Transcript Microorganisms (The Coliform Group Bacteria)

Introduction

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
Introduction
 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
water:
 Bacteria
 Virus
 Protozoa
 Fungi
Bacterial Contamination
• Water borne Disease
can caused by
different type of
bacteria.
• Most of bacterial
pathogen may found
in distribution system
illustrate in this table.
organism
Salmonella typhi
Salmonella paratyphi
Salmonella typhimurium
Other salmonella sp.
Shigella
Vibro cholerae
Enterovirulent E.coli
Yersinia enterocolitica
Campylobacter jejuni
Legionella pneumophila
Major disease
Typhoid fever
Paratyphoid fever
gastroenteritis
Gastroenteritis(salmonell
osis)
Bacillary dysentery
cholera
gastroenteritis
gastroenteritis
gastroenteritis
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
water.
Primary Indication of Contamination
Total coliform bacteria
The organisms most commonly used as
primary bacterial indicators of faecal
pollution are the coliform group which .
are:

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
system.
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

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
- Chlorine dioxide
Quaternay-
Physical Treatment
- Ozone
--- Heat
- Mechanical removal
ammonium compounds.
- Anionic and
nonionic surface
active agent.
Methodology
Methodology
This is microbiologically and
chemically study to detect the quality
of water distribution system.
Methodology
Distribution of samples:
Type of sample
Number of sample before
treatment
Number of sample after
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
Methodology
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
Methodology
Reservoirs Sample Collection
- Sterilization of outer surface of bottle by
70%
ethanol
- Dipping of sterilized bottle inside
reservoirs
using long forceps.
Methodology
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%
ethanol.
- Water were allowed to run for 5
minutes
before the sample was taken.
Methodology
Microbiological analysis
Media and Reagent
- Nutrient Agar
-Endo-media
Total plate count analysis
- 100µl of sample were spread
on
NA by L- shaped glass
rod.
- Incubation at 37C for 24
hr
before counts was done.
- Colonies were counted
as
CFU/100ml.
Methodology
Total Coliform
Membrane Filtration Method:
-Filtration of 100
ml
water sample
on
membrane
filter.
-Picked up filter paper
on specified media.
- Incubation at 37C
for
for 24hr
Methodology
- Pink colony counted as
presumptive
total coliform.
- Green metallic colony counted
presumptive E.coli.
as
Methodology
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
Methodology
PH Measurement
Using pH meter
Dissolved Oxygen
Using DO meter
Result and
Discussion
Microbiological
Analysis
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
growth.
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
auroginosa
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
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
coliform.
Total coliform (TC)

23% of all reservoirs
sample were contaminated
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
samples were
contaminated with
Total coliform, and
only 20% of samples
were negative for TC.
Chemical Analysis
Chemical analysis
Chloride



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
Standard deviation
mun
115
572
372.7
68.38
filter
22
355
75.5
40.4
Geometric mean
365.5
68.82
Minimum
Maximum
Average
Chloride

Lowest percentage failure
chloride were found in N
and D building, while
highest percentage failure
were found in E and C
building.
Central filter has the
ability to decrease 70% of
chloride concentration
from the main sources
100
95
90
85
80
75
C
L
N
D
E
350
300
250
ppm

200
150
100
50
0
1
2
3
4
5
6
7
8
9
Nitrate analysis:
The efficiency of central filter to decease nitrate
concentration is about 40%.
70
60
50
ppm

40
30
20
10
0
1
2
3
4
5
6
7
8
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,
9:D000
9
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
PH

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
Minimum
Maximum
Average
Standard deviation
Geometric mean
MUN
3.4
FILTER
2.8
11.4
6.15
1.37
11.7
6.06
1.44
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
results.
120
100
80
60
40
20
%TC
%nitrate>70
%CL
0
C
L
N
D
E
120
1.E+05
100
1.E+04
ppm
80
1.E+03
60
1.E+02
40
20
1.E+01
0
1.E+00
1
2
3
4
5
6
nitrate
TPC
1: reservoir (main)
2: E: 507 bath, 3:407, 4:
E307, 5: E207, 6:E: 107,
7: E007
7
8
CFU/100m l
Analysis and correlation of
microbial and chemical test
Analysis and correlation of
microbial and chemical test
80
3500
3000
2500
2000
1500
1000
500
0
60
40
20
0
7
6
5
4
3
2
ml
Correlation between TPC and nitrate
concentration in C-building (mun)
CFU/

1
TPC
nitrate
1: mun source, 2: reservoir (mun), 3: mun out let C5011
4: C401, 5: C301, 6: C101
Result
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
concentration.
This elevation may be due to many
reasons including:

Inadequate or insufficient treatment were
applied

Biofilm sloughing from pipes.

Disinfectant resistant may be another cause
of increase the contamination after
treatment.
Tc of ref samples before and after
treatment
400
300
200
tc
100
tc*
0
7
6
5
4
3
ref sample
2
1

The following figure show the correlation
between chloride and nitrate concentration and
total coliform count were tested for refrigerator
sample after treatment
350
300
250
200
150
100
50
0
TC
nitrate
cl
before
after
Total coliform, nitrate and chloride concentration in
refrigerator sample (N100)
Total coliform, nitrate, and chloride concentration
in central filter
250
200
150
100
50
0
1
2
3
1: Central filter before treatment
2: Central filter after treatment
3: Central filter after change the station
nitrate
tc
cl
Conclusion and
Recommendation
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
consumers.
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
considered.
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