EPISCOPAL DIOCESE OF SOUTHERN PHILIPPINES

BRENT HOSPITAL AND COLLEGES INCORPORATED

R.T. LIM BOULEVARD ZAMBOANGA CITY
P.O. BOX 33, ZAMBOANGA CITY 7000 PHILIPPINES
School Tel: 992-4447 Fax: (062) 991-5358

BASELLA RUBRA AND IPOMOEA BATATAS EXTRACT AS A BIOLOGICAL STAIN

FOR MICROSCOPIC URINE ELEMENTS

A Thesis Proposal

Presented to

The Faculty of the College of Medical Technology Department

Brent Hospital and Colleges Incorporated

Zamboanga City

In Partial Fulfillment

of the Requirements for the Degree

BACHELOR OF SCIENCE IN MEDICAL TECHNOLOGY

Researchers:

Hassan, Hanisa A.

Ali, Areej-zadeh A.

Kamlani, Sarah Grace G.

Mohadali, Ayman I.
 TABLE OF CONTENTS

Page

TITLE PAGE …………………………………………………………………….……. i

TABLE OF CONTENTS………………………………………………………………. ii

CHAPTER 1: INTRODUCTION

1.1. Background of the study…………………………………………………………. 1

1.2 Statement of the problem…………………………………………….…………… 3

1.3 Hypothesis…………………………………………………………….………... 4

1.4 Significance of the study………………………………………………………..…. 5

1.5 Scope and delimitations of the study………………………………….………….. 6

1.6 Definition of Terms…………………………………………………….……...... 6

CHAPTER 2: REVIEW OF RELATED LITERATURE

2.1 Basella rubra…….……………………………………………………….………… 8

2.2 Sweet Potato (Ipomoea batatas)………………………………………...…...... 10

2.3 Urine…………………………………………………………………….……… 13

2.4 Urine Analysis……………………………………………………….…………. 14

2.5 Related Studies……………………………………………………….……… 15

2.6 Conceptual Framework……………………………………………………….. 16

CHAPTER 3: METHODOLOGY

3.1 Research Design…………………………………………………….................. 17

3.2 Research Sample……………………………………………………................... 17

3.3 Research Locale………………...……………………………………………….. 20

3.4 Research Instrument…………….……………………………………….…… 20

3.5 Preparation of Plant…………………………………………….…................ 20

3.6 Preparation of different concentrations of Ethyl alcohol………………........ 21

3.7 Preparation of Pure extract of the leaves………………………………......... 21

3.8 Extraction of Plant using distilled water………………...………………...... 22

3.9 Extraction of the plant using ethyl alcohol…….………………………........ 22

3.10 Collection and Preparation of Semen Sample…….……………………….. 22

3.11 Application of Plant Extract to the Collected Sample……………….......... 22

3.12 Proper Waste Disposal…………………………………….…........................ 24

3.13 Rubrics………………………………………………….……………………. 25

3.14 Statistical Analysis…………………………………….…………………... 25

BIBLIOGRAPHY……………………………………………………………... 27
 CHAPTER I

1.1 Background of the study

Clinical microscopy is a method that utilizes scientific techniques to examine non-blood

bodily fluids, including urine. A test called a urinalysis examines many elements of a urine

sample. A full urinalysis includes visual, chemical, and microscopic examinations. Urinalysis is

widely performed to check for diabetes, kidney, liver, and urinary tract infections (UTIs). A

urinalysis could be carried out as part of a normal examination, to assess specific symptoms, or

after a patient is admitted to the hospital (Kampfrath, 2022). Due to its popularity, quick

turnaround time, and low cost, urinalysis is a common screening test utilized in a variety of

inpatient and outpatient clinical settings. It can also aid in the diagnosis and progression of a

variety of illnesses, including renal calculi, metabolic problems, strokes, and cancer (Advani, S.

et. al 2021).

When urinary sediment components need to be confirmed, recognized, or distinguished

from similar components, a variety of acceptable staining techniques may be helpful. Basic

staining solutions include Sternheimer staining (S staining) and Sternheimer–Malbin staining

(SM staining). Other stains like Sudan III can stain fat globules, fatty casts, and oval fat bodies

yellowish red. Prescott-Brodie can stain peroxidase-containing cells such as neutrophils,

eosinophils, and monocytes blue or blackish blue, while lymphocytes and other cells stain red.

And lastly, Lugol’s solution stains the epithelial cells which stain the cytoplasm brownish red

(Japanese Association of Medical Technologists, 2017).

Malabar spinach (Basella rubra) is a jungle vining plant that is often referred to as

spinach but is not a true spinach. It is a member of the Brassica family and has a flavor and

texture similar to spinach, growing well in hot summer months. There are two types, one with
green and red stems, and both produce small, globe and pinnate-shaped flowers that turn into

purple berries. The plant grows in long vines, making it an attractive ornamental plant and its

leaves are heat tolerant as long as the soil is kept moist. All parts of the plant are edible, with the

leaves, stems, and flowers often enjoyed raw or cooked. The roots have medicinal uses, and the

berries are used as a dye (Specialty Produce Network, n.d).

Sweet potato (Ipomoea batatas [L.] Lam.) is a staple food grown worldwide, particularly

in Sub-Saharan Africa, East Asia, and Oceania. It is known for its versatility and ability to grow

in various climates. Sweet potatoes contain various phytochemicals, including beta-carotene,

which is present in high amounts in white-fleshed purple-skinned sweet potatoes and is being

used to combat vitamin A deficiency in some countries. Purple-fleshed sweet potatoes are also

enriched with anthocyanins, phenolic compounds responsible for the intense color and potential

health benefits ( Lim, S. et.al 2013).

Natural elements including fruits, roots, leaves, and insects are used to make natural

colors. They provide natural alternatives to synthetic dyes, which are frequently made from

petroleum and can be hazardous to the environment and people's health due to chemical exposure

and toxic waste. Natural dyes promote regional economies and conventional dyeing methods.

Natural dyes, however, can have a detrimental influence on the environment and are more costly

and frequently less colorfast than synthetic dyes. However, the usage of natural dyes in the

fashion and textile sectors is expanding because of the rising demand for sustainable and

ecologically friendly goods (Bell, 2021).

Plant pigments can be divided into four main groups: chlorophylls, anthocyanins,

carotenoids, and betalains. These are the major sources of naturally occurring colors from plants.

Most natural pigments are sensitive to pH and their color can change or degrade when exposed to
an incompatible pH. The color can also vary based on the pH level. Some natural colorants, such

as carotenoids found in fruit and vegetable juices, can be damaged by heat (Dikshit &

Tallapragada, 2019). Anthocyanins are an important type of plant pigment, belonging to the

flavonoid family. Over 500 different anthocyanins have been found. They provide plants,

flowers, and fruits with vibrant colors ranging from pink to scarlet, purple, and blue. They are

soluble in water ( Sudhakar, P. et al. 2016).

The researchers conducted a study to investigate the use of Malabar Spinach (Basella

rubra) and Sweet Potato (Ipomoea Batatas) as potential eco-friendly enhancement stains for

sediments only present in the urine sample. The results of the study somehow provide a new

stain alternative and are used to compare its effectiveness to current methods.

1.2 Statement of the Problem

The main objective of this study is to determine the staining potential between Malabar

Spinach (Basella rubra) and Purple Sweet Potato (Ipomoea batatas) extracts as a screening stain

for the observation of urine sediments.

Specifically, this study seeks to answer the following questions:

1. Determine the staining capacity of Basella rubra and Ipomoea Batatas plant extract in

urine sediments at different concentrations and solution?

1a .Pure Extract

2a. Distilled Water

2a1. 25%

2a2. 50%
 2a3. 70%

3a. Ethyl Alcohol

3a1. 25%

3a2. 50%

3a3. 70%

4a. Normal Saline Solution

4a1. 25%

4a2. 50%

4a3. 70%

2. Determine the significant difference in the staining capacity of Basella rubra and

Ipomoea Batatas extract to urine sediments in using different concentrations of solutions?

3. Determine the ideal staining capacity of the plant extracts in assessing the urine

sediments?

1.3 Hypothesis

1. There is no staining capacity of Basella rubra and Ipomoea Batatas plant extract in urine

sediments at different concentrations and solutions.

2. There is no significant difference in the staining capacity of Basella rubra and Ipomoea

Batatas extract to urine sediments in using different concentrations of solutions.

3. The staining capacity of the plant extracts is not ideal for assessing urine sediments.
1.4 Significance of the Study

This study will benefit the following:

Academe. This research will provide additional information about the efficacy of a natural dye

that will be used as a screening stain for urine sediments.

Future Researchers and Future Studies. This study will provide a wide range of information

and insight into the effectiveness of a natural dye in the field of clinical method studies.

Manufacturer. This research will benefit manufacturers of environmentally friendly or

"eco-friendly" products by providing them with new ideas for their products, which will enable

them to target new markets and expand their customer base.

Medical Laboratory Scientists. This study will be particularly useful for enhancing the

visualization of urine sediment and those working in rural or remote areas where access to

commercially available stains may be limited.

Medical Technology Students. This research will provide information that may help the

importance of the section specifically the clinical microscopy and the clinical importance of

urine sediments. This study will provide students with a deeper understanding of their field of

study and the practice of clinical microscopy, specifically in regard to alternative, eco-friendly

staining methods.

1.5 Scope and Limitations of the Study

To achieve the objectives, the researcher mainly focused on the determination of the

staining properties of the Basella rubra (Malabar Spinach) and Ipomoea batatas L. (Purple
Sweet Potato) extracts, and to enhance the visualization of urine sediments that may be clinical

significant findings. The researchers obtained the two plants and randomly collected urine

samples at Suterville, San Jose Gusu, Zamboanga City and Laboratory, Brent Hospital, RT Lim,

Boulevard, Zamboanga City, respectively.

The plant, Basella rubra, and Ipomoea batatas extracts were prepared in different

concentrations and used it as a dye for staining the urine sediment. The urine samples were

evaluated microscopically by (3) three Registered Medical Technologists from different

hospitals. Each evaluator has a rubric form for assessment.

1.6 Definition of terms

For clarity in the statements, the following terminologies are defined operationally:

Clinical microscopy - this section is where urinalysis is performed. This section applies

to the study conducted.

Malabar spinach - it is the plant that is used for the experiment which is added to the

urine sample, and prepared in different concentrations and solutions.

Purple sweet potato - it is the plant that is used for the experiment which is added to the

urine sample, and prepared in different concentrations and solutions.

Urine - this sample is used to detect sediments or cells in the urine.

Urinalysis - this method is used to examine and assess urine and its sediments
 CHAPTER 2

REVIEW OF RELATED LITERATURE

This chapter presents the research on Ipomoea batatas (Sweet Potato Leaves) and Basella

rubra (Malabar spinach) leaf and stem extracts as substitute stains for detecting the presence of

urine sediments. This chapter was give an insight into the staining capacity of Ipomoea batatas

and Basella rubra which served as an alternative stain. The conceptual framework, synthesis, and

overview were stated in this chapter which fully supports the study.

2.1 Basella rubra (Malabar Spinach)

The vegetable family Basellaceae includes the nutrient-rich Malabar spinach (Basella

spp.). It is indigenous to South Asia's tropical regions, which include New Guinea and the Indian

subcontinent. In China, tropical Africa, Belize, Brazil, Colombia, Fiji, French Polynesia, and the

West Indies, it most likely became a native species (Baksh-Comeau et al. 2016). Currently, this

species may be found in the tropics of Southeast Asia, South America, and Africa. Indian

spinach, Ceylon spinach, vine spinach, and climbing spinach are some of the other names for

Malabar spinach ((Deshmukh and Gaikwad 2014, Singh et al. 2018).

In the study of Chaurasiya et al., which was conducted in the year 2021, they stated that

Malabar spinach is a thick, succulent, slender, smooth, and brilliant stem with spirally arranged

leaves. The stem also shows side branches. The stem measures 8 to 10 meters. Because the

length of the leaf is greater than its breadth, the leaf is oblong and has a short stem. Depending

on the cultivar, Basella rubra has white, red, or pink flowers. Red or black are the colors of fruits.
The seed's exterior is brilliant and rough, and it is colored black and brown. The seed is

surrounded by thick testa.

Basella rubra L. is one of the inexpensive and indigenous vegetables being promoted by

the Bureau of Plant Industry in the Philippines. This plant has heart-shaped leaves, sapid taste,

branched stems, and non-woody vines. It offers a multitude of potential health benefits but is an

underutilized crop in the country. Additionally, it contains bioactive non-nutrient compounds

including its vasodilatory actions, anticancer, antioxidant, and anti-inflammatory properties

(Tongco et al., 2015).

Basella rubra leaves and fruits contain betacyanin and flavonoid pigments. The major red

pigment in its dye extract is gomphrena-I, which is a betalain. The red-violet color of its leaves,

stalks, petioles, and fruits is due to betalains. The plant also contains the natural pigment

anthocyanin in its stem, leaves, and flowers. Betalains are red or yellow pigments found in many

plants, including beetroot and amaranthus, but the content in Basella is lower than in beet tubers.

Betalains extracted from Basella and other plants are used in food, pharmaceutical, and cosmetic

industries as an alternative to synthetic colorants. The fruit extract of Basella rubra can be used

as a natural fabric dye due to its pigments (Chaurasiya A., 2021). Bari and Kanon (2022)

concluded that Malabar spinach leaves natural dye can be a possible substitute for synthetic dyes.

A number of methods for using Basella rubra L. dye were provided in the study by Mitra

and Das (2015). Malabar spinach or (Alugbati) could work as an alternate stain. For example, it

was discovered that the plant's fruit can be efficiently utilized in the paper sectors. Synthetic hair

dyes and food coloring can be replaced with the red, blue, and purple anthocyanin pigments that

were extracted from this spinach. Its safety is assured by the fact that the extracted dye is natural.
2.2 Purple Sweet Potato (Ipomoea batatas)

According to Pakkies N. & Keesler N. (2018), the nomenclature of Sweet Potato

(Ipomoea batatas L.; Lam.) is a dicotyledonous vegetable belonging to the family

Convolvulaceae. The sweet potato plant is a creeping, branching vine with white or lavender

blooms and lobed, spirally arranged leaves in the shape of hearts. The plant has tubers, which are

larger roots that serve as an energy reservoir for the plant. The sweet potato plant is typically

grown as an annual and is harvested after one growing season. Sweet potato vines can grow up to

4 m (13 ft) in length. Sweet potatoes, which are native to Central America, are sometimes known

as yams or Spanish potatoes. It is the seventh most-produced crop worldwide after wheat, rice,

maize, potato, barley, and cassava, and the fifth in developing countries (Laveriano-Santos et al.,

2022). In many countries, its culture and production are essential, because it contributes to

reducing food shortages in times of crisis (natural disasters or wars). It is amongst the world's

most important, versatile, and under-exploited food crops with more than 90 million tons in

annual production, contributed mostly by Asian and African countries (Alam, 2021).

Due to the crop's flexibility and phenotypic and morphological diversity, it was able to

successfully spread over Asia, Africa, and Latin America in the 17th and 18th centuries and

could be modified to satisfy a range of nutritional needs. The majority of the world's sweet

potato production—roughly 95% of it—is grown in developing nations; it is also the most widely

farmed root crop there. Due to its adaptability and high nutrient content, it has a long history of

being utilized as a lifesaving, famine-relieving crop and is known in some regions of East Africa

as the "protector of children" for these reasons (Motsa, 2015).

 Sweet potato plants (Ipomoea batatas L.) consist of 3 varieties: yellow, red, and purple

sweet potatoes. Based on these varieties, sweet potatoes have a variety of colors, namely white,

yellow or orange, and purple. Purple sweet potato is a variety that has advantages over other

sweet potato varieties, namely that it contains higher anthocyanin pigments (Rustanti, 2018).

Leafy sweet potato is a new type of sweet potato in China consumed for its nutritious

stems and leaves, which are rich in proteins, fiber, vitamins, and minerals. A study found that the

leaves contain high levels of flavonoids and anthocyanins, including several cyanidin

derivatives. The genes DFR4, F3H1, anthocyanin synthase (ANS), UDP-glucose flavonoid

3-O-glucosyltransferase (UFGT3), and MYB1 were found to be important for regulating the

accumulation of anthocyanins in sweet potato leaves (Li et al., 2019). In a comparative study

done by Vishnu et al, (2019), it is concluded that cyanidin, an anthocyanin subset, is higher in

leaves than in its tubers.

Meanwhile, anthocyanins are water-soluble pigments responsible for the red, blue, and

purple appearance of diverse fruits and vegetables. The use of this pigment as a natural dye is

claimed to give pharmaceutical ingredients beneficial for an individual’s health due to its

medicinal values specifically antidiabetic, anti-obesity, antimicrobial, and antioxidant properties

(Azlan et al., 2017).

In relation to the pigments, the tint, and stability of the colorant were distinguished and

summarized by Azlan et al. (2017). They observed that the color and the lasting nature of the

dye are influenced by several factors enumerated as the acidity and basicity level, light,

temperature, and structure. The color of anthocyanins depends on the temperature - it affects the

color of the pigment. Anthocyanins are not very stable in hotter solutions. It was reported that a
heat treatment with a maximum of 35⁰ C lessened the amount of anthocyanin content in a

common grape compared to the effect of heat treatment at 25⁰ C. Mild heat treatment is

recommended to avoid the oxidation of pigments.

Anthocyanins have a growing demand in the food industry as natural colorants due to

their vibrant colors and health benefits. Despite their high reactivity and destabilizing

interactions, attempts have been made to stabilize anthocyanins for food applications through

co-pigmentation, metal ion complexation, acylation, and other methods. The application of

anthocyanins as food colorants has been limited but has been reported to produce various colors

within a pH range of 2-8. Companies are working on producing stable anthocyanin-based natural

colors which have been reviewed in the literature (Alappat et al., 2020).

2.3 Urine Analysis

Approximately 6,000 years ago, the analysis of urine specimens started laboratory

medicine. Urinalysis was still termed as ‘uroscopy’ which is derived from two greek words

namely; “ouron” which means urine and “skopeoa” which means ‘behold, contemplate,

examine, and inspect.’. Physicians of the ancient times believed that urine was a window to the

body's inner workings and is a reflection of different diseases. One of the earliest known records

of urine tests is pouring urine on the ground and observing whether the ants were attracted to it

or not. (Milani and Jialal, 2022)

Today, urine analysis or urinalysis is a physical, chemical, and microscopic examination.

It is done to diagnose diseases and check for abnormalities. Dipstick urinalysis is a common

method for urine analysis and is convenient but false-positive and false-negative results can

occur. Urine is commonly collected midstream and should be examined within 1-2 hours of

collection. There are three examinations done: physical, chemical, and microscopic. Physical
examination includes the color, clarity, volume, odor, and the urine's specific gravity. By

physical means, a cloudy urine is usually a result of precipitated phosphate crystals in alkaline

urine or it can also be pyuria. A strong odor can be the result of concentrated specimen or a

urinary tract infection. Chemical examination checks for pH, RBC, WBC, proteins, glucose,

urobilinogen, bilirubin, ketone bodies, leukocyte esterase, and nitrites. In depth examination of

urine specimens can be done by microscopic means. It can check for casts, cells, crystals, and

microorganisms. For example, an abnormal amount of urine sediment can be closely examined

such as the increase of calcium oxalates which can be an indication of kidney stones. (Simerville

et al. 2005, Milani and Jialal, 2022.)

Aside from diagnosing diseases and checking for abnormalities, urinalysis is also used

for detecting drugs in criminal justice setting. Although urinalysis is mostly useful, it does not

ensure that all instances of drug use are detected. There are varying periods of time where

metabolites stay in urine. An active ingredient in marijuana called THC, can be detected for as

long as 5 weeks after chronic use while opiate use will remain for only 1-2 days in the urine.

(MacPherson, 2001.)

Urinalysis is a major screening clinical laboratory test that is performed most frequently

on random specimen. In collecting the specimen, a clean and dry wide-mouthed plastic container

with tight-fitting, sterile cap are provided to the patients. The patients must be instructed to pee

directly into a clean plastic container or a clean and dry bedpan which will then be transferred

into a clean plastic container. All patient must be instructed to bring a clean-voided specimen and

to immediately cover and deliver it to the laboratory. A mid-stream specimen is commonly used

for routine analysis. (Kassa et al. 2002)

 The diagnosis and treatment of diseases are becoming more and more dependent on

medical tests. The three main regular examination procedures used in modern medicine—urine,

blood, and stool—account for the majority of medical tests. The human body generally processes

urine as a bodily fluid. Urine offers various benefits over blood when used as a test sample in

medicine, including being non-invasive and easy to collect (Zhang et al., 2022).

Urinalysis, or the examination of urine, may be required in the assessment of kidney and

urinary tract conditions. It can also be used to assess conditions that affect the entire body, such

as diabetes or liver issues. Most frequently, a clean-catch technique or another sterile technique is

used to collect a urine sample. For instance, inserting a catheter through the urethra into the

bladder is one way to get a sample of clean urine (Chung, 2022).

2.4 Urine

Abebayehu (2023) stated in their study that urine, a waste product of the body, can give

us immense information about health conditions or disorders of certain organs. It is a biological

fluid interminably produced and excreted from the body and provides important information on

the states of disease or body dysfunction before another fluid composition is altered to a

significant extent. Therefore, the examination of urine is an aid in the diagnosis and monitoring

of the course of treatment of certain diseases.

The elimination of urine is very important for different bodily functions. It regulates the

balance of water in the body, for example, and also gets rid of substances that are produced

during metabolic processes and are no longer needed by the body. These include toxic substances

in food or medicines. Urine tests can help detect diseases of the urinary system as well as

metabolic diseases like diabetes or liver disease (DeGuyter, 2017).

 The correct specimen collection, processing, and management by the healthcare

practitioner is the most crucial phase in urine analysis. By identifying the potential reasons that

could aid in the patient's diagnosis, it is one approach to learn about the patient's health state

(Wayne, 2016). Delanghe and Speeckaert (2014) cited in their study that the correct information

on the appropriate patient preparation and sample technique must come from the laboratory.

Only when these requirements are met is it possible to interpret test findings. Informing the

patient entails much more than merely outlining the technicalities of the urine sample. The

impact of potential biological confounders, such as nutritional intake, diuresis, exercise, and

other interfering factors, should be highlighted in more detail. If required, illustrative guidelines

for sampling might be given

A clean, sterile, clear, wide-mouth, flat-bottomed container with a screw cover is required

for collecting urine. To ensure the validity of test results, the technique is gender-specific. Male

patients who have not had their foreskins circumcised should wash their hands with soap and

water and retract their foreskins. Wipe the tip of the penis with a tissue. When you start to pee,

let a small amount of urine enter the toilet bowl to flush out urethral impurities. The generated

urine stream should be transferred into a sterile, screw-cap plastic cup. It should contain roughly

50 milliliters, or half of the cup. Pour the remaining urine into the toilet. The cover of the cup has

to be securely fastened on. Send the sample soon away. While procedures for women are mostly

the same as for men, there are significant differences as well. Spread the skin folds (labia) apart

with one hand until the urine drains into a sterile screw-top container. From the front to the rear,

clean the urethral meatus. Allow the initial portion of the urine to pass until the urine stream is

properly established before catching the specimen in the sterile container without interrupting the
flow. Holding the sterile container should be done to prevent contact with the legs or clothes

(Atef, 2022).

In 2021, Nabili stated that, after receiving the sample in the lab, the macroscopic

inspection must be completed before the microscopic urine analysis. Observations that can be

seen with the naked eye and do not need to be examined under a microscope are referred to as

macroscopic. Urine's physical appearance is examined as part of the macroscopic analysis

process. Normal urine is clear and pale yellow in color. In addition to noting the quantity, color,

and clarity of the urine, a macroscopic urinalysis also records any additional features of the urine

that are visible, such as the presence of blood or blood clots, precipitates, or sediments.

2.5 Urine sediments

Sediments are defined as the matter that settles at the bottom of the liquid. Urine

sediments can cause the urine to look cloudy rather than its normal clarity. These urine sediments

are often found in urinalysis specifically in microscopic examinations. It becomes a problem

when the number exceeds its normal amount or it shows an abnormally high concentration.

(Mukherjee et al. 2022)

There are two elements of urine sediment, namely; organized and unorganized elements.

Biological components like sperm, leukocytes, erythrocytes, epithelial cells, casts, bacteria,

fungus, and parasites make up organized urine sediment. Crystals of various salts such as

oxalate, phosphate, urate, and amorphous salts, can be found in unorganized urine sediment.

These components of urinary sediment plays a significant role in the diagnosis and treatment of

renal patients. (Bunjevac et al. 2018)

One of the urine sediments are the epithelial cells. These cells comes from every surface

of the body, outside and within. It can come from your skin, blood vessels, and urinary tract, or
organs. Their role is to protect the body from viruses. There are three types of epithelial cells

commonly found in the urine. These are renal tubular, squamous, and transitional epithelial cells.

Renal cells are the most necessary epithelial cells. An increased number might be an indication

for kidney disorder. Squamous epithelial cells are the largest type that comes from the vagina and

urethra. Lastly, transitional epithelial cells are found in a male's urine and are most common in

older adults. They shed from the male urethra and the renal pelvis. (Stephens, 2018)

2.6 Related studies

A study demonstrated that turmeric possesses the ability to color microscopic urine

components, primarily due to its high curcumin content, which acts as an active coloring agent.

However, casts, which have a dense protein structure, did not stain adequately across different

concentrations of the turmeric extract due to their limited affinity for stains. Crystals and

epithelial cells, on the other hand, exhibited enhanced visibility in urine samples containing 2%

and 3% concentrations of the extract. Red blood cells (RBCs) showed the best staining results

with a 2% concentration, while urates and phosphates were observed most effectively at either

1% or 3% concentrations. Other urine elements, such as white blood cells (WBCs), yeast cells,

and hyphal elements, did not exhibit optimal staining reactions at any of the concentrations

tested but were moderately stained across the different extract concentrations.

Crystals and RBCs displayed the highest staining affinity at a pH level of 6, while yeast cells and

hyphal elements exhibited optimal staining reactions at pH levels 5 and 6. Urates and phosphates

yielded better staining results at pH 5. WBCs and epithelial cells did not show an ideal staining

response at a specific pH level but were moderately stained across various pH levels. Casts

displayed insufficient staining capacity regardless of the pH level used.

 Epithelial cells, RBCs, WBCs, yeast cells, hyphal elements, urates, phosphates, and casts

demonstrated improved staining outcomes at higher concentrations, particularly at 3%, using

either a 5-minute or 10-minute staining time. Crystals showed optimal staining reactions

regardless of the concentration used, with both a 5-minute and 10-minute staining time.

2.6 Conceptual Framework

Plate 1. The conceptual framework

The conceptual paradigm explains how the Basella rubra L. and Ipomoea batatas will

become a biological stain, this shows the input, which means what material is to be used as a

biological stain and the sample that is subjected for testing. Next is the Process and procedure on

how we create the stain down to microscopic evaluation to see the staining capacity of the

concentration and solution. And lastly the output determines the efficacy of the biological stain.
 CHAPTER III

METHODOLOGY

This chapter presents the research design, research sample, research locale, and research

instruments or methods utilized in the experiment from the collection of the materials up to

testing the efficacy of the extracts of the materials and observing the staining effects on the urine

sediments which are viewed under the microscope.

3.1 Research Design

The researchers assessed the effectiveness of Purple Sweet Potato (Ipomoea batatas) and

Malabar Spinach (Basella rubra) extracts as an alternative stain for urine sediments assessment,

which is then exposed to various concentrations of the extracts and compared to see which of the

two plants stains the best. This study used a quantitative and cross-sectional experimental

research design that employs a systematic, scientific, and comparative approach to research. This

research design used one or more variables, control, and numerical data analysis to determine the

relationship and difference of this study. This research compared which of the concentrations are

potent in staining the urine sediments which contributes to its visibility when viewed in the

microscope. It is quantitative since it uses the methods of collecting data which are numerical

and is based on the result of the experiment. (definition of experimental design)

3.2 Research Sample

The researchers used Purple Sweet Potato (Ipomoea batatas) and Malabar Spinach

(Basella rubra) extracts which were utilized as an alternative stain. The sweet potato plant is a

creeping, branching vine with white or lavender blooms and lobed, spirally arranged leaves in

the shape of hearts. The plant has tubers, which are larger roots that serve as an energy reservoir
for the plant. The color and shape of the tubers can vary, and they might be red, yellow, brown,

white, or purple. Since it is a component of many regional specialties, sweet potatoes are a staple

food in the Philippines. It can be grown throughout the entire year. Malabar spinach produces

modest pink blooms with a white tinge in the leaf axils and reaches heights and widths of eight to

ten feet. The blossoms turn into single-seeded, tiny, highly decorative purple berries after

fertilization. It requires full sun to partial afternoon shade, the soil should be well-drained and

requires support, such as a trellis since other plants will be swiftly surpassed by it.

Plate 2. Purple Sweet Potato (Ipomoea batatas L.) and Malabar Spinach (Basella rubra)

3.3 Research Locale

The study was conducted at Brent Hospital and Colleges Incorporated which is located at

R.T. Lim Boulevard, Zamboanga City. The researchers conducted the experimentation at the

Medical Technology Laboratory of Brent Hospital and Colleges Incorporated.

 Figure 3. Brent Hospital and Colleges Inc., R.T. Lim Boulevard, Zamboanga City

3.4 Research Instrument

All the laboratory equipment used are clean and sterilize such as Food processor,

centrifuge, Grade 1 Whatman paper, sterile containers, glass slides, microscope, Erlenmeyer

flask, funnel, beaker, test tubes, Muslin cloth, laboratory gown, facemask, Pasteur pipet, and

gloves.

3.5 Preparation of Plant

The Purple Sweet Potato (Ipomoea batatas L.) and Malabar Spinach (Basella rubra)

plant was collected from the public market of Zamboanga City which is located in Magay and in

Barangay Suterville, Zamboanga City. The plant exhibits a purplish-red color and bright or dark

green and is not wilted. The plant was washed with tap water to remove the dirt present. After

washing, the plant was placed on a filter and was air-dried for at least 3 minutes. After the plant
has dried, the plant is pulverized into powder using a food processor or blender. After the plants

have been pulverized, it is placed into a sterile container.

3.6 Preparation of Pure Extract

3.6.a Ipomoea batatas

The pulverized leaves of the plant were extracted using a Muslin cloth which was

squeezed to obtain the pure extract. The extracts were then stored in the Erlenmeyer flask and

covered to avoid any contamination. After extracting, the leftovers of the plant were disposed

into its appropriate waste bin.

3.6.b Basella Rubra

The plant was squeezed using a Muslin cloth after grinding it. The extracts were

then placed in an Erlenmeyer flask. The pure extract was then centrifuged for at least 10 minutes

at high speed to get rid of the sticky substances that could interfere with the results. After

centrifuging, it was stored in a container and covered.

3.7 Preparation of Different Concentrations of Ipomoea batatas

3.7.a Ethyl Alcohol

To create 25% ethanol concentration, 7.5 mL of absolute ethyl alcohol was placed

in an Erlenmeyer flask. 2.5 mL of pure extract of Ipomoea batatas were added to the

alcohol solution.

To create 50% ethanol concentration, 5 mL of absolute ethyl alcohol was placed

in an Erlenmeyer flask. 5 mL of pure extract of Ipomoea batatas were added to the

alcohol solution.
 To create 70% ethanol concentration, 3 mL of absolute ethyl alcohol was placed

in an Erlenmeyer flask. 7 mL of pure extract of Ipomoea batatas were added to the

alcohol solution.

3.7.b Distilled Water

To create 25% distilled water concentration, 7.5 mL of distilled water was placed

in an Erlenmeyer flask. 2.5 mL of pure extract of Ipomoea batatas were added to the

distilled water.

To create 50% distilled water concentration, 5 mL of distilled water was placed in

an Erlenmeyer flask. 5 mL of pure extract of Ipomoea batatas were added to the distilled

water.

To create 70% distilled water concentration, 3 mL of distilled water was placed in

an Erlenmeyer flask. 7 mL of pure extract of Ipomoea batatas were added to the distilled

water.

3.7.c Normal Saline Solution

To create 25% NSS concentration, 7.5 mL of normal saline solution was placed in

an Erlenmeyer flask. 2.5 mL of pure extract of Ipomoea batatas were added to the NSS

solution.

To create 50% NSS concentration, 5 mL of normal saline solution was placed in

an Erlenmeyer flask. 5 mL of pure extract of Ipomoea batatas were added to the NSS

solution.

To create 70% NSS concentration, 3 mL of normal saline solution was placed in an

Erlenmeyer flask. 7 mL of pure extract of Ipomoea batatas were added to the NSS

solution
3.8 Preparation of Different Concentration of Basella rubra

3.8.a Ethyl Alcohol

To create 25% ethanol concentration, 7.5 mL of absolute ethyl alcohol was placed

in an Erlenmeyer flask. 2.5 mL of pure extract of Basella rubra were added to the alcohol

solution.

To create 50% ethanol concentration, 5 mL of absolute ethyl alcohol was placed

in an Erlenmeyer flask. 5 mL of pure extract of Basella rubra were added to the alcohol

solution.

To create 70% ethanol concentration, 3 mL of absolute ethyl alcohol was placed

in an Erlenmeyer flask. 7 mL of pure extract of Basella rubra were added to the alcohol

solution.

3.8.b Distilled Water

To create 25% distilled water concentration, 7.5 mL of distilled water was placed

in an Erlenmeyer flask. 2.5 mL of pure extract of Basella rubra were added to the

distilled water.

To create 50% distilled water concentration, 5 mL of distilled water was placed in

an Erlenmeyer flask. 5 mL of pure extract of Basella rubra were added to the distilled

water.

To create 70% distilled water concentration, 3 mL of distilled water was placed in

an Erlenmeyer flask. 7 mL of pure extract of Basella rubra were added to the distilled

water.

3.8.c Normal Saline Solution

 To create 25% NSS concentration, 7.5 mL of normal saline solution was placed in

an Erlenmeyer flask. 2.5 mL of pure extract of Basella rubra was added to the NSS

solution.

To create 50% NSS concentration, 5 mL of normal saline solution was placed in

an Erlenmeyer flask. 5 mL of pure extract of Basella rubra was added to the NSS

solution.

To create 70% NSS concentration, 3 mL of normal saline solution was placed in an

Erlenmeyer flask. 7 mL of pure extract of Basella rubra was added to the NSS solution.

3.9 Collection and Preparation of Urine Sample

The researchers obtained the sample at Suterville, San Jose Gusu, Zamboanga City and

Brent Hospital Laboratory with the affirmation of the person entrusted and respondents.. It was

collected using a clean, sterile container with a flat bottom and screw cap The respondents for

this study were five females from the institution and were instructed to catch the midstream urine

to avoid contamination and altering of results. The urine sample was then transferred to test tubes

of the same size and same volume of the urine sample and was centrifuged for at least 5 minutes

at high speed. After centrifuging, the supernatant was decanted and only the sediments at the

bottom were left behind. Tapping the bottom of the tube was performed to mix the sediments and

to not make them adhere too much on the test tube wall.

3.10 Application of Ipomoea batatas and Basella Rubra Pure Extract to the sample

In a clean glass slide, three drops of urine were placed leaving an even space between the

three of them. The extracts were then dropped onto the urine drop and it was mixed in a gentle

manner.
3.11 Application of Purple Sweet Potato in Different Concentrations

3.11a Ethyl Alcohol

Using a clean glass slide, two drops of urine were placed. One for the control and

one for the stained urine. The 25% ethanol solution of Ipomoea batatas was dropped onto

the urine and mixed. The sample is then observed microscopically.

Using a clean glass slide, two drops of urine were placed. One for the control and

one for the stained urine. The 50% ethanol solution of Ipomoea batatas was dropped onto

the urine and mixed. The sample is then observed microscopically.

Using a clean glass slide, two drops of urine were placed. One for the control and

one for the stained urine. The 70% ethanol solution of Ipomoea batatas was dropped onto

the urine and mixed. The sample is then observed microscopically.

3.11b Distilled Water

Using a clean glass slide, two drops of urine were placed. One for the control and

one for the stained urine. The 25% distilled water solution of Ipomoea batatas was

dropped onto the urine and mixed. The sample is then observed microscopically.

Using a clean glass slide, two drops of urine were placed. One for the control and

one for the stained urine. The 50% distilled water solution of Ipomoea batatas was

dropped onto the urine and mixed. The sample is then observed microscopically.

Using a clean glass slide, two drops of urine were placed. One for the control and

one for the stained urine. The 70% distilled water solution of Ipomoea batatas was

dropped onto the urine and mixed. The sample is then observed microscopically.
 3.11c Normal Saline Solution

Using a clean glass slide, two drops of urine were placed. One for the control and

one for the stained urine. The 25% NSS concentration of Ipomoea batatas was dropped

onto the urine and mixed. The sample is then observed microscopically.

Using a clean glass slide, two drops of urine were placed. One for the control and

one for the stained urine. The 50% NSS concentration of Ipomoea batatas was dropped

onto the urine and mixed. The sample is then observed microscopically.

Using a clean glass slide, two drops of urine were placed. One for the control and

one for the stained urine. The 70% NSS concentration of Ipomoea batatas was dropped

onto the urine and mixed. The sample is then observed microscopically.

3.12 Application of Basella rubra in Different Concentrations

3.12a Ethyl Alcohol

Using a clean glass slide, two drops of urine were placed. One for the control and

one for the stained urine. The 25% ethanol solution of Basella rubra was dropped onto

the urine and mixed. The sample is then observed microscopically.

Using a clean glass slide, two drops of urine were placed. One for the control and

one for the stained urine. The 50% ethanol solution of Basella rubra was dropped onto

the urine and mixed. The sample is then observed microscopically.

Using a clean glass slide, two drops of urine were placed. One for the control and

one for the stained urine. The 70% ethanol solution of Basella rubra was dropped onto

the urine and mixed. The sample is then observed microscopically.

 3.12b Distilled Water

Using a clean glass slide, two drops of urine were placed. One for the control and

one for the stained urine. The 25% distilled water solution of Basella rubra was dropped

onto the urine and mixed. The sample is then observed microscopically.

Using a clean glass slide, two drops of urine were placed. One for the control and

one for the stained urine. The 50% distilled water solution of Basella rubra was dropped

onto the urine and mixed. The sample is then observed microscopically.

Using a clean glass slide, two drops of urine were placed. One for the control and

one for the stained urine. The 70% distilled water solution of Basella rubra was dropped

onto the urine and mixed. The sample is then observed microscopically.

3.12c Normal Saline Solution

Using a clean glass slide, two drops of urine were placed. One for the control and

one for the stained urine. The 25% NSS concentration of Basella rubra was dropped onto

the urine and mixed. The sample is then observed microscopically.

Using a clean glass slide, two drops of urine were placed. One for the control and

one for the stained urine. The 50% NSS concentration of Basella rubra was dropped onto

the urine and mixed. The sample is then observed microscopically.

Using a clean glass slide, two drops of urine were placed. One for the control and

one for the stained urine. The 70% NSS concentration of Basella rubra was dropped onto

the urine and mixed. The sample is then observed microscopically

3.13 Microscopic Analysis

The stained urine slides were then observed microscopically. Observe using the low

power objective to check for urine sediments present in the urine and observe the staining
capacity of each plant solution and concentrations in the urine sediments. Observe at least 10

fields and write down the results.

3.14 Proper Waste Disposal

The slides used were washed using tap water and a bleach solution. After it has been

cleaned, the sink was also washed with a bleach solution so there won’t be any residues left. The

slides and the container used for the collection of urine were placed into a biohazard container.

Cleaning the area used for the experiment should also be cleaned with a bleach solution to get rid

of any potentially harmful substances. Proper waste disposal of hazardous or human secretions is

important because the waste could cause potential diseases that could harm the population.

3.15 Rubrics

RATING SCALE INTERPRETATION QUALITATIVE

DESCRIPTION

0 – 1.0 Poor No distinct color stained to

Epithelial cell, RBC, WBC,

Hyphal element, Crystal, Cast,

Urates/Phosphates

1.1 -2.0 Fair Slightly visible color stained to

Epithelial cell, RBC, WBC,

 Hyphal element, Crystal, Cast,

Urates/Phosphates

2.1 – 3.0 Good Visible color stained to

Epithelial cell, RBC, WBC,

Hyphal element, Crystal, Cast,

Urates/Phosphates

3.1 – 4.0 Excellent Clearly visible color stained

Epithelial cell, RBC, WBC,

Hyphal element, Crystal, Cast,

Urates/Phosphates

Figure 2. Rubric chart of urine sediments visibility using Basella rubra and Ipomoea batatas L.

plant extracts as an Alternative Stain.

3.16 Statistical Analysis

This study employed the use of the following statistical tools to evaluate and analyze the

data. Mean was used to compute the average results of each concentration of each plant in terms

of its staining capability. One Way ANOVA was used to compute the significant difference

between the two plants in terms of staining effects. And One Way ANOVA to determine the

difference between each concentration of extractions to the control.

Figure 5. Flow chart of Basella rubra and Ipomoea batatas L. plant extracts as an Alternative

Stain for Sperm Morphology Assessment.

 The researchers collected the required materials and equipment for the experiment. They

also obtained Basella rubra and Ipomoea batatas L. plants, which were carefully cleaned and

air-dried for at least 3 minutes. The dried plants were then pulverized into powder using a food

processor or blender. The resulting powder was placed in a clean sterile container with a tight lid.

To prepare the pure extract of Ipomeoea batatas, researchers extracted the pulverized leaves of

the plant by squeezing them through a Muslin cloth. Then it was stored in an Erlenmeyer flask,

covered to prevent contamination. In the preparation of Basella rubra pure extract, the plant was

grounded and squeezed through a Muslin cloth. The obtained extract was placed in an

Erlenmeyer flask. To remove sticky substances that could interfere with the results, the pure

extract was centrifuged at high speed for at least 10 minutes. Next, it was stored in a covered

container. Different concentrations of extracts from Ipomoea batatas and Basella rubra were

prepared using different solvents. For Ipomoea batatas, ethanol, distilled water, and normal saline

solution (NSS) were used as solvents. To create the desired ethanol concentrations (25%, 50%,

and 70%), specific amounts of ethanol and extract were mixed together. The same process was

followed for distilled water and NSS concentrations. For Basella rubra, the same procedure was

used to create ethanol, distilled water, and NSS concentrations. The application of Ipomoea

batatas and Basella rubra pure extracts to urine samples was carried out, drops of urine were

placed on a glass slide, and extracts from Ipomoea batatas and Basella rubra were added to the

urine drops. The mixture was gently mixed. The slides were then observed under a microscope to

examine the urine sediments and the staining effects of the plant extracts. The used slides were

cleaned with water and bleach, and the waste materials were disposed of properly to prevent any

potential harm or spread of diseases.