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Chapter 1

THE PROBLEM AND ITS SCOPE

Introduction

The occurrence of water contaminants such as microorganisms specifically

Escherichia coli and other bacteria in drinking water presents a detrimental effect to an

individual’s health. Accoording to Livestrong (2016), the health effects of drinking non

potable water can range from no physical impact to severe illness. Symptoms include

gastrointestinal and stomach illnesses like: nausea, vomiting, cramps, and diarrhea. Thus, the

evaluation for the presence of water contaminants in drinking water is very essential to

ensure a safe and quality drinking water to the public.

The study of Zoleta et al. (2016) on drinking water from well and spring water

sources revealed the presence of bacterial contamination especially at the treatment source. It

is very alarming to know that a lot of people could get sick even with the simple act of

drinking water. People will never know how safe the water they drink unless they have it

tested and monitored on a periodic basis by the City Health Sanitation Officer.

In order to minimize water contaminants, it is a necessity for the water to be

disinfected. One of the disinfectants that is widely used is chlorine. However, the presence of

chlorine that would exceed 4mg/L in the drinking water could cause cancer (Home Plus

Products 2016). As indicated by the ​National Primary Drinking Water Regulations (2016),
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drinking water contaminated with the byproducts of chlorine, could lead to liver, kidney or

central nervous system problems and increased risk of cancer. According to Dr. Group

(2016), Trihalomethanes (chlorination byproducts), or THMs is formed when chlorine is

added to water as it combines with other natural compounds. As a result, these chlorine

byproducts triggers the production of free radicals in the body, causing cell damage, and are

highly carcinogenic. Although concentrations of these carcinogens (THMs) are low, it is

precisely these low levels that cancer scientists believe are responsible for the majority of

human as determined by the Environmental Defense Fund (2016).

In addition, the pH of the water is also one of the risk factors in producing quality

drinking water. Drinking water with a pH < 6.5 could be acidic, soft, and corrosive. Acidic

water is associated with an increase level of toxic metals such as iron, manganese, copper,

lead, and zinc. Thus, acidic water pose health risks associated with these toxic metals. On

the other hand, water with a pH > 8.5 denotes that the water is hard. Hard water does not

pose a health risk, but can also cause aesthetic problems. (A​PEC Water, 2016).

In order to minimize the health risks from drinking contaminated water, it is a

necessity that water refilling stations should adhere to the Department of Health (DOH)

Implementing Rules and Regulations of the Sanitation Code of the Philippines, stipulated

in Presidential Decree 856. As outlined in the DOH regulations, water refilling stations must

be located at least 25 meters away from any direct source of pollution, and not in an area

subject to flooding. Proprietors are required to have water samples tested by a

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DOH-accredited laboratory to comply with the Philippines National Standards for Drinking

Water (PNSDW) (Rappler, 2014).

It is a requirement that a water refilling station should ensure the potability of the

drinking water they produce. A quality drinking water should have a pH of 7 ​(A​PEC Water,

2016)., a chlorine content of >4mg/L ​(Home Plus Products 2016), and microorganisms such

fecal coliform and E. coli should have a maximum contaminant level of 5% in ​mg/L

(EPA,2008) and should not be detectable in every 100 mL sample (DOH, 2008).

In addition, t​here are cases of death here in the Philippines such as the case in

Pampanga wherein 20 children died due to the consumption of water contaminated with E.

coli (Manila Times, 2011). It is said that the source of the contaminated water is from a poso.

Thus, it is recommended to have the drinking water disinfected. To avoid such incidence, a

lot of people consume bottled water from water refilling stations since it is expected that

these water refilling stations are producing safe and clean water for it had undergone steps to

purify water.

However, ​70 percent out of the 630 licensed water refilling stations in two cities in

Metro Manila did not fully comply with the strict guidelines of Department of Health (DoH)

Administrative Order 2007-012 or the Philippine National Standards for Drinking Water

(Philippine Star, 2015). More than 80 percent of the water refilling stations did not comply

with DoH requirements that their personnel wear proper attire such as mask, hair net, gloves
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and scrub suit to prevent water contamination while 35 percent of water refilling stations

were not using the proper procedure for thoroughly cleaning and sanitizing the water

containers. (Philippine Star, 2015).

With the cases presented wherein a number of water refilling stations violated the

strict guidelines of Department of Health, the assurance of having a clean and safe drinking

water is not totally attained.

The researchers were driven by the fact that there are some water refillling stations

especially in the Philippines who didn't apply the guidelines of DOH. The researchers

assumed that water contaminants may be detected in drinking water. To help the community,

the researchers concluded to conduct a study concerning about the potability of bottled water

in water refilling stations around the locality. In this manner, it would be very beneficial for

the individuals in the community.

Conceptual Framework

This study is anchored on the concepts of:

Concept #1 Non potable water may be contaminated with microorganisms and

chemicals which is harmful for people who consumes it. As stated by wisegeek, the safety of

water needs to be assessed with tests which look for potential harmful contaminants.

Concept #2 Adding chlorine to drinking water as one of the standard in water

treatment. Thus, its byproduct can trigger the production of free radicals in the body that can
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cause cell damage and are highly carcinogenic as supplemented by Chlorine, Cancer and

Heart Disease (2011).

Concept #3 A drinking water with a pH of <6.5 that is associated with an increase

level of toxic levels, whereas a pH of >8.5 is associated with aesthetic problems as supported

by Kulthanan et al (2013).

Concept #4 E.coli as one of the parameters in checking if the drinking water is

potable or not. As what the Apec Water Company states that E.coli can produce a powerful

toxin and can cause severe illnesses.

INDEPENDENT VARIABLE DEPENDENT VARIABLE

Figure 1. An interplay of Independent and Dependent Variables

Objectives

The study aims to determine the incidence of contamination by coliform bacteria

among select water-refilling stations around Cagayan de Oro City. Particularly, it seeks to

attain the following objectives:

1. To determine potability of bottled water from select water refilling station in

terms of:
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1.1 Bacterial Contamination

1.2 Chlorine

1.3 pH

To determine the significant difference between the number of contaminated drinking

water and the number of non-contaminated water.

2. To compare the severity of contamination among the subjects.

Null Hypothesis

Ho1: In terms of Bacterial Contamination, pH and Chlorine, there is no significant

difference in the quality of drinking water.

Significance of the study

This section will provide a brief description on the various significances of the study

given the five categories: homeowners; water refilling station owners and employees; local

government agency; international health agency; and educational.

Homeowners. The study will give homeowners assurance that the drinking water

from refilling stations are safe and uncontaminated.

Water refilling station owners​. This study will be beneficial to water refilling

station owners in establishing good reputation in providing safe, quality, and clean drinking

water. It will also help their business excel and gain more trust to the people that will make

their water refilling station, a reliable one.

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Water refilling station employees​.This study will benefit the workers to be

completely informed about the purity of the drinking water they produced and to be more

cautious in their service. It can also help the workforce to be more confident in their daily

amenity and produced more satisfying products to the community.

Department of Health. ​This study helps their agency in assuring that all water

refilling stations provides a safe and clean drinking water to the individuals in the community

Future researchers. ​The study will benefit the future researchers and guide them in

their research in determining the incidence of coliform contamination around the city of

Cagayan de Oro.

CDC. ​The Center for disease control and prevention agency can benefit in this study

through the prevention of coliform borne diseases.

Scopes and Limitations

The study aims to investigate on the potability of bottled water that covers the

detection of water contaminants such as microorganisms, disinfection byproducts and as

well as the pH of the water only. But does not include the detection for the presence of

inorganic and organic chemicals, minerals, and radionuclides.

In this study, the specific coliform that will be studied are the total coliform bacteria

and fecal coliform specifically Escherichia coli and the particular disinfection byproduct is

the Total Trihalomethanes (TTHMs), a chlorination byroduct.

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The study includes in its scope those select water refilling stations located within the

area of Cagayan de Oro City. Additionally, the study will focus only on small water refilling

station businesses. The method of subject determination that will be applied is the random

sampling. Since the drinking water from the select water refilling stations, are randomly

selected. A total of twelve (12) samples shall be collected from the four (4) water refilling

stations selected. The research is only limited to identifying the presence of microorganisms,

chlorination byroduct and pH of the water but does not include preventive measures to inhibit

the bacteria from causing effects on the people drinking the contaminated water.

Definition of Terms

Bacteria. ​This refers to a large member of a large group of unicellular

microorganisms that have cell walls but lack organelles and an organized nucleus, including

some that can cause disease.

Coliform​. This refers to a type of bacteria that could cause possible intestinal illness.

Coliform inhabits the intestinal tract of warm blooded animals, as well as in plants, soil, air,

and the aquatic environment

Chlorine. ​This refers to ​a substance used for disinfecting any contaminants in

drinking water

Contamination. When a certain substance ( in this case drinking water) contains or

is infected with Coliform bacteria of any sort. The substance is known to be what we call

contaminated.
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Disease​. This refers to a disorder caused by coliform bacteria that affects the structure

or function in a human, especially one that produces specific signs or symptoms or that

affects a specific location and is not simply a direct result of physical injury.

Escherichia Coli​. This refers to a specie of rod shaped, facultatively anaerobic

bacteria in the large intestine of humans and other animals, sometimes pathogenic, which can

cause diarrhea and vomitting.

Fecal coliforms. ​This refers to coliforms found in the feces of various warm-blooded

animals.

Incidence. This refers to the number of cases of coliform contamination in the area

covered by the study.

Potable​. This refers to a quality of water of being drinkable and free from water

contaminants.

Purification. ​This refers to the process of removing undesirable contaminants ( in

this case coliform bacteria). The goal of this process is to produce water fit for a specific

purpose.

Total coliforms. This refers to atotal large collection of coliform which are generally

harmless. If presence is detected it would indicate environmental contamination rather than

fecal contamination.
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Chapter 2

REVIEW OF RELATED LITERATURE AND STUDIES

This chapter presents the review of related literature and studies that have an

important direct bearing the present study.

Foreign Literature and Studies

Water and life are intrinsically connected. Just like air, that is very essential so as

water in the human body. As claimed by Listovative (2016), there are about 4 million people

who die each year because of ingestion of contaminated water. One of the most common

water contaminants that is responsible for gastrointestinal diseases is the coliform bacteria.

Coliform bacteria including E.coli is enteropathogenic associated with gastroenteritis,

cholera, meningitis, typhoid, paratyphoid fever and other illness caused by E.coli. a study
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conducted by, J App Pharm, Syed at el., 2012 the WHO/UNICEFF estimates that 500 million

diarrhea cases reportedly take place each year in children less than five years in Asia, Africa

and Latin America (WHO/UNICEF, 2010). This is because humans have limited access to

water and other factors may also include the water sources which is contaminated with

coliform bacteria, and unacceptable levels of undissolved chemicals.

The presence of coliform form bacteria in water refilling stations is essential in our

study. The most common test done to determine the safety of drinking water is by testing the

presence of coliform bacteria in water. Coliform bacteria include a large group of many types

of bacteria that occur throughout the environment (© The Pennsylvania State University

2016) they may live and inhabit in soil, in surface water and in human skin. but most type of

coliform bacteria cause no harm to humans if there is no presence of |E. coli; but there are

still some that may bring mild to chronic illness to humans such as Gastrointestinal illness

which diarrhea, vomiting, cramps are example of this illness caused by the human and

animalwaste. Total coliform used as an indicator that other potentially harmful bacteria mayb

present whereas the Coliform are naturally present in the environment and the fecal coliform

and E.coli may come from human and animal waste and disease-causing microbes may pose

a special health risk of infants, young children, and people with severely compromised

immune system. The presence of bacteria from each progressively smaller subset heightens

the concern that disease causing organisms may also be present in the water just like the fecal

coliform. Fecal coliform is a subset of the total coliform group. Fecal coliform bacteria

inhabit the intestines of mammals. They have a relatively short life span compared to other
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coliform bacteria. Whenever this organism is present, it would entail that there is an improper

disposal of sanitary waste. Immediate public notice and a boil order to the users (within 24

hours) are required due to the higher likelihood of disease organisms also being present in

water. Another subset in the fecal coliform is the microorganism Escherichia coli (E. coli).

E.coli originate only in the intestines of animals including humans. Just like other fecal

coliform, they have a relatively short life span compared to nonfecal coliform bacteria. Their

presence indicates a strong likelihood that human or animal wastes are entering the water

system. Immediate public notice and a boil order (within 24 hours) are required due to a

higher likelihood of disease organisms also being present in the water (©New Hampshire

Department of Environmental Services 2010).

E.coli are easily controlled with chlorination, but can cause deadly outbreaks given

conditions of inadequate or no disinfection. While protecting against microbial

contamination is the top priority, water systems must also control disinfection byproducts

(DBPs), chemical compounds formed unintentionally when chlorine and other disinfectants

react with natural organic matter in water. In the early 1970s, EPA scientists first determined

that drinking water chlorination could form a group of byproducts known as trihalomethanes

(THMs), including chloroform. EPA set the first regulatory limits for THMs in 1979. While

the available evidence does not prove that DBPs in drinking water cause adverse health

effects in humans, high levels of these chemicals are certainly undesirable. There are

chlorine byproducts that trigger the production of free radicals in the body that will cause cell

damage and they are highly carcinogenic. It is a pesticide and its sole purpose is to kill living
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organisms. When people consume water containing chlorine , it destroy cells and tissues

inside the body. Dr. Robert Carlson, highly respected University of Minnesota researcher

whose work is sponsored by Federal Environment Protection Agency, sums it up, “the

chlorine problem is similar to that of pollution” and adds that “chlorine is the greatest

crippler and killer of modern times!” In such a way, several diseases like “Breast Cancer”,

that affects one in every eight women in North America, has recently been linked to the

accumulation of chlorine compounds in the breast tissue. A study carried out in Hardford

Connection the first of its kind in North America found that; “women with breast cancer have

50% to 60% higher levels of organochlorines (chlorination byproducts) in their breast tissue

than women without breast cancer.” Some cases also acclaimed that “Chlorine Inhalation” is

harmful. Exposure to such substance inhaled while showering can contain up to 50 times the

the level of chemicals and most other contaminants vaporize much faster and at a lower

temperature than water. The inhaled chlorine gas (chloroform) directly goes to the

bloodstream causing so much harm to the body. It may be filtered by the kidneys and

digestive system but only partial. Chlorine vapors, known to be a strong irritant to the

sensitive tissue and bronchial passage inside the lungs; it was used as a chemical weapon in

World War II. Suspected illness such as asthma and brochitis, especially in children, that

increased 300% in the last two decades.

According to WHO, improvements in the quality of drinking water appear to be of

significant benefit to health when the improvement is done close to the point of use, that is in

the household. In a report, the UNICEF said that treating water at the household level is one
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of the most effective and cost-effective means of preventing waterborne disease, especially in

development and emergency settings. The United states Environmental Protection Agencys

states that there are two classification of public water drinking system these are the

community and non-community system.

It is a necessity for public water systems to deliver safe and reliable drinking water to

their customers 24 hours a day, 365 days a year. If the water supply becomes contaminated,

consumers can get seriously ill. Fortunately, public water systems take many steps to make

sure drinking water is safe. One of the most important steps is the regular testing for

coliform bacteria. Coliform bacteria is as referred to as "indicator organism" because they

indicate the potential presence of disease-causing bacteria in water (© The Pennsylvania

State University 2016).

The total coliform is considered an indicator, because it’s presence would indicate the

possibility that an organism might be be present in the drinking water. But as stated by

Extension Organization (2016), if a water sample is tested to be positive for total coliform

bacteria, it does not necessarily mean that it is unsafe for consumption. Drinking water would

only become unsafe if the water sample is tested to be positive for fecal coliform bacteria

which would indicate recent fecal contamination. Detection for fecal contamination is

possible with the use of the standard total coliform test. Thus, when total coliforms are

absent, disease causing organisms are less likely to be found in the drinking water.

Practically, it is impossible to frequently check every type of disease causing

organism thus, the reliability of the total coliform test is very critical in determining bacterial
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safety relative to the hundreds of possible diseases. However, Giardia and Cryptosporidium

being an exception to this generalization, can be present in water even when the total

coliform test reveals a negative result for the presence of organisms. When Giardia and

Cryptosporidium are present in water, illness could occur. Nevertheless, the total coliform

test remains the most commonly used test in determining the bacterial quality of drinking

water in the US and also the world (©New Hampshire Department of Environmental

Services 2010).

The features of coliform bacteria in water has no smell, no taste and no color, the only

way to know if water is contaminated by these bacteria is through laboratory testing.

according to Canadian Drinking Water Quality Guideline, maximum Acceptable

Concentration for Drinking Water = none detectable per 100 mL so this means that for every

100 ml of drinking water tested, there should be no E. coli detected (© The Pennsylvania

State University 2016).

There are proper procedures on water purification that the Department of Health

requires and water refilling stations must abide on these rules to prevent contamination.

Water must be filtered first to trap and remove large impurities and if the water is cloudy, it

can be made to stand for half a day, after which, clear water can be scooped and then filtered.

Boiling water kills all potential germs you may get from contaminated water. Heat water and

allow it to boil for two (2) minutes then let it cool with the boiled water that has been cooled

can be used immediately. Stirring it or putting it from one clean container to another

container several times or adding a pinch of salt or powdered juice can improve taste. Water
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can be disinfected by adding 2 drops of 5% chlorine solution (unscented bleach) for every

one (1) liter of unrefrigerated water. Allow it to stand for an hour. If a faint chlorine smell is

detected after an hour, then it is safe to drink and repeat the procedure if you cannot detect

the smell after your first tries. If it does not have the faint chlorine smell on the third try then

the water must be discarded since it may contain many germs. To improve taste, allow it to

air some more or transfer it from one clean container to another several times. The National

Primary Drinking Water Regulations (NPDWR) are legally enforceable primary standards

and treatment techniques that apply to public water systems. Primary standards and treatment

techniques protect public health by limiting the levels of contaminants in drinking water. The

Maximum Residual Disinfectant Level Goal (MRDLG), Maximum Residual Disinfectant

Level (MRDL), and Maximum Contaminant Level (MCL) measures the byproducts chlorine

(Cl2), total trihalomethanes (TTHs) and total coliform. For chlorine, the MRDLG is 4 mg/L

and its MRDL is 4.0 mg/L. The effect for long term exposure is eye or nose irritation and

stomach discomfort. For total trihalomethanes, the MCL is 0.080 mg/L and long term

exposure may cause liver, kidney or central nervous system problems; increased risk of

cancer. For the total coliform the MCL is 5% and long term exposure can bring no harm to

health and can be used to indicate whether other potentially harmful bacteria may be

present.5

E.coli​ are easily controlled with chlorination, but can cause deadly outbreaks given

conditions of inadequate or no disinfection and more precise indicator of fecal pollution, the

count of thermotolerant coliform bacteria is an acceptable alternative. If necessary, proper

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confirmatory tests must be carried out. Total coliform bacteria are not acceptable indicators

of the sanitary quality of rural water supplies, particularly in tropical areas where many

bacteria of no sanitary significance occur in almost all untreated supplies.

It is recognized that, in the great majority of rural water supplies in developing

countries, fecal contamination is widespread.

Local Literature and Studies

There are about 50 percent of the region’s underground water is contaminated with

fecal coliform, a type of bacteria from feces of humans and warm-blooded animals based on

the results of the study conducted by the DOH-6(2014). DOH-6 said fecal coliform can cause

skin allergies and waterborne diseases that can lead to intestinal illness such diarrhea,

cholera, abdominal pain and etc.

According to Villalobos (2014), most of the water stores get their water from deep

well, shallow tube-well and Metro Iloilo Water District (MIWD). The primary cause of

contamination, according Villalobos, are the septic tanks built less than 25 meters away from

deep wells and water pumps. In Iloilo City, there are around 200 water refilling stations

operating but some of them have no sanitary permits. The city health office issues a sanitary

permit to a water refilling station after the submission of an operational permit issued by the

DOH.

There are more than 20,000 water refilling stations in the Philippines ,where up to 40

% of households nationwide and 60% of Metro Manila residents buy drinking water, this is

emphasized by Healthy family (2015). From the research of the Pediatric Infectious Disease
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Society of the Philippines, they focused on the characteristics of water refilling stations such

as disinfection, frequency of monitoring, personnel hygiene, attire and practices may affect

the safety and portability of the production of clean and safe drinking water.

In addition, the data gathered by an independent research agency showed that during

the time of sanitary inspection, 70 percent out of the 630 licensed water refilling stations in

two cities in Metro Manila did not fully comply with the strict guidelines of Department of

Health (DOH) Administrative Order 2007-012 or the Philippine National Standards for

Drinking Water (Tribune Wires, 2015).

More than 80 percent of the water refilling stations did not comply with DOH

requirements that their personnel wear proper attire such as mask, hair net, gloves and scrub

suit to prevent water contamination while 35 percent of water refilling stations were not

using the proper procedure for thoroughly cleaning and sanitizing the water containers.

(Tribune Wires, 2015).

The presence of coliform bacteria can also alter the quality of drinking water.

Coliform bacteria is typically present in the environment ( water and vegetation ) and is

harmless like E.coli but some strains can cause illness. If this will be seen in drinking water

in the laboratory, it is probably from environmental sources or fecal contamination. Argued

by DOH (2016) , the most outbreaks of E.coli 0157:H7 a subgroup of coliform group

received a lot of media coverage. Boiling or disinfecting contaminated water can also help in

destroying all forms of E.coli , including 0157:H7. In addition, confirmation of E.coli in a

water system will most likely indicates fecal contamination , that may be pose an immediate
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health risk to anybody who consumes the water. Clean, drinkable water that is consumed by

Filipinos daily are from water refilling stations. According to (The Pediatric Infectious

Disease Society of the Philippines), although water from water refilling stations are not

contaminated with microorganisms, other risk factors can affect the purity of the water.

through unsanitized containers, poor personnel hygiene and improper handling, storage and

transport of refilled water. Inappropriate clothing and attire is also included.

Sanitation Code of the Philippines, stipulated in Presidential Decree 856 requires that

water refilling stations must adhere to the rules and regulations implemented by the

Department of Health (DOH).

The DOH requires that water refilling station personnel’s must wear proper protective

gear. such as gloves, face masks, hair nets, and scrub suits to prevent purified water from

coming into contact with any part of the body.

The DOH also demands that containers should be properly sanitized and cleanse

before filling them with purified water.

Chapter 3

MATERIALS AND METHODS

This chapter presents the research setting, design, instruments, protocol, and

speficic methods employed by the researchers in the data gathering and analysis.

Research Setting
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The research will be conducted within the city of Cagayan de Oro specifically in

water refilling stations situated at Nazareth, Carmen,Canitoan and Patag for the testing of the

potability of bottled water.

Research Design

The researchers applied a descriptive with diagnostic research design to test potability

of the drinking water samples from select places in Cagayan de Oro City.

Respondents and Sampling Procedure

The water refilling stations are the researchers target specially located at Nazareth,

Zayas,and Canitoan. Purposive sampling will be utilized in this research, since the

researchers are looking for water refilling stations that are small enterprise not bigtime to

really check and help the owners in improving business. Thus, the study targets on small and

unlicensed water refilling stations selected by the researchers. Subjective sampling is easier it

can help the researchers in detecting the presence of coliform bacteria in select water refilling

station that are small enterprise, thus bigtime enterprises are not qualified already for the

presence of coliform bacteria because they are already assured in their sanitation and

registered.

The (4) locations are selected for some reasons, there are reported cases of the

presence of coliform bacterias in that particular refilling stations. Also, the said refilling

stations , are one of the distributor of purified water in some areas and many people are

involved.

Research Instrument
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The researchers used a check list upon observation by the time the sample is acquired

from the selected water refilling stations. During the collection of sample, a 120 ml bottle is

used. A 0.1 ml of a 3% solution of sodium thiosulfate (Na2S2O3) for water that has been

chlorinated is added. The equipments used in the identification coliform are as follows: One

(1) broth ampule, one (1) m-Endo sterile buffered dilution water, one (1) membrane filter (45

micron), one (1) petri dish with absorbent pad (47-mm), one (1) Filtration apparatus with

aspirator or pump, one (1) sterilized forceps, one (1) incubator, one (1) Microscope

(low-power), one (1) Pipet(s) for dilution or for sample volumes less than 100 mL, if

necessary. Spa and Pool Master kit for testing the pH and chlorine of the water sample

Research Protocol

To ensure the quality of the research output, the researchers employed the following

research protocol:

1. The approval of the adviser after thoroughly reviewing the research proposal.

2. The Registered Medical Technologist uses Proper Protective Equipment (PPE) in

conducting the experimental study.

3. The researchers will give letters and secure permission from the heads of office to

allow them to conduct the study.

4. The researchers will secure the respondents’ consent to participate in the study.

Moreover, the respondents will be assured that all their responses are to be treated with

utmost confidentiality. Provision of the final manuscript.

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5. The researchers will provide the adviser the final copy or the research proposal for

assessment and review of the quality and relevance of the paper.

Data Gathering Procedures

Volume of sample

The volume of the water sample is ideally not less than 100 ml for it to be sufficient

to carry out all the tests required.

Sample container

Samples will be collected in a 120 ml clear bottle that have been carefully rinsed and

cleansed with distilled water. As instructed in the standard method of analysis for water and

wastewater, sterilize the sample. Sampling container should be covered with plastic screw

caps or ground glass stoppers protected by a paper or a thin aluminum foil. The neck of the

bottle should also be covered. Before sterilization, add 0.1 ml of a 3% solution of sodium

thiosulfate (Na2S2O3) for water that has been chlorinated to a clean sample bottle

Sample Collection, Handling and Storage

Keep away from contamination during collection and before examination of water

sample. The water sample should be a representative of the water under examination. The

tap should be cleaned and free from attachments and fully opened with water allowed to

waste for a sufficient time to permit the flushing/clearing of the service lines. Flaming is not

necessary. Taps that had been contaminated should be disinfected with hypochlorite

solution (NaOCl 100 mg/L). Leaking taps are avoided. No samples shall be taken from it.
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Keep the sample bottle unopened until it is ready for filling. Avoid any contamination on the

inner surface of the cap or stopper and the neck of the sample bottle. The sample bottle

should be filled without rinsing it. Enough space (at least 2.5 cm) should provided to enable

mixing of the water. Mix the water sample by shaking. Immediately replace stopper or cap.

The processing of the water sample should be done promptly within six (6) hours after

collection. If beyond six (6) six hours, it is recommended to use ice coolers for storage of

water samples during the transport of the sample. The collection and processing should not

exceed 24 hours.

Identification of Samples

Equipped with complete information, the sample bottles must be labeled with the

accurate identification and description. For analysis of water quality, the information about

the samples obtained can be recorded in a request form.

Frequency of Sampling
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Source: Department of Health, Philippine National Standards (2007)

The minimum number of samples to be collected and examined periodically must be based

on the mode and source of water supply (Table 1). For water refilling stations as the source

and mode of supply, the minimum frequency of sampling is one (1) sample monthly.

However, there are factors that should be put into consideration in the frequency of sampling

Factors such as the number of raw water sources, the adequacy of treatment and capacity of

the treatment plant, the risk of an epidemic and the practice of disinfection , the past

frequency of records yielding unsatisfactory results, risks of contamination at the source and

in the distribution system, the quality of raw water treated, and the size and complexity of the

distribution system are to be assessed.

Before sample dilution

First, set the temperature of the incubator to 35 ± 0.5 °C (95 ± 0.9 °F). Let the

incubator temperature become stable, then add the samples. Make sure that the water must
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have no coliform bacteria. Do not dilute water samples. Then wash hands thoroughly with

soap and water. Use a germicidal cloth, bactericidal spray, weak bleach solution or weak

iodine solution to clean the work area. Make sure that all of the materials that come in

contact with samples are sterile. During filtration, remove the vacuum as soon as the funnel

is empty so that the membrane filter does not become dry. As an alternative to the filter

assembly with flask, use a sterile, disposable filter unit. As an alternative to the m-Endo

broth, use m-Endo agar plates.

Sample dilution

Dilute samples that contain a high level of bacteria so that approximately 20 to 200

bacteria colonies grow on the membrane filter. First, wash hands thoroughly with soap and

water. Then invert the sample container for 30 seconds (approximately 25 times). After

inverting open a bottle of sterile buffered dilution water. Add 11 mL of sample into the

dilution water bottle using a sterile pipet. Then put the cap on the dilution water bottle and

invert for 30 seconds (25 times). This is a 10x dilution (sample is diluted by a factor of 10).

Next, add 11 mL of the 10-fold dilution to another dilution bottle (100x dilution). Mix well.

Finally add 11 mL of the 100-fold dilution to the third bottle (1000x dilution) and mix well.

Presumptive test for total coliforms

In the presumptive test for total coliforms, first invert one m-Endo broth ampule 2 to

3 times. Next, open the ampule After opening the ampule, lift the lid of a petri dish and

carefully pour the contents equally on the absorbent pad. Then set up the membrane

filtration apparatus. Use a sterile forceps to put a membrane filter in the assembly. Make sure
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that the grid side is up. Following this, invert the sample or the diluted sample for 30 seconds

(25 times) to make sure that the sample is mixed well. Then, pour or use a pipet to add the

sample into the funnel. If the volume is less than 20 mL, add 10 mL of sterile buffered

dilution water to the funnel. After that,apply the vacuum until the funnel is empty. Stop the

vacuum. And then, rinse the funnel with 20 to 30mL of sterile buffered dilution water. Apply

the vacuum. Rinse the funnel two more times. After rinsing, stop the vacuum when the

funnel is empty. Remove the funnel from the filter assembly. Using sterile forceps, lift the

membrane filter. Next step is to put the membrane filter on the absorbent pad. Let the

membrane filter bend and fall equally across the absorbent pad to make sure that air bubbles

are not caught below the filter. Then put the lid on the petri dish and

invert the petri dish. Then incubate the inverted petri dish at 35 (± 0.5) °C (95 (± 0.9)

°F) for 22–24 hours. And lastly remove the petri dish from the incubator. In counting the

number of bacteria, use a 10 to 15X microscope

Confirmation of total coliforms

For potable water samples, do the confirmation procedure on typical colonies to make

sure that they are coliforms. Confirm sheen colonies to a maximum of five. Move growth

Coliforms, Total, Fecal and E. coli, m-Endo 3 from each colony to inoculate parallel tubes of

Lauryl Tryptose (LT) single-strength (SS) broth and Brilliant Green Bile (BGB) broth.

Growth and gas production in the two tubes makes sure that the organisms are coliforms.

Most Probable Number (MPN) coliform tubes are recommended for this procedure. Use the

swabbing technique for fecal coliforms or E. coli as follows:

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1. EC or EC/MUG media

2. Lauryl Tryptose (LT) single-strength broth

3. Brilliant Green Bile (BGB) broth

Confirmation test of total coliforms

First step is to touch a sterilized inoculating needle or a sterile disposable needle to

the coliform (sheen) colony growth. Put the needle in a Lauryl Tryptose broth tube. Next step

is to touch the sterilized inoculating needle again to the same coliform (sheen) colony

growth. Put the needle in a Brilliant Green Bile (BGB) broth tube. Then, invert the tubes to

remove air from the inner vials. Gently swirl, if necessary. Followed by examining the tubes

to make sure that the inner vial is full of liquid with no air bubbles. And then, icubate the

inoculated confirmation media at 35 ± 0.5 °C (95 ± 0.9 °F) for 1 hour. Bubbles that form in

the inner vials during the first hour are not from bacteria. After 1 hour, invert the tubes to

remove air from the inner vials. Make sure that there are no bubbles and keep the tubes in a

vertical position. Loosen the caps only a little, then put the tubes in the incubator. Next step

is to incubate the inoculated confirmation media at 35 ± 0.5 °C (95 ± 0.9 °F) for 24 (±2)

hours. It is necessary to keep the tubes in a vertical position for the remainder of the test.

After 24 (±2) hours, remove the samples from the incubator. Tap each tube gently and

examine the inner vials for gas. If the broth is cloudy and the inner vials contain gas bubbles,

coliform bacteria are likely in the sample. Any gas that shows is an indication of coliform

bacteria. If no gas can be seen, put the tubes in the incubator for 24 (±2) hours (48 (±3) hours

total) and examine the tubes again. After 48 (±3) hours, gently tap each tube and examine the
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inner vials for gas. If the inner vial contains gas bubbles, the test is positive for total coliform

bacteria. If none of the tubes contain gas, then the test is negative for total coliform bacteria.

Then confirm positive results. If growth and gas occur in the Lauryl Tryptose broth tube but

not in the Brilliant Green Bile (BGB) broth tube, inoculate another Brilliant Green Bile

(BGB) broth tube from the gaspositive Lauryl Tryptose broth tube. Repeat steps 3–9 again on

the Brilliant Green Bile (BGB) broth tube. If growth and gas occur within 48 (±3) hours, the

colony is confirmed as coliform.

Confirmation test for fecal coliforms

First, use a sterile cotton swab or inoculating loop to touch all of the surface of the

membrane filter that is positive for total coliforms. Second, swirl the cotton swab or

inoculating loop in an EC Medium Broth tube to move the colonies collected from the filter

to the tube. Third, do steps 1–2 again for each test to be verified. Use one broth tube for each

test. Use the same cottom swab. Fourth, invert the tubes to remove air from the inner vials.

Gently swirl, if necessary.Fifth, examine the tubes to make sure that the inner vial is full of

liquid with no air bubbles.And then incubate the inoculated confirmation media at 44.5 ± 0.2

°C (112.1 ± 0.5 °F) for 1 hour. Bubbles that form in the inner vials during the first hour are

not from bacteria. After 1 hour, invert the tubes to remove air from the inner vials. Make sure

that there are no bubbles and keep the tubes in a vertical position. Loosen the caps only a

little, then put the tubes in the incubator. Next, incubate the inoculated confirmation media at

44.5 ± 0.2 °C (112 ± 5 °F) for 24 (±2) hours. After 24 hours (±2) hours, remove the samples

from the incubator. Gently tap each tube and examine the inner vials for gas. If the inner vial
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contains gas bubbles, the test is positive for fecal coliform bacteria. If none of the tubes

contain gas, then the test is negative for fecal

Confirmation of E. coli

To confirm E. coli, first step is to use a sterile cotton swab or inoculating loop to

touch all of the surface of the membrane filter that is positive for total coliforms. And then

swirl the cotton swab or inoculating loop in an EC/MUG Broth tube to move the colonies

collected from the filter to the tube. Repeat steps 1–2 again for each test to be verified. Use

one broth tube for each test. Use the same cottom swab. Followed by the inversion of the

tubes to remove air from the inner vials. Gently swirl, if necessary. Then, examine the tubes

to make sure that the inner vial is full of liquid with no air bubbles. After examining the

tubes, incubate the inoculated confirmation media at 44.5 ± 0.2 °C (112.1 ± 0.5 °F) for 1

hour. Bubbles that form in the inner vials during the first hour are not from bacteria. After 1

hour, invert the tubes to remove air from the inner vials. Make sure that there are no bubbles

and keep the tubes in a vertical position. Loosen the caps only a little, then put the tubes in

the incubator. Again, Incubate the inoculated confirmation media at 44.5 ± 0.2 °C (112 ± 5

°F) for 24 (±2) hours. And then, apply UV light to the incubated sample that contains MUG

broth with a long-wave UV lamp. Lastly, examine the tubes in a dark area. Look at the tube

90° from the UV light. Compare the fluorescence of the sample tubes to a tube that contains a

known E. coli positive confirmation. If the sample fluoresces, E. coli bacteria are in the

sample. If the sample does not fluoresce, the test is negative for E. coli (HACH Company,

2016).
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DOH. (2012). DOH advisory on safe drinking water. Retrieved from

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“Ensuring the quality of the drinking water, by detecting the presence of Chlorine

and E.coli in the water sample from the select water refilling stations in Cagayan de Oro

City for the safety and awareness of the consumer.”

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