Final Examination

In EducSci Microbiology and Parasitology

2nd Semester, SY 2020-2021

Name: __________________________ Date:____________________

Course, Year, Section:_________________ Score:___________________

Scenario

A new waterborne disease occurs in a village affecting half of its population. This was caused
by the contamination of water from a single source after the village was hit by the blood. Scientists
confirmed that the pathogenic microorganism that caused the disease is a novel species of
bacterium. Emerging symptoms are also new to affected individuals. Unfortunately, no drugs are
available to treat new disease. The challenge is to identify the novel species and to find treatment
for waterborne diseases.

Your Task

Main Goal: To design experiments on the identification of a novel waterborne bacterium and the
inhibition of the bacterial growth.

Tasks:

 Brainstorm with your classmates. Discuss the need the need to call for a project. Discuss
original ideas from each of you. To come up with an original idea, each student has to do a
critical evaluation of scientific literature and thorough research for the appropriate
laboratory techniques and required skills for the project. Write and record your discussions
in group chats, video call, etc.
 Design experiments. Design experiment based on appropriate laboratory techniques
learned from your class. The designed experiment should address the following:

1. Identification of waterborne pathogen through morphological and molecular basis.

Classify whether the pathogen is a gram positive or gram negative, then determine the
appropriate techniques to use in microscopy. (You may choose the taxonomy of your
hypothetical bacterium). Identification of bacterium through molecular basis doesn’t need
to be discussed in detail but you may state the appropriate techniques to use. Justify why
you are using those techniques. Justify why the identified bacterium is a gram positive or
gram negative based on its morphological structure.
Note: Assume the general classification of your bacterium whether a gram positive and
gram negative. Also, assume on what genus it belongs. Describe the general characteristics
of this genus and your hypothetical newly identified bacterium.

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 2. Inhibit the growth of the bacterium. Design an experiment on the test of bioactive
compounds from either one of the source (e.g., animal, plants, environmental sources, etc.)
that may inhibit the growth of your hypothetical newly identified bacterium. You have to
have a thorough research of related literatures on the use of bioactive com[ounds from
different sources that may inhibit specific bacterial growth.
Use appropriate laboratory techniques to use in the control of these bacteria. The following
techniques might or might not be applicable based on the nature of your project.
 Techniques in culturing specific bacteria (note that there are specific culture
medium for a particular type of microorganisms)
 Application of bioactive compounds to inhibit the growth of microbes
 Measuring microbial count

Justify why you will use your identified laboratory techniques for your experiment.

3. Communicate your Project Proposal

 Make an audio-recorded powerpoint presentation or video report to discuss your
project.

Note: You are not required to write a full research project proposal; this requires
enough time. However you are required to come up with a frameworks of your
proposed project highlighting the design of your experiment. The following is a
suggested formal, but you are free to add necessary headings based on the nature of
your project.

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 TITLE OF THE PROJECT:

Lemon (Citrus Limon) Juice as Antibacterial Potential Inhibitor against E.coli bacteria causing
Diarrhea

ABOUT THE PROJECT:

Waterborne diseases have become a major public health issue globally, which affect more than half
the population of the developing world. Approximately one billion people depend on contaminated
water sources amounting to 2.2 million annual deaths caused predominantly by diarrheal diseases
(Miagostovich et al. ) to which World Health Organization (WHO) estimates around 4% of the global
disease burden (WHO 2011). UNICEF (2010) survey estimates that the children under the age of five
are more prone to diarrheal diseases which account for more than 90% of annual deaths with about
5000 children die per day. Waterborne diseases are not only the problems of developing nations but
also a great challenge to the developing nations. Furthermore, this project designs an antibacterial
bioactive compounds to be a potential inhibitor against Escherichia coli which causes waterborne
diseases that will help the society and it's people to combat the said health problems. Lemon Juice
as a fruit extract is selected as the inhibitor of the e.coli which causes certain waterborne diseases.

OBJECTIVES:

The purpose of this project is to address the following questions: How should waterborne pathogen
through morphological and molecular basis be identified? How the growth of the newly identified
bacterium be inhibited through bioactive compounds such as in plants, animal, environment sources
etc. How is Lemon juice as Antibacterial inhibitor against E.coli bacteria effective in preventing
waterborne diseases?

SIGNIFICANCE OF THE PROJECT:

Waterborne pathogens have appeared again and again for a number reasons including:
contaminated water, increase in sensitive population, changes in drinking water treatment
technology, globalization of commerce and travel, and by the development of molecular methods
for detection and source tracking. Thus this study is significantly intended for the public to secure
the health and to lower the mortality rate that is caused by this water borne diseases. So they can
also have an access on how to combat this waterborne pathogen by the inhibition of the lemon
juice. Moreover this is also intended for the future researcher for further study and expansion of
knowledge as they based this as a literature.

DESIGN OF THE EXPERIEMNT:

1. Identification of of waterborne pathogen through morphological and molecular basis.

A. Selecting Specimen

The specimens were sent for microbiological examination from patients with suspected waterborne
diseases specifically diarrhea. The common specimen were selected such as the faeces. The samples
for bacteriological culture were obtained before antibiotic therapy is started because this maximizes
the sensitivity of the investigations and reduces false-negative results. Specimens is accurately
labelled and accompanied by a properly completed requisition form, indicating the nature of the

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 specimen, the date of sample collection, relevant clinical information, the investigations required,
and details of antibiotic therapy.

B. Bacterial Isolation Procedure

The principal method used for the detection of bacteria from clinical specimens is by culture on solid
culture media. Bacteria grow on the surface of culture media to produce distinct colonies. For
obtaining the isolated colonies streaking method is used, the most common Method of inoculating
an agar plate is by streaking

Streaking plates

1. A small amount of sample is placed on the side of the agar plate (either with a swab, or as a drop
from an inoculating loop).

2. A sterile loop is then used to spread the bacteria out in one direction from the initial site of
inoculation. This is done by moving the loop from side to side, passing through the initial site.

3. The loop is then sterilised (by flaming) again and the first streaks are then spread out themselves.

4. This is repeated 2-3 times, moving around the agar plate as shown in the figure.

In this method single bacterial cells get isolated by the streaking, and when the plate is incubated,
forming discrete colonies that will have started from just on bacterium each.

For quantitative analysis or semi quantitative analysis of the sample for example in case of urinary
tract infection. In fact E.coli is implicated as the causative organism in urinary tract infection only if
there are >105Colony forming units per millilitre of urine. The method of inoculating the solid
culture media is as shown in the figure. Different options are available for the isolation of E. coli. The
choice depends on target strain and objective of isolation. The ability to ferment lactose gives an
option to use MacConkey agar to discriminate E. coli from other nonlactose fermenting coliforms
from fecal, stool, food, water, and soil samples. Sample suspension (for solid samples) is made at any
concentration, for example, 5% in normal saline or phosphate buffer solution and inoculated onto
MacConkey agar followed by 18–24 h incubation at 37°C. Pink, round medium-sized colonies are
picked as E. coli suspect colonies. All E. coli strains can be captured on MacConkey agar, and this
approach gives a wide spectrum of strains to work on. Incubation of inoculated culture media at
45°C selects for thermophilic E. coli strains.

The concentration of sample suspension may be set at different levels such as 1 g of solid sample in
19 ml of normal saline or phosphate buffer solution (5%), 1 g in 9 ml (10%) or 1 g in 4 ml of diluent
(20%). However, the concentration of sample suspension will affect the number of colonies on the
culture plate. This is well evidenced in bacteria count procedures whereby higher dilution, like 105,
will give lower number of bacteria than low dilutions, for example, 101. This is because the bacteria
growth rate depends on initial cell density in the sample [8].

Sample suspension can be enriched by 24 h incubation at 37°C in nondifferential broth such as

Muller-Hinton or nutrient broth. This procedure will allow multiplication of E. coli and hence

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 increase the chance of E. coli isolation especially when infrequent strains, such as pathogens, are the
target. The generation (doubling) time for E. coli at 37°C incubation is 17–18 min [8], therefore, in
18–24 h incubation there will be 60–80 E. coli cell generations. However, clonal variability will
decrease when samples are enriched because same bacteria increase in number. Therefore, this
procedure is suitable when the research aims at a mere presence of a single specific strain and not
its variants.

The weight of the sample and the volume of diluent used in making the sample suspension may
affect the probability of bacteria recovery. Large sample weight normally increases the sensitivity of
the isolation procedure. For example, in E. coli studies to isolate nonsorbitol-fermenting Shiga toxin-
producing E. coli (NSF STEC) whereby E. coli broth was used to enrich fecal samples, different
prevalence measure was obtained. When 10 g of sample was suspended in 90 ml of E. coli broth, the
prevalence of Shiga toxin-producing E. coli (STEC) obtained was 1.3% [9], while the suspension of 20
g in 180 ml of same diluent resulted into a prevalence 11.1% NSF STEC [10].

Purification of E. coli colonies can be done in nondifferential media such as blood or nutrient agars.
Depending on the degree of colony density, a series of inoculations can be desired until pure, single,
or solitary colonies are obtained

C. Smearing Procedure

1. Label a clean glass slide as demonstrated by your instructor.

2. Add a small drop of saline to the slide (you will usually put two bacteria on one microscope
slide- Follow your instructor’s specific instructions). This can be done by placing a drop of saline
onto your inoculation loop and then transferring it to the slide. If you use the saline dropper directly
on the slide, do not release a full drop.

3. With an inoculation loop or needle, pick up a small amount of bacteria. Mix it well with the
saline and spread the mixture over a wider area of the slide.
• Be careful not to have the two smears run into each other.
4. Air dry the bacterial specimen on the slide (slide warmers may also be used).
5. When slides are completely air-dry, heat fix the bacterial specimen by passing the slide
slowly over the flame twice (your instructor will demonstrate this).
• Heat fixing kills cells, and adheres them to the slide.
• Cells will be rinsed off the slides if they are not heat fixed properly.
• Be careful not to overheat the slides in this procedure
After heat-fixing is complete, you are ready to gram stain your slide.

D. Staining Procedure

1. Add a few drops of crystal violet (primary stain) to the smear and let it sit for 60 seconds.

2. Rinse the slide with water. All cells are purple after this step. Stopping here would be a simple
stain.

3. Add a few drops of Gram's iodine (mordant) to the smear and let it sit for 60 seconds. Gram’s
iodine forms a complex with crystal violet

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 4. Rinse the slide with water. All cells are purple after this step.

5. Decolorize with 95% ethanol: let the alcohol run over surface of slide until no more crystal violet
color comes out of the smear (time varies—no more than 5-10 seconds). Gram positive cells retain
crystal violet and remain purple. Gram negative cells lose crystal violet and are now colorless
6. Rinse with water. Water rinse stops the decolorization process
7. Add a few drops of safranin (counterstain) and let it sit for 60 seconds. Safranin is a
pink/red dye
8. Rinse with water. Blot dry on bibulous paper. Be careful not to wipe off the bacteria. Gram
positive cells remain purple; gram negative cells are now pink/red
9. Observe your slide under the microscope. For each organism, determine morphology,
arrangement and Gram reaction

2. Inhibition of the growth of E.coli Gram negative bacterium through Lemon (Citrus Limon) Juice

Sample Collection
Lemon (Citrus limon) was collected from the traditional market in Larangan Market Sidoarjo, East
Java, Indonesia. The sample was placed in a plastic bag and was transported immediately to
Bacteriology Laboratory Faculty of Health Science, University of Maarif Hasyim Latif, Sidoarjo, Jawa
Timur, Indonesia. The sample was processed immediately in the laboratory.
2.2.Instruments, Reagents, and Medium
The instruments used in this study were a test tube, rack tube, scale pipette, spatula, drop pipette,
petri dish, ose, autoclave, bunsen, beaker glass, erlenmeyer, Mc. Farland 0.5 standard, steel pickers,
filters, tweezers, measuring cups, beaker glass, micropipette, yellow tip, incubator, stirrer, hot plate,
sterile swabs, digital scales, orange squeezer, aluminum foil and sterile gauze. The material used in
this study was lemon (Citrus limon) juice, enterotoxigenic Escherichia coli, Mac Conkey Agar medium
(Merck, Germany), Eosin Methylen Blue medium (Merck, Germany), Nutrient Broth medium (Merck,
Germany), Kliger Iron Agar medium (Merck, Germany), biochemical reactions medium (Merck,
Germany), sterile physiological zouth, sterile aquadest, MHA medium, ciprofloxacin 500 mg and
alcohol 70%.
2.3.Purification of ETEC Suspension
The stock of test bacteria, in this case, was Enterotoxin Escherichia coli (ETEC) planted in NB medium
and then incubated at 370 C for 24 hours. The bacteria that grew in NB medium were planted on
MCA and EMB medium and then incubated at 370 C for 24 hours [15]. Colonies grown on EMB
medium were planted in KIA medium and then incubated at 370 C for 24 hours. Furthermore, it was
planted in the biochemical reaction medium and incubated in 370 C for 24 hours. The colonies were
taken and planted on the NAS medium as the stock of bacteria
2.4.Preparation of ETEC Suspension
The bacterial colonies that have been grown for 24 hours on NAS medium were inserted into 10 mL
of sterile physiological zouth and then homogenized. The turbidity level of ETEC suspension was
equalized using McFarland 0.5 standard which had a density equivalent to 1.5 x 108 CFU/ml [16].
2.5.Preparation of lemon juice dose
Lemon (Citrus limon), that has been washed with sterile aquadest, was cut transversely using a
sterile knife and then squeezed using an orange squeezer. The juice was filtered using a sterile
gauze, as much as 100 mL was taken and diluted with 100 mL of sterile aquadest to obtain a

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 concentration of 1000 mg/ml dilution. From the dose of 1000 mg/ml, several dilutions were made to
obtain doses of, 200, 300, 400, 500, 600, 700, 800, and 900 mg/ml.
2.6.Preparation control
Prepared 1 mL of sterile aquadest and taken as much as 100 µl for inclusion in the well for negative
control [17]. Prepared of ciprofloxacin 500 mg was dissolved in 100 mL of sterile aquadest and then
100 µl was taken to be put into the well for positive control [17].

2.7.Lemon juice antimicrobial potential test

Prepared two types of tubes and labeled A and B. In tubes labeled A containing 5 ml MHA medium
And tube labeled B containing 15 ml MHA medium. Five mL of MHA medium in tubes labeled A was
Poured into a petri dish and allowed to solidify, then three steel pickers were placed with a spacing
Between 1.5 cm. One mL of ETEC bacterial suspension was put into 15 mL of MHA medium in tubes
Labeled B and homogenized, then poured into a petri dish containing a steel tray as a second layer
and Allowed to solidify. After the medium solidify, steel pickers were aseptically removed from the
petri Dish so that the wells formed. Each of the wells was filled with 100 µl of lemon (Citrus limon)
juice in various doses and Controls, then incubated at 370 C for 24 hours. Observation was done for
the formed inhibitory zone.

EXPECTED RESULT:

After doing all the process and procedures it therefore expected that Lemon (Citrus limon) juice can
inhibit the growth of pathogens that cause diarrhea, in this case, the Enterotoxin Escherichia coli
(ETEC) bacteria. This results showed that the optimum dose of lemon (Citrus limon) juice in inhibiting
diarrhea-causing pathogenic bacteria was 900 mg/ml.

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