Coliform Count and Detection of Escherichia Coli on

Pork Sold at Wet Markets and Supermarkets in

Parañaque City

Bonceli, Roana Gwyn

Cagna-an, Paulene Joy
Castro, Raya Samantha
Ladines, Eunice Katheryn
Nicdao, Krissy Danielle
Que, Paulyn Joy
Tegrado, Kenneth Renz

FEBRUARY 2019

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 ABSTRACT
This research describes the difference in coliform count and detection of Escherichia coli
between raw pork bought in supermarkets and those bought in wet markets, and their relation to
the environment they were bought in. The researchers used several methods that were done at the
Natural Sciences Research Institute in University of the Philippines Diliman in order to find the
coliform count and presence of E. coli. It was found that all of the samples from supermarkets
were within the FDA limit for coliform count, while those from wet markets were above the limit,
and one was found to contain E. coli. The strict standards and quality control of supermarkets
contributed to the small amount of coliform detected and lack of E. coli, whereas the lack of
regulation and sanitation standards led to more coliform and E. coli on the pork bought in wet
markets.

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 TABLE OF CONTENTS

ABSTRACT …………………………………………………………………………………… 2
INTRODUCTION …………………………………………………………………………… 4-6
METHODOLOGY ………………………………………………………………………….. 6-10
RESULTS AND DISCUSSION …………………………………………………………….10-11
CONCLUSION ………………………………………………………………………………... 11
INTERVENTION …………………………………………………………………………....12-14
RECOMMENDATION ………………………………………………………………………... 15
ACKNOWLEDGEMENT …………………………………………………………………........14
BIBLIOGRAPHY……………………………………………………………………………15-16
APPENDIX…………………………………………………………………………………..16-22

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 INTRODUCTION
A. BACKGROUND OF THE STUDY
Raw pork has always been part of the daily meal of the Filipinos, and pork is usually
seen sold in wet markets and in grocery stores. Pork has been part of the meal plans of humans
since the start of human life. This has been because of the abundance of pork in the surroundings
of people back then because this could be taken from pigs. This animal tends to thrive where water
and food are present and in the past humans were also trying to survive.

In 2016, according to an article in the Inquirer News, the Philippines is among the top pork
consumers in the whole world and this was based on consumption increases from Food and
Agricultural Policy Research Institute-Iowa State University in 2012. For the compound manual
growth from 2011 to 2021 was estimated to be 30 percent each year, making the Philippines part
of the world’s top pork consumers.

Pork has been widely enjoyed in various families in the Philippines, though the
consumption of pork needs thorough identification of the right meat to buy for it could develop
bacteria whenever it is being stored, especially if it is being stored in places where it is not sealed
and is exposed to various contaminants such as polluted air and flies that could cause problems to
the consumers.

The source of pork, which are pigs, naturally carry Escherichia coli in their intestines, the
process of slaughtering the animal, as well as the procedures needed to be done with the use of
instruments and tools can contaminate the pork being reproduced and the means of transporting
and distributing the pork to various markets could contaminate the product being reproduced.

This can be detected with a high presence of coliform bacteria, an indicator of a possible
presence of a microorganism that is harmful to our health.

According to the Food Safety Focus in an article written in 2017, "These pathogenic
bacteria are able to invade our bodies or produce toxins to cause illness. They cannot be seen or
smelled on the pork, but can generally be killed by normal cooking conditions,” though there are
multiple cases that pork is not properly cooked and that could be the root cause of bacteria to enter
the bodies of the different consumers.

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 The number of bacteria present in the pork in wet markets could be doubled since the
product is more exposed to contaminants present in the different wet markets in the Philippines
especially in markets near roads and polluted rivers. This could pose a greater risk to the people
buying the pork in wet markets and could cause various complications to the consumers.

B. STATEMENT OF THE PROBLEM

This study intends to detect the presence coliform as well as the measurement of the said
colony in raw meat found in wet markets and supermarkets in Paranaque.

Specifically, this aims to answer the following questions:

1. Does the exposure of pork in wet markets increase the risk of growing coliform bacteria as well
as contain Escherichia coli (E. coli) in the pork?

2. (If yes), is there a specific reason why the coliform count is different in the pork sold in wet
markets compared to those sold in supermarkets?

C. HYPOTHESES

Ho1: The exposure of the pork in wet markets do not increase the risk of growing coliform bacteria
and Escherichia coli.

Ho2: There is no specific reason on why there is more coliform count in the pork found in the wet
markets than those in supermarkets.

Ha1: The exposure of the pork in wet markets increase the risk of growing coliform bacteria and
Escherichia coli.

Ha2: There is a reason why the coliform bacteria count is higher in the pork found in the wet
markets than those in supermarkets.

D. SIGNIFICANCE OF THE STUDY

Pork is part of the daily meals of Filipino families and usually, people tend to buy these
products from the nearest wet market near them. But due to lack of studies and awareness of the
people as to what the pork’s bacterial content can cause to them makes the Filipino families in
danger when it comes to harboring serious illnesses from contamination of the raw pork they

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bought. With this study, the people will be able to know what they should look out for and what
the authorities from the wet markets look out for.

With the Philippines being labeled as one of the countries that consume pork often, more
people are at risk in getting illnesses from the pork. This research would not only help the
community, but it will surely change the system of the wet markets when it comes to sanitation
and prevention of contamination and exposure of the food products to various pollutants and
contaminants. This could specify the root causes of the future illnesses caused by coliform bacteria,
with these doctors are able to study and know the cure to the illnesses that Filipino consumers
could get.

E. SCOPE AND LIMITATION

This study focuses on the detection of Escherichia coli and identification of the coliform
count present in pork found in wet markets and supermarkets in Parañaque City.

METHODOLOGY

RESEARCH DESIGN

The researchers used Convergent Qual-Quant Research design wherein they gathered
qualitative data through testing then they supplemented it with a quantitative data gathered from
surveys and were able to triangulate a finding.

RESEARCH LOCALE

The research was conducted in Parañaque City, Philippines, throughout January to

February 2019. The samples used in the study were bought at several wet markets and
supermarkets within different barangays. The stores where the samples were sold are held
confidential as per the agreement with the vendors. The researchers chose the city of Parañaque
because of its accessibility to the researchers and their target population.

RESPONDENTS OF THE STUDY

The researchers chose random Parañaqueño students, using random sampling, as their

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respondents for their research and were asked if they were aware of the bacteria E. coli, what
diseases they can acquire from it, how many times they eat pork per week, and do they boil their
pork first before cooking it.

DATA GATHERING PROCEDURE

The following procedure is according to Bacteriological Analytical Manual.

Equipment and materials:

 Covered water bath, with circulating system to maintain temperature of 44.5 ±
0.2°C. The temperature for water baths for the shellfish program is 44.5°C ± 0.2°C.
Water level should be above the medium in immersed tubes.
 Immersion-type thermometer, 1-55°C, about 55 cm long, with 0.1°C subdivisions,
certified by National Institute of Standards and Technology (NIST), or equivalent
 Incubator, 35 ± 1.0°C. The incubator temp for the shellfish program is 35°C ± 0.5°C
 Balance with capacity of>2 kg and sensitivity of 0.1 g
 Blender and blender jar
 Sterile graduated pipets, 1.0 and 10.0 mL
 Sterile utensils for sample handling
 Dilution bottles made of borosilicate glass, with polyethylene screw caps equipped with
Teflon liners. Commercially prepared dilution bottles containing sterile Butterfield's
phosphate buffer can also be used.
 Quebec colony counter, or equivalent, with magnifying lens
 Longwave UV light [~365 nm], not to exceed 6 W.
 pH meter

Media and regents:

 Brilliant green lactose bile (BGLB) broth, 2%

 Lauryl tryptose (LST) broth
 Lactose Broth
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  EC broth
 Levine's eosin-methylene blue (L-EMB) agar
 Tryptone (tryptophane) broth
 MR-VP broth
 Koser's citrate broth
 Plate count agar (PCA) (standard methods)
 Butterfield's phosphate-buffered water or equivalent diluent
 Voges-Proskauer (VP) reagents
 Gram stain reagents
 Methyl red indicator
 Violet red bile agar (VRBA)
 VRBA-MUG agar
 EC-MUG medium
 Lauryl tryptose MUG (LST-MUG) broth
 Peptone Diluent, 0.5%

Procedure:

Weigh 50 g of food into sterile high-speed blender jar. Frozen samples can be softened by
storing for <18 h at 2-5°C, but do not thaw. Add 450 mL of Butterfield's phosphate-buffered
water and blend for 2 min. If <50 g of sample is available, weigh portion that is equivalent to
half of the sample and add sufficient volume of sterile diluent to make a 1:10 dilution. The total
volume in the blender jar should completely cover the blades. Prepare decimal dilutions with
sterile Butterfield's phosphate diluent or equivalent. Number of dilutions to be prepared depends
on anticipated coliform density. Shake all suspensions 25 times in 30cm arc or vortex mix for 7s.
Using at least 3 consecutive dilutions, inoculate 1 mL aliquots from each dilution into 3 LST-
MUG tubes for a 3 tube MPN analysis (other analysis may require the use of 5 tubes for each
dilution). Lactose Broth may also be used. For better accuracy, use a 1 mL or 5 mL pipet for
inoculation. Do not use pipets to deliver <10% of their total volume; e.g. a 10 mL pipet to deliver

8
0.5 ml. Hold pipet at angle so that its lower edge rests against the tube. Not more than 15 min
should elapse from time the sample is blended until all dilutions are inoculated in appropriate
media. Incubate LST-MUG tubes at 35°C ± 0.5°C. Examine tubes and record reactions at 24 ± 2
h for gas, i.e., displacement of medium in fermentation vial or effervescence when tubes are
gently agitated. Re-incubate gas-negative tubes for an additional 24 h and examine and record
reactions again at 48 ± 3 h.

Perform confirmed test on all presumptive positive (gas) tubes. Be sure to inoculate one
tube of LST-MUG with a known GUD-positive E. coli isolate as positive control (ATCC 25922).
In addition, inoculate another tube with a culture of Enterobacter aerogenes (ATCC 13048) or a
Klebsiella pneumoniae strain as negative control, to facilitate differentiation of sample tubes that
show only growth from those showing both growth and fluorescence. Incubate tubes for 24 to 48
± 2 h at 35°C. Examine each tube for growth (turbidity, gas) then examine tubes in the dark
under longwave UV lamp (365 nm). A bluish fluorescence is a positive presumptive test for E.
coli. Studies by Moberg et al. show that a 24 h fluorescence reading is an accurate predictor of E.
coli and can identify 83-95% of the E. coli-positive tubes.

After 48 h of incubation, 96-100% of E. coli-positive tubes can be identified. Perform a

confirmed test on all presumptive positive tubes by streaking a loopful of suspension from each
fluorescing tube to L-EMB agar and incubate 24 ± 2 h at 35°C. To perform the completed test for

E. coli, gently agitate each gassing EC tube, remove a loopful of broth and streak for isolation on
L-EMB agar plate and incubate for 18-24 h at 35°C ± 0.5°C. 14. Examine plates for suspicious E.
coli colonies, i.e., dark centered and flat, with or without metallic sheen. Transfer up to 5 suspicious
colonies from each L-EMB plate to PCA slants, incubate them for 18-24 h at 35°C ± 0.5°C and
use for further testing.

Perform Gram stain. All cultures appearing as Gram-negative, short rods should be tested
for the API20E or the automated VITEK biochemical assay reactions below and also re-inoculated
back into LST to confirm gas production. Inoculate tube of tryptone broth and incubate 24 ± 2 h
at 35°C ± 0.5°C. Test for indole by adding 0.2-0.3 mL of Kovacs' reagent. Appearance of distinct
red color in upper layer is positive test. Voges-Proskauer (VP)-reactive compounds. Inoculate tube

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of MR-VP broth and incubate 48 ± 2 h at 35°C± 0.5°C. Transfer 1 mL to 13 × 100 mm tube. Add
0.6 mL α-naphthol solution and 0.2 mL 40% KOH, and shake. Add a few crystals of creatine.
Shake and let stand 2 h. Test is positive if eosin pink color develops. Methyl red-reactive
compounds. After VP test, incubate MR-VP tube additional 48 ± 2 h at 35°C± 0.5°C. Add 5 drops
of methyl red solution to each tube. Distinct red color is positive test. Yellow is negative reaction.
Citrate. Lightly inoculate tube of Koser's citrate broth; avoid detectable turbidity. Incubate for 96
h at 35°C ± 0.5°C. Development of distinct turbidity is positive reaction. Gas from lactose.
Inoculate a tube of LST and incubate 48 ± 2 h at 35°C ± 0.5°C. Gas production (displacement of
medium from inner vial) or effervescence after gentle agitation is positive reaction.

Calculate MPN of E. coli based on combination of confirmed fluorescing tubes in 3

successive dilutions.

INSTRUMENTATION

The researchers used testing and observations in order to collect qualitative data from the
samples. Then the researchers supervised a survey among their respondents to supplement the data
that they gathered from testing and observations. They analyzed the data gathered and were able
to triangulate both the qualitative and quantitative data in order to formulate a conclusion.

RESULTS AND DISCUSSION

Table 1. The number of Coliform and detection of E. coli per sample

Coliform Count,
Sample Detection of E. coli
CFU* per gram
A. Pork 1 (Supermarket A) 1.2 x 102 Not detected
B. Pork 2 (Wet Market A) 1.7 x 104 Present
C. Pork 3 (Supermarket B) 1.2 x 102 Not detected
D. Pork 4 (Wet Market B) 2.6 x 103 Not detected

*Colony-forming Units

The data above shows the difference in Coliform count and Escherichia coli detection from
pork bought from different supermarkets and wet markets. For supermarkets A and B, it is shown
that they have the same coliform count and Escherichia coli weren't detected. For the pork bought

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on wet markets A and B, it is observed that they have a higher coliform count than supermarkets
A and B. It was also shown that the pork from wet market A was the only one detected with
Escherichia coli.

Coliform and E. coli are often used as indicators of fecal contamination, so increased
presence of the said bacteria indicate that somewhere in the preparation process of the pork,
hygiene was not maintained enough to avoid contamination. The results showed that there was a
significant difference between the coliform count and E. coli presence in the pork bought at the
wet market and the pork bought at the supermarket. Since the wet markets in the study were busy
wet markets with the pork displayed in arm’s reach, it can be inferred that the continuous influx
of people touching the pork along with the environmental factors like the lack of regulated
cleanliness standards contributed to the increased amount of coliform, as well as the presence of
E. coli found in one sample. The strict sanitation standard, closed glass display, and available
tongs for pork handling in pork sections of supermarkets may be the reason why the samples
from supermarkets have a lower value of coliform and no presence of E. coli. Pork has a high
degree of variability in how it was butchered, packaged, and how many additives may have been
introduced. Because of the variables, the control specification for coliform counts are generally
1,000 CFU/g, and only the samples from the supermarkets met this regulation. It is worth noting

though that with thorough cooking and enough heat of at least 72 degrees Celsius, most E. coli
and coliform die, so it does not pose much of a health issue to people.

CONCLUSION
There is a difference in the amount of bacteria present in the pork being sold in wet
markets and supermarkets. Due to the given conditions as to where the pork is exposed, such
environment has affected the quality of the raw pork. Supermarkets tend to store their products in
sanitized areas hence the low amounts of coliform, while those sold in wet markets are often
exposed to areas with unfavorable conditions.

The lack of regulation, the environment, and handling of the pork in wet markets affected
the amount of coliform in the raw pork. Therefore, we can conclude that when it comes to
cleanliness and sanitation, supermarkets are more reliable.

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 INTERVENTION

No Yes
47.31% 52.69%

Fig 1. People who are aware or not about the bacteria E. coli
The researchers asked 167 students that were chosen through random sampling if they are
aware of the bacteria E. coli, and 88 of them said yes while 79 said no.

Yes
11.98%

No
88.02%

Fig. 2. People who are aware or not about the diseases a person can get from a pork with E. coli
The researchers asked 167 students that were chosen through random sampling if they
know what diseases that a person could get if they ate a pork contaminated with E. coli, and 20 of
them said yes while 147 said no.

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 7 servings 0 servings
6 servings 0.6% 1.8%
1.2% 1 serving
8.38%
5 servings
13.17%

4 servings 4 servings
15.57%
17.37%

3 servings
41.92%

Fig. 3. Servings of pork per week

The researchers asked 167 students that were chosen through random sampling how many
times do they eat pork per week, and 3 of them said they don’t eat pork, 14 said they eat pork once
a week, 26 said twice a week, 70 said thrice a week, 29 said four times a week, 22 said five times
a week, 2 said 6 times a week, and 1 said once a day.

No
12.57%

Yes
87.43%

Fig. 4. People who boil pork or not before cooking

The researchers asked 167 students that were chosen through random sampling if they boil
the pork that they eat before they cook it, and 146 of them said yes while 21 said no.

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 ACKNOWLEDGEMENT

First, thanks to God for giving us everything that we need in order to survive. As time
passes by, we, researchers are called to dig in deeply to all of these resources and innovate. Also,
we praise God for allowing us to finish our research successfully and making this journey indelible.

We would like to acknowledge our parents for supporting us in doing this research. As well
as helping us financially, giving care and understanding. We're really grateful to have someone
like them to support us these past few months.

We would like to sincerely express our gratitude to our research adviser and adult sponsor,
Mrs. Ma. Theresa G. Baita, and our science school head, Mrs. Ma. Cristina Villareal, for providing
us information, being there for us like our second parents, and helping us in enhancing our research.
We are also thankful to the scientists in National Sciences Research Institute (NSRI)
especially to the ones in the Biological Research and Services Laboratory (BRSL). Without their
help, this research wouldn't have been possible.
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 RECOMMENDATIONS

It is highly recommended to use this research for further study in regards to improving the
health sanitation of raw meats, either from wet markets or supermarkets, for the well-being of the
consumers. In the presence of E. coli, it is stated that there are other bacteria that could also be
present and could be far more dangerous than E. coli. Further research on the bacteria present as
well as thorough identification of these bacteria are encouraged.

BIBLIOGRAPHY

1) Compendium of Methods for the Microbiological Examination of Foods. 3rd ed.

Washington, DC: American Public Health Association, 1992.
2) Doyle, M.P. and Schoeni, J.L. Isolation of Escherichia coli O157:H7 from retail meats and
poultry. n.p., 1987.
3) Entis, P. Hydrophobic grid membrane filter/MUG method for total coliform and
Escherichia coli enumariton in foods. n.p., 1989.
4) Feldsincd, P.T., Falbo-Nelson, M.T. and Hustead, D.L. ColiComplete Substrate-supporting
disc method for confirmed detection of total coliforms and Escherichia coli in all foods.
n.p., 1994.
5) Feng, P.C.S and Hartman, P.A. Fluorogenic assays for immediate confirmation of
Escherichia coli. n.p., 1983.
6) Grant, M.A. A new membrane filtration medium for simultaneous detection and
enumeration of Escherichia coli and total coliforms. n.p., 1997
7) Hartman, P.A. The MUG (glucuronidase) test for Escherichia coli in food and water. In
Rapid Methods and Automation in Microbiology and Immunology, edited by A. Balows,
R.C. Tilton, and A. Turano, 290-308. Brescia, Italy: Brixia Academic Press, 1989.
8) Manafi, M. Fluorogenic and chromogenic enzyme substrates in culture media and
identification tests. n.p., 1996.
9) Moberg, L.J., Wagner, M.K., and Kellen, L.A. Fluorogenic assay for rapid detection of
Escherichia coli in chilled and frozen foods. n.p., 1988.

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10) Neill, M. A., Tarr, P.I., Taylor D.N., and Trofa, A.F. Escherichia coli. In Foodborne
Disease Handbook, edited by Y. H. Hui, J. R. Gorham, K. D. Murell, and D. O. Cliver,
169-213. New York, NY: Marcel Decker, Inc., 1994.
11) Weagant, S.D. and Feng, P. Comparative Analysis of a Modified Rapid Presence-Absence
Test and the standard MPN Method for Detecting Escherichia coli in Orange Juice.n.p.,
2002.
12) Feng, P., Lum, R., and Chang, G. Identification of uidA gene sequences in beta-D-
glucuronidase (-) Escherichia coli. n.p., 1991.
13) Frampton, E.W. and Restaino, L. Methods for E. coli identification in food, water and
clinical samples based on beta-glucuronidase detection. n.p., 1993.
14) Recommended Procedures for the Examination of Seawater and Shellfish. 4th ed.
Washington, DC: American Public Health Association, 1970.

APPENDIX

Fig.1. Plagiarism check for Background of the Study

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Fig.2. Plagiarism check for Statement of the Problem

Fig.3. Plagiarism check for Significance of the Study

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Fig.4. Plagiarism check for Scope and Limitation

Fig.5. Plagiarism check for Research Design

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 Fig.6. Plagiarism check for Research Locale

Fig.7. Plagiarism check for Respondents of the Study

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 Fig.8. Plagiarism check for Instrumentation

Fig.9. Plagiarism check for Results and Discussion

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 Fig.10. Plagiarism check for Conclusion

Fig.11. Excuse letter for going to UP Diliman during school hours

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Fig.12. Another excuse letter for going to UP Diliman during school hours

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