride Analysis
 Silver nitrate titration was performed to determine
 chloride concentration as mg/l.

 silver nitrate titration for chloride determination

PH Measurement

Using pH meter

Dissolved Oxygen

Using DO meter
Result and
Microbiological analysis
Total plate count (TPC)
   The range of TPC in
    tested samples varied from
    as low as 1000CFU/100ml
    to TNTC ,with the
    exception of 14 samples
    which did not show any
   TPC value increased were
    water flow from main
    sources to finished taps.
Total plate count (TPC)
   Some pathogenic bacteria
    with distinctive
    appearance were isolated
    and defined from some
    samples, such as Serratia
    and Pseudomonas

                                Red colonies of Serratia were observed
                                   on NA in filter reservoir sample
Total plate count (TPC)

Green colony of Pseudomonas auroginosa
   observed on NA in different samples
Total plate count (TPC)
    It observed that highest levels of TPC correlated with the
     age of building; older building such as (teeba, admission,
     medical service, student and academic affairs buildings)
     showed higher levels than new building.

                              TPC count CFU/100ml

    Building       L           C        TEEBA       Administration

    Minimum        0           0          4000           1000
Maximum           2000        4000      500*10³        142*10³
    Average       750         1285      303.4*10³      31.5*10 ³
Total plate count (TPC)
   During the study period, some reservoirs in
    these building were found open or their
    cover not completely secure which indicate
    that there is a shortage of inspection to
    prevent such contamination that may result
    from dust or other sources (e.g., animals,
    insects and birds).
Total coliform (TC)
   Samples collected showed      absance fromTC
                                  contamination with TC
    that approximately 76%
    were contaminated with
    total coliform with a range
    varying from one
    CFU/100ml to TNTC. The
    rest of the samples (24%)
    were negative for Total
Total coliform (TC)
   23% of all reservoirs          contamination
    sample were contaminated       with tc
                                   absance from tc

    with TC

   100% of all filter reservoir
    samples were
    contaminated with TC

   33%of all municipality
    reservoir samples were
    contaminated with TC
Total coliform (TC)
   Highest main sources contamination observed in
    well. The following table illustrate total coliform
    levels in the three main sources in the university
    during the study period.
      SOURCE                     TC count CFU/100ml

                       January       February         March
Municipality line(1)      -             1               6
Municipality line(2)      4             -               -
        Well             48             13              -
    Central filter        2             2              200
Total coliform (TC)
    80% of refrigerator
                            absanc from
    samples were            TC

    contaminated with       with TC

    Total coliform, and
    only 20% of samples
    were negative for TC.
Chemical Analysis
Chemical analysis
   It observed that:
   93% of municipality samples did not comply with WHO
    standard, it exceeded 250mg/l
   The following table summarize the result for filter and
    mun samples

                         mun                 filter
    Minimum              115                  22
    Maximum              572                 355
     Average            372.7                75.5
Standard deviation      68.38                40.4
 Geometric mean         365.5                68.82
   Lowest percentage failure    100

    chloride were found in N      95

    and D building, while         90

    highest percentage failure
    were found in E and C         75
                                             C           L       N       D           E
   Central filter has the             350

    ability to decrease 70% of         300

    chloride concentration             200


    from the main sources              100
                                                 1   2       3   4   5       6   7       8   9
Nitrate analysis:
   The efficiency of central filter to decease nitrate
    concentration is about 40%.

                          1    2    3    4     5    6    7    8   9

                        1: mean of reservoir before filtration.
                        2: central filter, 3: mean of reservoir
                            after filtration, 4: filter out let
                                     (kitchen) D500
                         5:D400, 6:D300, 7:D200, 8:D100,
Nitrate analysis:
   Summarize of nitrate result for filter and
    municipality samples illustrate in this table:

                         MUN             FILTER
        Minimum          41.75            15.07
        Maximum          120.6            82.34
         Average         69.03            36.33
    Standard deviation   16.87            13.66
     Geometric mean      67.16            34.33
  For all samples were tested for PH measurement,
  results were showed in normal range according to
  Palestinian standard (6.5-8.5).
 Summary of pH result for filter and mun samples

                       MUN             FILTER

      Minimum           6.8              7.05
      Maximum           8.4              8.21
       Average          7.83             7.53
 Standard deviation     0.28             0.25
    Geometric mean      7.83             7.52
Dissolved oxygen
   Dissolved oxygen measurement results were showed
    significant change for all samples, and this variation may
    be due to change in temperature during study period.
   Normal range for DO is (6-8ppm)
   summary of DO result for filter and municipality samples

                                   MUN                         FILTER
     Minimum                        3.4                          2.8
    Maximum                         11.4                        11.7
     Average                        6.15                        6.06
Standard deviation                  1.37                        1.44
 Geometric mean                     6.01                        5.91
Analysis and Correlation of
Microbial and Chemical test
Analysis and correlation of
microbial and chemical test
   In this study tests were performed showed different
    correlation between chemical and microbial analysis

         %TC            0
         %CL                 C   L   N   D   E
Analysis and correlation of
microbial and chemical test

          120                                             1.E+05
          100                                             1.E+04

                                                                   CFU/100m l

          20                                              1.E+01

              0                                           1.E+00
                  1    2     3    4    5     6    7   8

                      1: reservoir (main)
                      2: E: 507 bath, 3:407, 4:
                      E307, 5: E207, 6:E: 107,
                      7: E007
Analysis and correlation of
microbial and chemical test
   Correlation between TPC and nitrate
    concentration in C-building (mun)
                    80                               3500
                    60                               2500


                    20                               1000
                    0                                0
                         7   6   5   4   3   2   1

1: mun source, 2: reservoir (mun), 3: mun out let C5011
              4: C401, 5: C301, 6: C101
After Treatment
Result after treatment
Randomly    collected samples were re-tested to
measure the efficacy of water treatment and the
integrity of the distribution system

Treated  units were retested showed an
elevation of total coliform and total plate count
per 100 ml, and related changes in nitrate
This elevation may be due to many
reasons including:

   Inadequate or insufficient treatment were

   Biofilm sloughing from pipes.

   Disinfectant resistant may be another cause
    of increase the contamination after
Tc of ref samples before and after

      tc                                 100
      tc*                                0
            7   6   5   4    3   2   1
                    ref sample
   The following figure show the correlation
    between chloride and nitrate concentration and
    total coliform count were tested for refrigerator
    sample after treatment
            TC        nitrate     cl     before

    Total coliform, nitrate and chloride concentration in
    refrigerator sample (N100)
Total coliform, nitrate, and chloride concentration
in central filter






            1            2            3       tc

 1: Central filter before treatment
 2: Central filter after treatment
 3: Central filter after change the station
 Conclusion and
Conclusion and Recommendation
 It is not meaningful or practicable to strive for a
  sterile drinking-water network devoid of all
  microorganisms. The principal objective is to
  remove pathogenic organisms from the water
  supply and prevent contamination during
  distribution system.
 Chemical, physical and microbial parameter
  should meet WHO and Palestinian standard to
  avoid adverse health effect that may cause for
Conclusion and Recommendation
   The result in this research showed high average
    number of heterotrophic plate count and total
    coliform in drinking water and this is not safe for
    students and workers consumption as its levels of
    contamination are high and exceed by far the
    allowable limits, and this poses a health risk for
    the consumers.

   The High regrowth of heterotrophs and total
    coliform occurring after chlorination indicates the
    inefficiency of chlorination steps or the levels of
    chlorine in treated water were low.
Conclusion and Recommendation
   The occurrence of bacterial regrowth within
    distribution system is dependent upon a complex
    interaction of chemical, physical and operational
    parameters. No single factor could account for all
    the coliform occurrences, all these parameter in
    devising a solution to regrowth problem must be
Conclusion and Recommendation
 Sanitary inspections should be carried out
  regularly by specialist worker on all water
  distribution system in university and not merely
  the points were analyzed.
 Periodically maintenance of Reverse Osmosis
  purification system should be taken to check the
  efficiency of R\O in filtration of water to prevent
  microbial contamination that may be occurred in
  the distribution system.
Conclusion and Recommendation

   Educate students for good use of the
    refrigerators which is used for drinking in
    the university and use it carefully with good
    hygiene to prevent contamination that may
    be occur inside
                   Thanks TO

                 Dr.Abdelraouf Elmanama

         Medical Technology Department

   Environmental and Rural Research Center

             Administration of University

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