Exercise 1: Microbial Load of Buko Juice Sold in

Abdullah A, Beldia SR, Diamoda K, Fantonalgo JL, Gerona JK, Nalicat O, Sespeñe R
MCB103 Laboratory Report No. 1 Group 4 .
2 March 2024

ABSTRACT
Aerobic plate count (APC) is a fundamental method used in microbiology
laboratories to assess the microbial load in various samples, particularly food products,
playing a crucial role in determining their sanitary quality and evaluating handling and
storage conditions. This exercise aimed to assess the microbial quality of "Buko Juice"
sold in Mindanao State University - General Santos campus using the Aerobic Plate
Count (APC) method, focusing on the pour plate technique following BAM guidelines.
Results revealed a high microbial load, with colony counts exceeding the countable limit
in the undiluted sample. Dilutions 10-3 and 10-4 yielded countable plates, with the 10-3
dilution producing an average colony count of 7.75 x 104 CFU/mL. Factors such as pH
and temperature variations influenced microbial proliferation. The exercise highlighted
the need for improved food handling practices and quality control measures to ensure
consumer safety. The exercise provides valuable insights into microbial contamination in
"Buko Juice" and underscores the importance of stringent food safety protocols in
beverage production and distribution.

INTRODUCTION

In recent years, the prevalence of foodborne illnesses attributed to microbial contamination has
heightened concerns surrounding food safety and quality assurance practices. The assessment of microbial
quality in food products is essential to ensure their safety and shelf-life. Aerobic plate count (APC) is a
widely used method for quantifying viable microorganisms present in a sample, providing valuable
insights into the overall microbial load and sanitary conditions of the product. In this laboratory exercise,
our focus is on the APC pour plating technique, following the methods outlined in the Bacteriological
Analytical Manual (BAM), to analyze the microbial content of "Buko Juice" purchased within Mindanao
State University.
"Buko Juice," a popular beverage made from coconut milk and water, presents unique challenges
in terms of microbial quality control. Intrinsic factors such as pH levels, moisture content, and nutrient
availability, alongside extrinsic factors like temperature fluctuations, packaging materials, and storage
conditions, play pivotal roles in creating an environment conducive to microbial growth and survival.
In this lab exercise, the students aim to utilize aerobic plate count technique to compare the
bacterial load of different drinks sold in various stores at Mindanao State University-General Santos.
Investigate the influence of intrinsic and extrinsic factors on the microbial load of drink samples. Through
detailed calculations using standard formulas for colony-forming units (CFU) per milliliter, students will
delve into the detailed calculation of microbial load. Understanding these factors is essential for assessing
the microbiological quality of beverages and ensuring food safety standards are met.
MATERIALS & METHODS

Media Preparation

This experiment utilizes Plate Count Agar (PCA) for bacterial enumeration. A class group (group
1) prepared the main stock solution,TM MEDIA Plate Count Agar, in 1 liter of distilled water. The
solution was heated to dissolve the ingredients, dispensed into suitable flasks or tubes, and autoclaved for
15 minutes at 121°C to ensure sterility. After autoclaving, the groups prepared their working solutions by
suspending 3.525 g of PCA powder in 150 mL of sterile distilled water.

Sampling
Sterile gloves and masks were donned for proper aseptic technique before sampling. The group
was assigned a specific fruit juice, buko juice, to collect from a vendor in Mindanao State University -
GenSan. Two 8-ounce (236.59 mL) subsamples were collected in clean, dry, wide-mouthed plastic cups
used by the vendor and were sealed with sterile aluminum foil to prevent spillage. Each sample was
labeled with a unique identifier (BJ for buko juice), date and time of collection, product information,
sampling location, analysis purpose, and sampler initials. During transport to the laboratory, the samples
were kept cool but not frozen (<10°C) within an icebox containing ice packs.

Pour Plating
Upon arrival at the laboratory, the prepared PCA media was melted and cooled to approximately
45-50°C. Aliquots of the melted media (typically 20-25 mL) were poured into sterile petri dishes and
were allowed to solidify. Once solidified, appropriate dilutions, 10-2, 10-3, and 10-4, of the collected juice
samples were prepared using a 0.85% saline solution. These dilutions were then plated onto the solidified
PCA media using sterile pipettes or a micropipette with disposable tips. The plated samples were allowed
to dry briefly before being inverted and incubated.

Modified Incubation Method

The experiment employed a modified incubation method to account for the potential presence of
acid-tolerant microorganisms in the fruit juices. Typically, PCA plates were incubated at 35°C for 48
hours. However, in this experiment, an additional incubation period at a lower temperature (25°C) was
included to encourage the growth of acid-tolerant bacteria. The total incubation time was divided, with an
initial incubation at 35°C for 24 hours followed by a secondary incubation at 25°C for an additional 24
hours.

Analysis
The petri dishes were then visually examined for the presence and number of colony-forming
units (CFUs) after incubation. CFUs represent individual viable bacterial cells that have multiplied into
visible colonies. The number of CFUs on each plate was counted, and the data was used to estimate the
total bacterial count in the original juice samples.
RESULTS

The results are classified in the following data tables: table 1 - ‘the pH level, internal temperature,
and ambient temperature of the buko juice’, table 2 -’ the buko juice vendor’s response regarding his/her
food handling practices and the ingredients of the buko juice’, and table 3 - ‘Aerobic Plate Count (APC)
in different dilution factors of buko juice sold in MSU-Gensan Campus after 24 hours of incubation’. The
data revealed are then further analyzed and discussed in the following section.

Table 1. The pH level, internal temperature, and ambient temperature of the buko juice.
Condition During the Sample Condition After the Sample
Collection Collection

pH - 5.46

Internal Temperature of the Buko 70C 7.80C

Juice

Ambient Temperature of the 290C 23.50C

Food

Based on table 1, the internal temperature of the buko juice during the sample collection was 70C
and increased to 7.80C when measured again at the laboratory before conducting the analysis. The
ambient temperature of the buko juice during the sample collection was 290C and decreased to 23.50C
when measured again at the laboratory before conducting the analysis. The pH level of the buko juice
which was 5.46 was only measured at the laboratory before conducting the analysis.

Table 2. The buko juice vendor’s response regarding his/her food handling practices and the ingredients
of the buko juice.
Survey Questionnaire Responses

Type of sample Buko juice

Source of Water Mineral water from water station

Source of Ingredients Buko powder from public market

Color of the Food Sample Creamy white

Handling Process (Scoop/Faucet) Scoop

Gloves or Without Without gloves

Storage of Reservoir Water jug

Ingredients “Pure buko”, evaporated milk, buko powder,

brown sugar
 Natural Product or Processed Process

Time of Preparation 9:00 AM

Location of preparation MSU Gymnasium (Outside)

During the sample collection of the buko juice, the group also gathered information regarding the
ingredients of the buko juice, materials and equipment used for the preparation and selling, and the time
and location of the preparation of the product. Table 2 summarizes the findings from a semi-interview
evaluating the buko juice vendor's food handling practices. The vendor offers fresh-buko juice, utilizing
water from a refilling station and market-sourced buko powder, sugar and evaporada. Notably, the
vendor's lack of glove usage during preparation and handling raises concerns about hygiene and potential
cross-contamination. These observations collectively provide an assessment of the vendor's adherence to
food safety regulations.

Table 3. Aerobic Plate Count (APC) in different dilution factors of buko juice sold in MSU-Gensan
Campus after 24 hours of incubation.
Number of Colonies
Dilution Average Count CFU/ml
R1 R2

10-2 TNTC TNTC TNTC TNTC

10-3 84 71 77.5 77,500 CFU/ml or

or 7.75 x 10-4
CFU/ml

10-4 18 20 19 TFTC

Table 3 summarizes the aerobic plate count (CFU/mL) of a buko juice sample obtained from the
MSU-Gensan campus using the pour plate method. The table shows the number of colonies per replicate
−2 −3 −4
for three dilution factors (10 , 10 , 𝑎𝑛𝑑 10 ) after 24 hours of incubation. The colonies obtained
from 10-2 and 10-4 are TNTC and 19, respectively, and fall outside the countable range of 25-250 CFU.
−3
The dilution factor of 10 yielded an average colony count of 77.5 colonies, which falls within the
countable range.

Calculating Aerobic Plate Count

An aerobic plate count (APC) was performed on buko juice samples to determine the
approximate number of viable aerobic bacteria present. The aerobic plate count (APC) is calculated by
multiplying the average number of colonies from countable plates (25-250 CFU) by the dilution factor
and then dividing by the volume plated. If all plates have excessive CFU (>250), the count is reported as
"too numerous to count" (TNTC) except for the plate closest to 250. Spreading colonies are also observed
 −4
in all plates except 10 R1 and R2 plates. In order to count colonies accurately, three distinct spreader
types are observed: 1) chains from bacterial clumps, 2) colonies within the agar-dish interface water film,
and 3) colonies on the agar surface water film. Plates exceeding 50% total spreader coverage or 25%
repressed growth area are classified as "spreaders" and excluded from counting. For countable spreader
plates, each of the three types is counted as one colony. For the first type, if a single chain exists, it's
counted as one. If multiple chains seem to originate from different sources, each source is counted as one
colony, excluding individual growths within the chains. Types 2 and 3 typically form distinct colonies and
are counted individually (Maturin and Peeler, 1998).
Due to limitations in the countable plate range, only the dilution factor of 10-3 was used for
calculating the APC. Based on the average colony count of 77.5 at this dilution, the estimated APC is
77,500 colony-forming units per milliliter (CFU/mL).

(𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠)(𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟)

𝑁= 𝑉𝑜𝑙𝑢𝑚𝑒 𝑝𝑙𝑎𝑡𝑒𝑑
1
(84 + 71/2)( −3 )
10
𝑁= 1 𝑚𝑙
1
(77.5 𝐶𝐹𝑈)( −3 )
10
𝑁= 1 𝑚𝑙
𝑁 = 77, 500 𝐶𝐹𝑈/𝑚𝑙 or 7.75 x 104

DISCUSSION

The experiment conducts a standard plate count (SPC) or Aerobic Plate Count (APC) to estimate
the total number of mesophilic aerobic bacteria in a sample, as a general index of the bacterial populations
and without differentiations among the various types. The result revealed an average colony count of
77,500 colony-forming units per milliliter (CFU/mL) tested from buko juice product from a stall near and
beside Mindanao State University - Gymnasium. Referring to the Food and Drug Administration -
Revised Guidelines for the Assessment of Microbiological Quality of Processed Foods, the acceptable
level of microorganisms (m) in powdered beverages must not exceed 3x10^3 in powdered beverages;
liquid milk (evaporated milk) is commercially sterile, pure ‘buko’ (coconut milk and /or water) must be
under Hazard Analysis and Critical Control Point (HACCP) systems for juices; and no specific guidelines
for brown sugar (Food and Drug Administration, 2013). The Food and Drug Administration employs the
Hazard Analysis and Critical Control Point (HACCP) systems for their processing operations, coconut
milk and/or water is covered by this regulation. The general guidelines for juices apply to coconut milk
and/or water. This ensures microbiological quality and consumption safety (Center for Food Safety and
Applied Nutrition (2003).
The aerobic plate count (APC) experiment aimed to quantify viable aerobic microbes in Buko
juice samples. Dilution series from 10-2 to 10-4 were utilized to reduce bacterial concentrations for
accurate enumeration. The 10-2 dilution yielded bacterial growth exceeding the countable limit, indicating
bacterial load deemed “too numerous to count” (TNTC). This suggests an initial bacterial concentration in
the undiluted Buko juice sample was exceptionally high.
The pour plate method relies on the principle that when a mixture of microorganisms and agar
medium is incubated, viable microorganisms will multiply to form distinct colonies. After incubation at
typically 37°C for 24 to 48 hours, these colonies become visible on and within the solidified medium. By
counting these colonies, one can determine the colony-forming units (CFU/mL) present in the original
sample. In Prashant Dahal's (2022) findings, it was noted that if a plate from the initial dilution (e.g.,
10^-2) where two replicates show colonies that are too numerous to count (TNTC), it suggests an
abundance of microorganisms in the sample. This occurrence needs re-sampling and repeating the process
with a higher dilution. The TNTC result indicates that the original sample contains an overwhelming
number of viable microorganisms, making it difficult to quantify them accurately without diluting the
sample further.
Subsequent dilutions, particularly 10-3 and 10-4, produced countable plates with discernible colony
formations. The 10-3 dilution showed an average colony count of 77.5, falling within the expected viable
cell count range. However, the 10-4 dilution resulted in a lower colony count of 19, falling below the
range.
Dilution 10-3 proceeded to the calculation of its colony-forming units (CFU) per milliliter and
resulted in a 77,500 or 7.75 x 104 CFU/ml. The Buko juice is considered a powdered beverage as one of
its ingredients is powdered Buko juice. In that category, According to the Revised Guidelines for
Processed Food Quality Assessment by the FDA of the Philippines circulated in February 2013, the
microbial load of the Buko juice samples from the stall near and beside Mindanao State University -
Gymnasium goes beyond the accepted microorganism level of 3x103.
Factors such as pH, temperature variations, and handling procedures influence microbial
proliferation and colony formation. In fact, with the Buko juice’s near-neutral pH of 5.46, favorable
conditions for microbial growth can be expected as neutral or near-neutral pH levels typically facilitate
microbial proliferation in food systems. Additionally, milk, a component of Buko juice, provides an ideal
environment for microbial growth due to its high nutrient content and water activity (Neto et al., 2019).
Also, dilutions 10-3 and 10-4 were noted to have appearances of a distinct black-colored microbial
growth which was considered as a presumptive fungal growth.
 CONCLUSION

The experiment aimed to quantify viable aerobic bacteria in buko juice samples obtained from a
stall near Mindanao State University - GenSan. The results revealed an average colony count of 77,500
colony-forming units per milliliter (CFU/mL) or 7.75 x 104 CFU/ml in the buko juice sample, which
exceeded the acceptable microbial load level of 3x103 CFU/mL for powdered beverages according to
FDA guidelines. Factors such as pH, temperature variations, and handling procedures likely influenced
microbial proliferation and colony formation in the buko juice samples. The near-neutral pH of the juice,
coupled with favorable temperature conditions during storage and handling, provided an ideal
environment for microbial growth. Additionally, the presence of milk as an ingredient further promoted
microbial proliferation due to its high nutrient content.
However, the experiment encountered limitations, particularly in the incubation process.
Incubation conditions, including temperature and relative humidity, were not adequately controlled,
potentially impacting the accuracy and reliability of the microbial plate counts. Additionally, the
appearance of spreaders in all plates suggested excess moisture during the pouring of agar media, leading
to difficulties in colony formation and enumeration.
Overall, the results of the study emphasize the need for continuous monitoring and enforcement
of food safety regulations to safeguard consumer health. Further improvements in experimental protocols,
particularly in incubation conditions and plate preparation, are necessary to enhance the accuracy and
reliability of microbial plate counts in future studies.

LIMITATIONS

On Incubation
The incubation was induced in large (25x20x15cm) plastic with a cover box, non-stacked, and
randomized order of petri dish. A proper incubator should have the ability to maintain temperature within
the unit in which the microbial growths are to be exposed. Incubation outside the incubator machine
affects the count accuracy and jeopardizes the reliability of the microbial plate counts. Temperature is a
critical factor that could influence and impact the validity of the results. Without a proper incubator,
temperature fluctuates in between periods. Time and incubation period are crucial in microbial growth
(Davis, Joseph, and Janssen, 2005).
Another concern is the differences in relative humidity in a makeshift incubator (box) that could
significantly affect microbial plate count incubation. A study by Qiu et al. (2022) suggests that a gain of
air temperature by 8°C due to relative temperature, elevates microbial growth by 50-60 folds in storage in
the cabinet. Bacteria are nourished in airtight environments with high humidity. This is characterized by
low ventilation and a highly humid environment, which is a characteristic of the box where the samples
are incubated. Thus, ventilation is an effective method for implementation in cabinet storage that could
limit or inhibit bacterial growth. Qiu et al. (2022) also found that high temperatures increase bacterial
growth, but ventilation in incubation reduces the degree of this phenomenon. As the relative humidity
equilibrium decreases, microbial metabolism is inhabited until growth is halted due to the condition.
Under high humidity, biofilm forms on the surface, providing protection, and under dry air,
microorganisms shrink and lose water, at RH 60% microbial replication stops. They have also found that
reducing humidity decreases bacterial growth at 26°C and 34°C. Thus, the study suggests that placing
charcoal or dehumidification bags inside a cabinet reduces humidity. Furthermore, air-conditioning
reduces humidity in cabinets, which is also found in the S6 room where the Petri dishes are stored in a
box.

On Appearances of Spreaders

All plates appeared to have a presence of spreaders. Spreaders usually form when there’s too
much moisture in the plates used for the cultivation of microorganisms upon inoculation of the microbes.
Moisture in the plates is due to the formation of condensation on the cover of the plate and usually forms
during the pouring of the agar media in the plates. Pouring of the media involves a partly high tolerable
temperature to keep the media pourable in its liquid state. This high-temperature solution upon reacting to
the ambient room temperature while pouring on the plate causes condensation. Accumulation of moisture
from the condensation is considered a “laboratory accident” and this will cause microbial growth of
spreaders. In physiological terms, moisture will somehow serve as a puddle on the plates where microbes
grown, particularly, flagellated microbes, may swim and spread its growth and will pose an inability to
form a single colony.
REFERENCE

Center for Food Safety and Applied Nutrition.(2003).Guidance for Industry: Questions and Answers on
Juice HACCP Regulation (2003).Food and Drug
Administration.https://www.fda.gov/regulatory-information/search-fda-guidance-documents/guid
ance-industry-questions-and-answers-juice-haccp-regulation-2003.

Davis, K. E., Joseph, S. J., & Janssen, P. H. (2005). Effects of growth medium, inoculum size, and
incubation time on culturability and isolation of soil bacteria. Applied and environmental
microbiology, 71(2), 826–834. https://doi.org/10.1128/AEM.71.2.826-834.2005.

Food and Drug Administration.(2013). REVISED GUIDELINES FOR THE ASSESSMENT OF

MICROBIOLOGICAL QUALITY OF PROCESSED FOODS. Department of Health. Retrieved
from
https://www.fda.gov.ph/wp-content/uploads/2021/03/FDA-Circular-No.-2013-010.pdf?fbclid=Iw
AR3UnJvyxckFXJ2A-pW_vtNAt70QJhKYarloJ7Hq2kXzU79pryXIa3GTSSU.

Maturin, L. and Peeler, J. T. (2001). BAM Chapter 3: Aerobic Plate Count. Bacteriological Analytical
Manual (BAM), Edition 8, Revision A. Retrieved from
https://www.fda.gov/food/laboratory-methods-food/bam-chapter-3-aerobic-plate-count

Neto, H. A., Tavares, W. L. F., Ribeiro, D. C. S. Z., Alves, R. C. O., Fonseca, L. M., & Campos, S. V. A.
(2019). On the utilization of deep and ensemble learning to detect milk adulteration. BioData
Mining, 12(1). https://doi.org/10.1186/s13040-019-0200-5

Prashant Dahal. (2024, January 2). Prashant Dahal- Author at Microbe Notes. Microbe Notes.
https://microbenotes.com/author/prashant-dahal/

Qiu, Y., Zhou, Y., Change, Y., Liang, X., Zhang, H., Lin, X., Qing, K., Zhou, X. & Luo, Z. (2022). The
Effects of Ventilation, Humidity, and Temperature on Bacterial Growth and Bacterial Genera
Distribution.International Journal of Environmental Research and Public Health (IJERPH)
19(22):15345. DOI:10.3390/ijerph192215345
APPENDIX A

Table 1. Interview Questionnaire for Buko Juice Vendor.

Survey Questionnaire Responses

Type of sample Buko juice

Source of Water Mineral water from water station

Source of Ingredients Buko powder from public market

Color of the Food Sample Creamy white

Handling Process (Scoop/Faucet) Scoop

Gloves or Without Without gloves

Storage of Reservoir Water jug

Ingredients “Pure buko”, evaporated milk, buko powder,

brown sugar

Natural Product or Processed Process

Time of Preparation 9:00 AM

Location of preparation MSU Gymnasium (Outside)

APPENDIX B

Sampling
 Figure 1. The images show the researchers gathering a sample from a vendor. The researchers gathered product
information from the vendor by giving an interview (upper left). Temperatures were recorded from the product on
the site (middle left), the environment on-site (upper right), and the temperature of the product inside the lab
(middle right). The pH was also recorded in the Chemistry Lab using the pH pen (below).

Pour Plating

Figure 2. The images show the different scenes in the pouring step of the experiment, including the 3 plates,
with the duplicates, with their label (upper left), the pouring (upper right), the aseptic technique (lower left),
and the putting of the sample to plates (lower right).
Analysis

Figure 3. Growth of Aerobic Bacteria from buko juice sample at 3-fold dilution (10-2, 10-3, and
10-4) inoculated through pour plating method incubated at 28 °C ± 2.0 °C for 24 hr on
Plate Count Agar.