DETECTION AND IDENTIFICATION OF GRAM-NEGATIVE
PATHOGENIC BACTERIA PRESENT IN TIMOGA
CHILDRENS SWIMMING POOL SURFACES
Researchers:
Larren Blaze O. De Guzman
Alta Faye D. Labella
Norhanifa T. Macatangcop
Research Adviser:
Helen T Rizabal, RMT, CAR MAEd
Adventist Medical Center College
ABSTRACT
Swimming pools are man-made
container filled with water intended
for swimming or other water-
based recreation. Contaminants introduced
by swimmers can dramatically influence the
parts of the swimming pool. Sources include
micro-organism from infected swimmers and
body such as sweat, cosmetics, lotions,
urine, saliva and fecal matter. Pathogenic
contaminants are of greatest concern in
swimming pools associated with numerous
water illnesses.
The samples were collected from the
surfaces of childrens swimming pool in
Timoga, Buru-un, Iligan City. The swab
from the childrens pool surfaces were
placed in the Thioglycolate as transport
medium then it will be planted on the Blood
Agar Plate and MacConkey Agar so that it
would form and isolate the colony. It was
incubated for 24 hours for fast growth. In
Gram Staining, the gram-negative bacteria
showed red color. Biochemical Test was
used if the organism is gram negative. It
shows the overall results of the
biochemical tests from Timoga
and that were E. coli, Klebsiella spp. and
Shigella spp.. These bacteria (except
Shigella spp.) are normal inhabitants of
specific areas of the body and it will be
Childrens Swimming Pool Surfaces. Triple
Sugar Iron (TSI) test is used to determine
whether a gram-negative rod utilizes glucose
and lactose or sucrose fermentatively and
forms hydrogen sulfide (H2S). Lysin Iron
Agar (LIA) is used to determine whether rod
decarboxylase or deaminates lysin and forms
hydrogen sulfide (H2S). Citrate Utilization
Test is used to determine the ability of an
organism to utilize sodium citrate as its only
carbon source and inorganic ammonium salts
as its only nitrogen source. SIM Test is used
to determine Hydrogen Sulfide production,
indole formation, and motility. Methyl Red
(MR) Test is used to determine the ability of
an organism to produce and maintain stable
acid end products from glucose
fermentation, to overcome the buffering
capacity of the system, and to determine the
ability of some organisms to produce neutral
end products from glucose fermentation.
Based on the findings of this study
and from the result of laboratory experiment,
this study concludes that there are gram-
negative pathogenic bacteria present in
Timoga Childrens Swimming pool surfaces
pathological if there is defective immunity
and or an increase in number of organisms.
Shigella spp. are found in humans at times of
infection and not is part of the normal flora.
The possible causes why bacteria are found
in Timoga Childrens Swimming Pool
surfaces are from the infected swimming
enthusiast and contaminated water.
The swimming enthusiast should be
aware of the possible diseases they will get
when they always swim in the public
swimming pool. Swimming enthusiast
should be hygienic in order for them not to
be infected with the pathogenic bacteria. The
management should be strict in maintaining
their swimming policies. The management
should apply strong disinfectant for in every
pools corner.
BACKGROUND
Recreational bathing includes spas
and hot tubs or wading/swimming in rivers,
lakes, ponds oceans or water parks or in
swimming pools. Swimming pools are man-
made container filled with water intended
for swimming or other water-
based recreation.
Contaminants introduced by
swimmers can dramatically influence the
parts of the swimming pool. Sources include
micro-organism from infected swimmers and
body such as sweat, cosmetics, lotions,
urine, saliva and fecal matter. Pathogenic
contaminants are of greatest concern in
swimming pools associated with numerous
water illnesses. It can be viruses, bacteria,
protozoa or fungi.
Microbes, also called
microorganisms, are minute living things
that individually are usually too small to be
seen with the unaided eye (Tortora, 2005).
Humans and other animals also depend on
the
Subsequent swab and water samples
consistently produced the organism.
OBJECTIVES
This study aims to determine the
microbes such as Escherichia coli found in
the intestinal tract of the human, for proper
digestion and some bacteria may help
synthesize vitamins that the body requires.
Humans encounter microorganisms
when they enter or exposed to the same
environment in which the microbial agents
live or when the infectious agents are
brought to the human host by indirect
means. The environment or place of origin
of the infecting agent is referred to as the
reservoir (Tille, 2013).
Microorganisms on surfaces can be
present as single, groups of cells or well
developed biofilms that may require
treatment. (Barkhudarov, et al., 2008)
Bacterial growth in natural environments is
often associated with attachment to surfaces.
This may involve adherence to surfaces of
material degraded by the bacteria (e.g.,
detritus particles) to provide nutrient, or to
inert surfaces (Brown, Ellwood, & Hunter,
1977).The attachment may often be strong
and while some bacteria have developed
specialized structures for this purpose (e.g.,
holdfasts in Caulobacter spp.), for most
organisms it has been suggested that the
production of a sticky extracellular
polysaccharide is responsible (Brown,
Ellwood, & Hunter, 1977). The reasons for
this preferential growth at surfaces of inert
material are not clear, but it is apparent that
bacterial growth is slow and is likely to be
nutrient-limited in natural aquatic
environments (Brown, Ellwood, & Hunter,
1977). There is an evidence of serious
outbreaks of violent stomach problems,
which are particularly dangerous in toddlers
and elders.
Previous routine laboratory
examination for coli forms organisms. Early
in the investigation, swab taken from the
stone surfaces, samples of silt from the
bottom, and water samples from the outlet
gate showed the presence of M. balnei in
large numbers (Mollohan & Romer 1961).
different gram-negative pathogenic bacteria
that are present on the Timoga Childrens
Pool Surface and state the possible diseases
of each identified gram negative bacteria that
may affect any individual.
 Specifically, it seeks answers to the
following questions:
1. Are there any gram negative
bacteria present in the children's swimming
pool surfaces?
2. What are the kinds of gram
negative bacteria present in the children's
swimming pool surfaces?
3. What are its pathogenicity?
METHODS
The study will use experimental
research in detecting and identifying the
possible gram negative bacteria in children's
swimming pool surfaces. This experimental
research will be purely on laboratory
experiment since this study is more one
specific approach on the possible gram
negative bacteria present in children's
swimming pool surfaces that cannot be seen
by the naked eye.
Materials
The materials used were the
following:
Swab
Thioglycolate
Incubator
MacConkey Agar
Inoculating Loop
Inoculating Needle
Slides
Alcohol Lamp
Match
Forceps
Phase IV. Biochemical Test
Bacteria can differ greatly in their
morphologies. These differences can help us
in identifying different species of bacteria.
This test is designed to identify various
metabolic properties of different bacterial
Test Tube Rack
Microscope
Gram Stain
Data Gathering Procedure
Phase I. Collection of Samples
The samples were collected from the
surfaces of childrens swimming pool in
Timoga, Buru-un, Iligan City. It was
collected before water replacement and
sterilization during night time, 6:00PM to
11:00 PM.
Phase II. Bacterial Inoculation and Culture
The swab from the Timoga childrens
pool surfaces was placed in the
Thioglycolate as transport medium then it
was planted on the MacConkey Agar so that
it would form and isolate the colony. It was
incubated for
24 hours for fast growth at 37C.
Phase III. Gram Staining
To prepare a smear for staining, an
isolated colony on the specimen was placed
on a clean microscopic slide.
Gram Stain is used to determine the
physical appearance of the bacteria whether
it is spherical-shaped, rod-shaped, or spiral-
shaped. Gram negative bacteria it shows red.
The first step was fixation of the
cultured specimen. Then, flood the fixed
slide with crystal violet stain and wait for 10
seconds and rinse with water. Next, flood
with grams iodine solution as a mordant and
wait for 10 seconds and rinse with water.
Decolorize with acetone alcohol only for 3
seconds and then rinse with water. Lastly,
flood with safranin for 10 seconds, rinse with
water, air-dry, and examine using the oil-
immersion lens. Organisms that have a gram
negative cell wall would decolorized and
would stain red with safranin counterstain.
species. Biochemical Test is used if the
organism is gram negative.
The biochemical tests used were the
following:
Triple Sugar Iron (TSI) Test
Lysine Iron Agar (LIA) Test
Citrate Test
 SIM Test Agar is used for isolation and differentiation
Methyl Red (MR) Test of lactose fermenting and non-lactose
fermenting enteric bacilli. This result gave
RESULTS the researhcers an idea that the bacteria
The result presents the analysis and present in the samples are Enterobacteriacae
interprets the data gathered by the spp.
researchers. Of the six samples collected, eight
colonies were isolated. Only 1 growth in
Table 1 Growths in each childrens each Pool1A, Pool1B, Pool2B, and Pool3A
swimming pool surfaces and 2 growths in each Pool2A and Pool3B in
Agar Pool 1 Pool 2 Pool 3 Timogo Childrens Swimming Pool
A B A B A B Surfaces. Isolation of bacteria is used to
MacConkey 1 1 2 1 1 2 obtain individual bacterial colonies present
Agar in a sample. As stated in the book entitle
Bailey and Scotts Diagnostic Microbiology,
Table 1 shows the growth of bacteria 12th Edition, MacConkey Agar contains
in MacConkey Agar from the six samples crystal violet dye to inhibit the growth of
collected by the researchers. The gram- positive bacteria and fungi, and
MacConkey allows many types of gram-negative bacilli
to grow. The differential capacity of this
medium is provided by the pH indicator,
neutral red, and gives the bacterial colonies a
pink to red color. Thus, strongly suggests
that the bacteria present in each samples are
Enterobacteriacae spp. and other types of
gram-negative species.
Isolated colonies are pure culture
which means only one species of bacteria is
present. Obtaining a pure culture is
mandatory to establish the identity and
susceptibility.

Table 2 Results from Biochemical Tests

Table 2 showsTSI
Pool the overallLIA
resultsCitrate
of utilizes MR
SIM glucose and lactose
Result or sucrose
the biochemical
1 tests from Timoga fermentatively and forms hydrogen sulfide
Childrens
A1 SwimmingA/A Pool Surfaces.
K/K The+ -(H
- 2-S). Lysin
- Iron Agar (LIA)spp.
Klebsiella is used to
streaked pattern
B1 indicates
A/A growth of
K/K bacteria+ -determine
-- - whether Klebsiella spp.
rod decarboxylates or
and the 2isolated colonies were obtained by deaminates lysin and forms hydrogen sulfide
pure culture.
A1 The isolatedA/A colonies
K/K were- +(H+ 2+S). Citrate
+ Utilization
E. coli Test
E. coliis used to
taken, subjected
A2 to Gram
A/A Stain, and
K/Kused for- +determine
++ + the ability of an spp.
Klebsiella organism to
microscopic examination
A/A + Gasof bacteria
K/K and+ utilize
--- sodium
- citrate as its only carbon
B1
identifies3 it is gram-negative cocci or gram- source and inorganic ammonium salts as its
negative bacilli. The bacteria that were seen
A1 A/A K/K + -only
- - nitrogen
- source.Klebsiella
SIM Test spp.is used to
during the examination were all identified as
B1 bacilli.K/A K/A + -determine
-- - HydrogenShigella
Sulfidespp.production,
Gram-negative The identified gram- Indole formation, and Motility. Methyl Red
K/A K/A + --- - Shigella spp.
negative B2 bacteria were subjected to (MR) Test is used to determine the ability of
biochemical tests for specific identification an organism to produce and maintain stable
of the bacteria present. acid end products from glucose fermentation,
Triple Sugar Iron (TSI) test is used to to overcome the buffering capacity of the
determine whether a gram-negative rod system, and to determine the ability of some
organisms to produce neutral end products
from glucose fermentation.
E. coli
Escherichia coli is a Gram-negative
bacilli, a lactose fermenter, citrate negative,
indole positive, methyl red positive, and L-
decarboxylase positive. Darland and Davis
reported that there are more than 200 H2S-
positive variants of E. coli characterized
biochemically and serologically. Because of
significant differences in some reactions and
Decarboxylation and no Fermentation of
glucose. As stated in Konemans Color Atlas
and Textbook of Diagnostic Microbiology,
the distribution of O and H antigens between
the variants and biochemically typical E.
coli, these investigators thought that the
variants represented a rather limited
subgroup of E. coli (Maker & Washington,
1974). E. coli is a facultative anaerobic that
is found in the GI tract of all humans (W.
Burton & Engelkirk,
2004).
In this sample, E. coli is positive in
Methyl Red (MR) Test, Indole Test, and in
Motility Test. LIA shows Alkaline
Slant/Alkaline Butt (K/K) which means
Lysine Decarboxylation and no
Fermentation of glucose. In Citrate Test, it
shows green in color which means negative.
Triple Sugar Iron (TSI) test shows Acid
Slant/Acid Butt (A/A) which means glucose,
sucrose, and/or lactose fermenter.
Figure 1 Methyl Red Reactions. Positive
results shows red color after adding Methyl
Red. Negative shows yellow color.
Klebsiella spp.
Klebsiella spp. exhibit mucoid
growth, large polysaccharide capsules, lacks
motility, and they usually gives positive test
for lysine decarboxylase and citrate (Brooks,
Butel, & Ornston, 1991). It is usually found
in GI tract of humans and animals.
In this bacteria, Klebsiella spp. is
positive in Citrate Test. In Methyl Red Test,
Indole Test, and Hydrogen Sulfide Test are
negative. LIA shows Alkaline Slant/Alkaline
Butt (K/K) which means Lysine
They produce acetyl methyl carbinol, which
interferes with the final alkaline pH shift,
leading to false-negative interpretations.
Triple Sugar Iron (TSI) test shows Acid
Slant/Acid Butt (A/A) which means glucose,
sucrose, and/or lactose fermenter.
exceptions do not ferment lactose but do
ferment other carbohydrates, producing acid
but no gas (E.A, 1982). This type of bacteria
does not belong to the normal GI flora.
In this type of organism, Shigella
spp. shows Alkaline Slant/Acid Butt (K/A)
which means it is glucose fermenter only.
LIA shows Alkaline Slant/Alkaline Butt
(K/K) which means Lysine Decarboxylation
and no Fermentation of glucose. Other tests
like Citrate Test is positive and in Methyl
Red Test, Indole Test, and Hydrogen Sulfide
Test are negative.

Figure 2 Citrate Test. Positive result

shows blue color.

Shigella spp.
Shigella spp. are non-motile, gram-
negative, aerobic rods, which with a few

Figure 3 SIM Test. 2A1 shows +++ SIM

Test. 1A1 shows --- in SIM Test.

Table 3 Habitant (Reservoir) and its Pathogenicity of the Bacteria

Bacteria Habitat Pathogenicity

E. coli 1. Extraintestinal [e.g. Uropathogenic Cytitis and Acute
E. coli (UPEC) and Pyelonephritis
Meningitis/Sepsisassociated E. coli Neonatal Meningitis
(MNEC)]

2. Intestinal [e.g. Enterohemorrhagic Hemorrhagic

E. coli (EHEC), Enterotoxigenic E. Diarrhea, Colitis, and
coli (ETEC), Enteroinvasive E. coli Hemolytic Uremic
(EIEC), Enteropathogenic E. coli Syndrome (HUS)
Mild Watery Diarrhea
 (EPEC), and Enteroaggregative E.
coli (EAEC)]
Klebsiella spp. 1. Nasopharynx (ex: K. pneumoniae)
2. Gastrointestinal Tract (ex: K.
oxytoca)
Shigella spp. Only found in humans at times of infections;
not part of normal bowel flora
Table 3 shows the location of the
bacteria. E. coli and Klebsiella spp. are
opportunistic human pathogens and Shigella
spp. are one of the primary intestinal
pathogens that are not part of the normal
flora of humans. Transmission is typically
person to person (Tille, 2013). It is also
transmitted via fecal-oral route with carriers
as the source. Shigella spp., Klebsiella spp.,
and E. coli may also be transmitted by flies,
fingers, and food or water contaminated by
infected persons.
E. coli
There are two isolates of
extraintestinal E. coli strains in this category.
Uropathigenic E. coli (UPEC) strains are the
major cause of E. coli associated urinary
tract infections. These strains contain variety
of pathogenicity islands that code for
specific adhesions and toxins capable of
causing disease, including Cytitis and Acute
Pyelonephritis (Tille, 2013).
Meningitis/Sepsisassociated E. coli
(MNEC) can cause Neonatal Meningitis.
This organism are spread to the meninges
from a blood infection and gain access to the
central nervous system via membrane-bound
Shigella spp.
All species found in this genus can
Watery to Bloody
Diarrhea
Prolonged,
Nonbloody Diarrhea,
Vomiting, and Fever
(infects infants or
children)
Inflammation and
accompanied by
Fever and Abdominal
Pain
Pneumonia
Hemorrhagic Colitis
Shigellosis
vacuoles in microvascular endothelial cells
(Tille, 2013).
As mentioned in Table 3, Intestinal
E. coli may be classified as
Enterohemorrhagic E. coli (EHEC) that can
cause Hemorrhagic Diarrhea, Colitis, and
Hemolytic Uremic Syndrome (HUS);
Enterotoxigenic E. coli (ETEC) produces a
heat-labile enterotoxin (LT) and a heat-
stable enterotoxin (ST) capable of causing
mild watery diarrhea (Tille, 2013);
Enteroinvasive E. coli (EIEC) causes a
watery to bloody diarrhea due to the direct
invasion of the epithelial cells of the colon;
Enteropathogenic E. coli (EPEC) does not
produce enterotoxins that causes prolonged,
nonbloody diarrhea, vomiting, and fever
that affects infants or children; and
Enteroaggregative E. coli (EAEC) shows
nonbloody and no white blood cells found in
stool and this strain causes inflammation and
accompanied by fever and abdominal pain.
Klebsiella spp.
This type of bacteria are found in the
nasopharynx and GI tract of the humans.
Isolates have been identified in association
with a variety of infections, including liver
abscess, pneumonia, septicemia, and urinary
tract infections (Tille, 2013).
cause bacillary dysentery but they vary in
epidemiology, mortality rate, and severity of
disease produced. Dysentery is characterized
by the presence of blood, mucus, and pus in
the stool. These organisms multiplied in the
small intestines move toward the colon
where they may be isolated 1-3 days after
the infection develops. Shigella dysentery
usually indicates improper sanitary
conditions and poor personal hygiene.
CONCLUSIONS
Based on the findings of this study
and from the result of laboratory experiment,
this study concludes that there are gram-
negative bacteria present in Timoga
Childrens Swimming pool surfaces and that
were Escherichia coli, Klebsiella species and
Shigella species. These bacteria (except
Shigella Species) are normal inhabitants of
specific areas of the body and it will be
pathological if there is defective immunity
and or an increase in number of organisms.
Shigella are found in humans at times of
infection and not is part of the normal flora.
RECOMMENDATION
The possible causes why bacteria are
found in Childrens Swimming Pool surfaces
are from the infected swimming enthusiast
and contaminated water. Thus, based on the
result, the following are recommended:
1. The swimming enthusiast should be
educated of the possible diseases they
will get when they swim in the public
swimming pool. They should practice
proper hygiene in order for them not
to be infected with the pathogenic
bacteria.
2. The management should be strict in
maintaining their swimming policies.
The management should apply
disinfectant in every pools surfaces.
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