IMPROVISED INCUBATOR AS MICROBIAL CULTURE

CHAMBER CULTIVATION SYSTEM

Presented to the Senior High School

Department of Heracleo Casco Memorial National High School

In Partial Fulfilment of the requirements in

Capstone Project

Angel Anne B.Tejana

Marielle B. Villanueva
Michelle Antialon
Veronica Gapusan
Jackelyn Capoc

MAY 2023
INTRODUCTION
 In the teaching-learning process of scientific inquiry, laboratory resources

are absolutely essential. It is vital to successful experimentation and the

identification and correction of mistakes. The incubator machine is one of these

necessary laboratory equipment. It is required for the culture method used in the

area of microbiology to grow the bacteria from the microbial samples that were

obtained. The researchers, however, suggested a different prototype to address this

issue because most schools in Davao Oriental do not have access to this

equipment.

In remote areas with few resources, there haven't been many culture

facilities. The samples collected by a researcher would therefore need to be

brought to the closest, well-equipped culture facility for processing, identification

of the causal bacterium, and assessment of its susceptibility to antibiotics. Since

mammalian cells and bacteria both grow best at a temperature of about 37 C, this

temperature must be maintained for specimens being transported from the

community to the lab (Thavaraj et al., 2015).

In the Philippines, there were 400 tertiary laboratories on the Health

Facilities and Services Regulatory Bureau list. In the assessment, only 17.3%

(60/347) of the laboratories implemented internal quality control procedures for

media, reagents, stains, and antibiotic disks; only 43.8% (152/347) of the

laboratories could perform Gram stain, acid-fast stain, negative stain, and wet

mount; and only 3.2% (11/347) used ATCC biological standards for quality

control. Moreover, only 35.7% (124/347) of the necessary major equipment and

instruments were completed. Similarly, only 5.8% (20/347) of the necessary

culture media and supplements for primary isolation, 19.3% (67/347) of the

necessary media for biochemical tests, and 3.3% (11/347) of the necessary
supplements for growth and identification were completed (Mondoy, 2017). This

study highlights the scarce lab equipment supplies that some students and

researchers in remote locations have very little access to.

Student-researchers from Heracleo Casco Memorial National High School

were having difficulties carrying out their research involving microorganisms that

must be cultured and observed under a microscope for identification. However,

the process took longer than anticipated, and researchers ran into problems

because the incubator equipment needed for bacterial culture was lacking.

The literature mentioned above emphasized the significance of the

improvised incubator as an alternative to an incubator machine that performs the

same function in cultivating bacteria that may be utilized for bacterial

identification, infection testing, and other research that involves bacteria.

Therefore, the proponent of this project investigated and constructed an

improvised incubator as microbial culture chamber cultivation system.

Statement of the Problem

This project aimed to construct an improvised incubator that will serve as

an alternative microbial chamber cultivating system. Specifically, this project

answered the following research questions:

1. What is the ideal temperature of the chamber for microbial culture?

2. What is the temperature (lumens) produce by the incandescent bulb

with:

2.1 25-wattage;

2.2 60-wattage; and

2.3 100-wattage?

3. What is the average of the colony count in each quadrant of the agar?
Beneficiaries

The findings of this study are important to students and researchers

who lacked access to facilities, laboratories, and equipment for cultivating

bacteria. Contrary assumptions will be addressed and clarified. This

research is significant to the following:

Students and Researchers. Students and researchers who will

engage in microbiology projects, such as microbial sampling, bacterial

culture, and bacteria observation, are the main beneficiaries of this project.
 METHODS

This section covered all of the methods utilized to address the problem

presented in this study. This section comprises three phases: Phase I- Gathering

of Materials and Assembling of Improvised Incubator Chamber. Phase II-

Preliminary Testing. Phase III- Microbial Culture Media Preparation. Phase

IV- Data Collection and Analysis.

Phase I- Gathering of Materials and Assembling of Improvised Incubator

Chamber

The improvised incubation chamber will be built using the components

listed below.

Chamber. The chamber is the primary and essential component of this

investigation. Boxes come in a range of sizes and shapes that can be utilized as an

alternate microbe chamber. The researchers evaluated a medium-sized styrofoam

box (refer to set up a) and a medium-sized, robust cardboard box attaching a foam

insulator (refer to set up b) while selecting the possible microbe chamber,

ensuring the anticipated outcomes in this investigation. The foam insulator will act

as a thermal barrier around the box's interior and a heat source that is enough to

keep the incubates warm as they grow and reproduce.

 Image 1. Alternative microbial chambers were chosen and constructed.

Electrical Components. Following the preparation of the microbe

chambers, the researchers constructed the bulb socket, wires, and plug that will

serve as the bridge of electricity that flows from the socket to light the bulb. To

ensure the safety during the whole duration of the project implementation, the

researchers assembled and connected the bulb socket, wires, and plug with the

assistance of an expert.

Image 2. Assembling of bulb socket, wire and plug.

The wired bulb socket was inserted after the researchers punched a hole in

the center of the box and Styrofoam box. Enough space should be left in the hole

to accommodate the socket, leaving no room for air to potentially leak into the

chamber to prevent possible contamination of the samples.

The researchers attached bulbs of different wattage ranging from 100

watts, 60 watts, and 25 watts bulbs while determining which bulb is sufficient to

cultivate a microbe at a specific period.

 Image 3. Finished Setup of the Improvised Incubator

Phase II- Preliminary Testing of the Improvised Incubator

As the incubator was custom-made by the researchers, it is vital to test

whether or not it will work. Initially, the researchers put the wired light bulb into

an electric socket. On top of that, it was critical to maintain the temperature within

the incubator in order to continue bacterial cultivation. The researcher used

thermometers in different locations of the incubator to determine whether each

regions received equal heat that can culture the bacteria and to record the heat

reflected off every light bulb (25 watts, 60 watts, and 100 watts) inside the

incubator at a given time, as shown in the picture below. Temperature were

recorded at a different time intervals.

Image 4. From right to left, the placement of the thermometers were indicated as

temperature 1, temperature 2, temperature 3 and temperature 4.

Phase III- Microbial Culture Media Preparation

Media preparation involves combining nutrients, buffering agents, and

other elements to assist an organism's growth and development. A general purpose

media was used by the researchers in this study.

Sterilization. The researcher set up an agar plate that would be used to

store the material before collecting the bacterial sample. Every material used to
create the agar plates was sterilized in a pressure cooker. Agar plates, a loop and

needle for inoculation, etc. were sterilized in the pressure cooker, and bleach

solution was used to sanitize storage items like the ice box and the improvised

incubator. The

Image 1. Sterilization process of the agar plates and Erlenmeyer flask

researcher sterilized these items by wrapping them in aluminium foil, sealing them

in zip-top bags, and placing them in a pressure cooker set to 380 degrees Celsius

for the duration of the sterilization procedure. This was carried out to get rid of

any contaminants and make sure the agar plates were sufficiently clean.

General Purpose Media. A pork cube was used as the main extract which

is the primary source of nutrients and a gelatin powder was used to produce the

Image 2. Cooking of Agar

main media. 9.5 grams of pork cubes were dissolved together with the gelatin

powder in a 300mL of distilled water. The mixture was put inside the Erlenmeyer

flask and cooked for 15 minutes at a temperature over 380 degrees Celsius.
 Afterwards, the mixture was poured and distributed into three (3) agar

plates. It was then placed inside a cooler to let the mixture set.

Collecting Microbial Samples. Cotton buds, inoculating loops and

needles, alcohol lamps, zip lock bags, and cotton buds were utilized to collect the

microbiological samples for the microbial culture.

Image 3. Collection of microbial samples by swabbing the surface of railings,

door knob, and faucet using a cotton swab

The researcher collected any potential bacterial samples by swabbing the

machine's nozzle with a cotton swab. The zip lock bag was used to store the buds

while they were being transported to the lab for culturing.

Microbial Culture

After gathering the samples of microorganisms, the researcher streaked the

microbes onto an agar plate and put it in an incubator for a week of observation

with a 25-watt light bulb attached that reflected 37 degrees Celsius, which was

enough to cultivate the bacteria.

 Image 4. Quadrant streaking method was applied in streak the sample into the
media

In order to prevent damaging the agar's surface, caution was taken while

swabbing the sample onto the media. Streaking was performed in the laboratory at

room temperature. The samples were also swabbed using a four-quadrant

streaking pattern. An overview of the streaking process was shown in the figure

below. In this procedure, the inoculum was made up of cotton bud samples (N. S.,

2021).

Inspection/Observation of Colonies. The setup was regularly checked by

the researchers. Observations were done 24 hours, 84 hours, and 168 hours after

inoculation. In each interval, the researcher took a sample from the agar plate,

placed it on a glass side, mixed it with one drop of distilled water, let it dry, and

examined it under a microscope with gram staining treatment.

Image 5. Gram-staining procedure and observation under microscope

 Bacterial Growth. To ensure that the microbial growth has been tracked

as suggested by the bacterial growth curve, repeated observations of the samples

were made (Alst et al., 2023). As mentioned, growth curves offer useful

knowledge on the kinetics of bacterial growth and cell physiology.

Figure 2. Bacterial Growth Curve

It enables the monitoring of the growth of bacteria under various growth

settings as well as the ideal growth conditions for a particular bacterium. Bacterial

growth curve suggests that culture growth progresses through four phases: lag

phase, exponential (log) phase, stationary phase, and death (Refer to figure 2).

Gram Staining Procedure. Gram staining was used to distinguish

between gram-positive and gram-negative bacteria in order to detect the presence

of bacteria. Crystal violet was used as a primary stain to first color the sample. It

is a fundamental dye that gives gram-positive bacteria a violet color by taking the

ionized forms of CV+ and Cl-. The sample was then treated with a mordant

(gram's iodine) to make a tissue stain more reactive. The contact between the

bacterial cells and the crystal violet tightened as a result. The sample has been

stained with a mordant and then decolorized.

Before performing a gram stain on any gram-negative bacteria on the slide,

the crystal violet stain must be removed. The researcher accomplished this by
treating gram-negative cells with a decolorizing solution. Lastly, the researchers

applied a counterstain (safranin) to the sample in order to remove the color of

gram-negative cells. Before using the following treatment, each one should only

be used for a minute before being gently rinsed away. The diagram below

provides a summary of the gram staining procedure.

Figure 3. Gram-staining Procedure

Phase IV- Data Collection and Analysis

This section provides how the researchers test the functionality and

effectiveness of the improvised incubator chamber as an alternative microbial

cultivating system. The following indicators will be observed:

Temperature. As mentioned by Thavaraj 2015 in his study, 37 degrees

Celsius is the ideal temperature to grow a bacteria. This temperature will be

investigated inside the incubator along the process of this study.

Agar and Agar Plates. Sanitary precautions should be meticulously

followed when making the agar and agar plates to prevent any contamination and

data manipulation.
 RESULTS AND DISCUSSION

This chapter answered the research problems raised earlier in this paper.

This section comprises data presentation and analysis, a summary of findings,

acceptable conclusions, and recommendations provided by the researcher based

on the findings. The result and discussion include the lumens of different wattage

within an incubator, the count of the bacteria colonies cultivated, and the

evaluation of the experimental and control group.

Lumens of Different Wattage within an Incubator.

The researchers assessed the heat reflected off each bulb to decide which

bulb would give off the proper quantity of heat that would be reflected inside the

incubator to culture the bacteria in the process.

One hundred watts bulb. First, the researchers used a 100- watt light and

measured the heat it reflected over time. To better establish and achieve ideal

findings, the researchers chose to test the lumens in both a sealed and unsealed

incubator.

As more than 25 minutes yield the same outcome, we have set our time

limit to 25 minutes. The heat reflected by the 100 watt increases as the recording

duration rises in this table, reaching a maximum temperature of 103 degrees

Celsius. Gutierrez et al. (2018) discovered that human infections often multiply

best between the temperatures of their human hosts (35°C and 37°C) in their

study.

In overall, the average temperature of sealed incubator with 100 wattage

bulb was equal to 85.40°C which is significantly higher than the unsealed
incubator with an average of 66.70°C with a t-statistics equivalent to 7.573 with a

p-value of 0.000 (Appendix A).

Table 1. Lumens of 100 Watts in a Sealed and Unsealed Incubator

Temperature

Duration 1 2 3 4 Mean

5 minutes (sealed) 71 78 81 68 75.67

5 minutes (unsealed) 70 68 76 62 69.00

10 minutes (sealed) 83 79 86 76 81.00

10 minutes (unsealed) 65 64 61 63 63.25

15 minutes (sealed) 87 88 90 80 86.25

15 minutes (unsealed) 68 66 71 66 67.75

20 minutes (sealed) 93 89 94 85 90.25

20 minutes (unsealed) 79 72 79 74 76.00

25 minutes (sealed) 93 92 103 92 95.00

25 minutes (unsealed) 56 56 61 57 57.50

However, the findings indicated that extreme heat may cause the bacteria

to be killed rather than produced. In order to get the necessary temperature, the

researcher proceeded with the assessment by decreasing the bulb's power (60

watts).

Sixty watts bulb. The 60-watt bulb illuminated heat over 37 degrees

Celsius, similar to the previous findings. According to the World Health

Organization (WHO), boiling water renders it safe from pathogens like bacteria,

viruses, and protozoa. Temperatures between 140°F and 150°F are sufficient to

destroy the majority of viruses. It is between 60 and 65 degrees Celsius.

 In overall, the average temperature generated by the sealed incubator with

60-watts bulb was equivalent to 63.30°C which is significantly higher than the

unsealed incubator with an average generated temperature of 48.40°C as indicated

by the t-statistics value of 8.614 with a corresponding p-value of 0.000.

Table 2. Lumens of 60 Watts in a Sealed and Unsealed Incubator

Temperature

Duration 1 2 3 4 Mean

5 minutes (sealed) 55 59 62 54 57.50

5 minutes (unsealed) 41 43 46 41 42.75

10 minutes (sealed) 59 63 66 58 61.50

10 minutes (unsealed) 41 42 46 39 42.00

15 minutes (sealed) 63 66 70 62 65.25

15 minutes (unsealed) 53 54 60 53 55.00

20 minutes (sealed) 66 69 74 63 68.00

20 minutes (unsealed) 50 46 53 49 49.50

25 minutes (sealed) 61 66 65 65 64.25

25 minutes (unsealed) 56 51 55 49 52.75

The results showed that the cultivation process can fail if the bacteria are

killed in specific areas of the incubator. To get the intended outcomes, the

researchers next switched to a lower wattage bulb.

Twenty-five watts bulb. In this table, 25 minutes in an open incubator

produced temperatures of 35°C at temperature 1, 36 °C at temperature 2, 37 °C at

temperature 3, and 35°C at temperature 4.

 In overall, the sealed incubator with 25-watts bulb produced an average

heat of 43.70°C which is significantly higher than the unsealed incubator with an

average heat of 34.90°C.

Table 3. Lumens of 25 watts in a sealed and unsealed incubator.

Temperature

Duration 1 2 3 4 Mean

5 minutes (sealed) 38 40 40 38 39.00

5 minutes (unsealed) 33 32 38 34 34.25

10 minutes (sealed) 39 40 46 40 41.25

10 minutes (unsealed) 33 34 39 34 35.00

15 minutes (sealed) 40 42 49 42 43.25

15 minutes (unsealed) 31 33 37 34 33.75

20 minutes (sealed) 45 48 50 45 47.00

20 minutes (unsealed) 35 36 38 34 35.75

25 minutes (sealed) 46 49 51 46 48.00

25 minutes (unsealed) 35 36 37 35 35.75

The 25 watt bulb generated enough heat to cultivate bacteria. In line to

existing literatures, the unsealed setup with 25 watts incandescent bulb is an ideal

chamber for bacteria cultivation. Hence, the researchers used this setup throughout

the entire culturing procedure.

Colony Count of the Bacteria Cultivated

 Upon placing the microbial samples into the incubator, frequent

examinations were made to make sure that the microbial growth was being

recorded as shown by the growth curve (Alst et al., 2023). As mentioned, growth

curves offer useful details on the conditions for a particular bacterium's growth.

The controlled sample and the experimental sample were placed in the incubator,

and illustration below shows the observation of bacteria colony growth. A

quadrant streaking technique was applied on the medium in the experimental

sample, as depicted in the figure below.

Figure 4. Quadrant Streaking Method

This method is a quick isolation technique. The microbial population size

in the initial sample is successively diluted. Organisms from mixed cultures are

separated via quadrant streaking. The results of the cultivation process is further

discussed.
 Experimental Group. Agar that belonged to the experimental group were

streaked with sample bacteria taken from a railing, door knob, and faucet.

Quadrant streaking method was applied in this media.

Table 1 shows an upsurge in microbial colonies throughout a 7-day culture

period inside the improvised incubator. As the quadrant streaking approach was

used in this media, bacteria colonies were isolated and visible in the media shown

above that simplified colony counting of this group.

After 7 days of culturing the bacteria, the microbial sample from the

railing revealed 154 bacterial colonies in quadrant 1, 100 in quadrant 2, 67 in

quadrant 3, and 34 in quadrant 4. The microbial sample from the surface of the

door knob

Table 1- Experimental Group

Microbial Samples
Source Day 1 Day 4 Day 7
Experimental

Railings
 Door Knob

Faucet

disclosed 154 bacterial colonies in quadrant 1, 100 in quadrant 2, 67 in

quadrant 3, and 34 in quadrant 4. The microbial sample from the surface of the

faucet revealed 154 bacterial colonies in quadrant 1, 100 in quadrant 2, 67 in

quadrant 3, and 34 in quadrant 4.

By streaking the growth plate four times, the inoculum is successively

diluted, causing the colonies to become thinner or to form isolated colonies. This

explains why a cluster of colonies was found in quadrant 1, whereas fewer,

thinner colonies were found in quadrant 4.

Table 2 presented the average colony count of the bacterial samples

obtained from railings, door knob, and faucet. All samples showed consistency in
the decrease of colony count along Quadrant 1 to Quadrant 4. Furthermore, an

Analysis of Variance (ANOVA) was done to test the significance of difference on

the average colony count of each quadrant. The findings revealed that the colony

count in Quadrant 1 (M = 203.33, SD = 76.540) is significantly greater than

Quadrant 3 (M = 72.67, SD = 18.23) and Quadrant 4 (M = 36.00, SD = 2.887).

However, no significant difference was observed between Quadrants 2 to 4. This

results implied that the isolation of the colonies was effective (see Appendix).

Table 2. Average Colony Count Per Quadrant

Microbial
Quadrant 1 Quadrant 2 Quadrant 3 Quadrant 4
Sample Source

Railings 175 138 89 36

Door Knob 290 155 76 41

Faucet 145 75 53 31

Control Group. This setup served as important basis whether the

improvised incubator was efficient. In the control group, no bacterial sample was

streaked.

Table 3. Controlled Group

Microbial Day 1 Day 4 Day 7
Sample Source

Railings

Door Knob
 Faucet

The control group samples were placed inside the incubator without

streaking any sample. As seen in Table 3, there was no observable colonies in the

agar plates for the first three days. However, as more day passed, there can be tiny

colonies that can be observed but without any pattern. The increase in microbial

colonies implied that there was a minimal or negligible contamination in the

preparation phases. However, it is important to note that the observed colonies

were not isolated and no comprehensible pattern just like from quadrant streaking

method can be seen. As a result, based from the initial observations, the

improvised incubator was working with efficiency.

Summary

The major purpose of this project is to construct an improvised incubator

as an alternative microbial chamber cultivating system. The analysis of the data

lead to the following findings:

Lumens of Bulb. It was determined after a thorough examination that the

25-watt incandescent bulb illuminated at 35–37 degrees Celsius, which is

sufficient heat to allow the bacteria to grow.

Colony Count of Bacteria Cultivated. After 7 days of culturing the

bacteria, the microbial sample from the railing revealed 175 bacterial colonies in
Quadrant 1, 138 in Quadrant 2, 89 in Quadrant 3, and 36 in Quadrant 4. The

microbial sample from the surface of the door knob disclosed 290 bacterial

colonies in Quadrant 1, 155 in Quadrant 2, 76 in Quadrant 3, and 41 in Quadrant

4. The microbial sample from the surface of the faucet revealed 145 bacterial

colonies in Quadrant 1, 75 in Quadrant 2, 53 in Quadrant 3, and 31 in Quadrant 4.

By streaking the growth plate four times, the inoculum is successively

diluted, causing the colonies to become thinner or to form isolated colonies. This

explains why a cluster of colonies was found in quadrant 1, whereas fewer,

thinner colonies were found in quadrant 4. This decrease was found to be

significant through an Analysis of Variance. Therefore, the isolation process was

effective.

Conclusion

Based on the aforementioned findings of the study, the following

conclusions are drawn:

The improvised incubator can cultivate the bacteria at temperatures

between 35 and 37 degrees Celsius. This suggests that the researchers' improvised

incubator is suitable as an alternative to the incubator machine used as the

microbial chamber cultivation system.

Recommendation

The following suggestions are made following a careful analysis and

conclusion on the findings and of the study:

Future researchers may install a temperature sensor to monitor the heat

inside the chamber and turn it into an electrical signal to track the consistency of

the heat illuminated inside the incubator.

 A system where the chamber can adjust the temperature inside by

releasing heat through a vent is highly recommended.