Laboratory Exercise 1:

Aerobic Plate Count

Prepared by:
GROUP 1
Abiso, Adrianne Legarde, Manuel
Custorio, Aubrey Olaver, Eunace,
Guinanas, Gessa Mae Pio, John Christian
Kudarat, Abdul Jalil
AEROBIC
PLATE
COUNT
Objectives
 Laboratory Exercise
In this exercise, the students will be able to:
utilize aerobic plate count technique to compare 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; and
delve in the detailed calculation of microbial load using
standard formula for colony forming unit (CFU) / ml.
What and Why?
 Aerobic Plate Count (APC)
intended to indicate the level of microorganism in a product
(Maturin & Peeler, 2001)
estimates the population of viable bacteria in a sample by creating
ideal conditions for their growth (Park & Kim, 2013).

HYGIENE SHELF LIFE QUALITY

 Limitations
does not specify microorganisms
only detects viable cells
range is not standardized
limited information
Materials and Methods
Media Preparation
Materials & Equipment
(to be borrowed and reserved)
For Media Preparation Reservation of Equipment:
Reservation: February 6, 2024 Autoclave-11-1 AM (Group 3)
Materials (per group)
Beaker (waste container) 0.85% Saline Solution (prepared for the class)
1 Dilution Bottle 650mL - 5.525g NaCl for 650 mL
2 Test tubes (w/ screw caps) Will be prepared by Group 1
1 Test tube rack
3 Glass pipette (5mL/10mL) or 1 Micropipette
(1000uL)
6 Petri dishes
Pipette tips (if micropipette)
1 Graduated cylinder (250mL)
1 Erlenmeyer Flask (500mL)
Plate Count Agar (150mL)-3.525g for 150mL
Distilled Water 2 500mL Beaker
Microbox 1 Glass pipette (10mL)
Aluminum foil
 Plate Count Agar (PCA)
used for the determination of the total number of live, aerobic
bacteria in a sample.
is a medium recommended for the standardized enumeration of
aerobic bacteria in water, dairy products and foods, cosmetics or
pharmaceuticals.
Composition of Plate Count Agar (PCA)

Ingredients Grams

Enzymatic Digest of
5.0 g
Casein/tryptone

Yeast Extract 2.5 g

Glucose 1.0 g

Agar 15.0 g
Preparation of Plate Count Agar (PCA) per
group:
Suspend 3.525g grams in 150mL distilled water.
Heat to boiling to dissolve the medium completely.
Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
Cool to 45-50°C.
 Saline Solution
is commonly used as a carrier for bacterial suspensions.
for the preparation of isotonic suspensions and dilutions of
microorganisms
 Composition of Saline Solution 0.85%
Ingredients Gms/L

NaCl 8.5 g

Distilled water 1L

Preparation of Saline Solution (prepared as a class)

Dissolve 5.525g NaCl in 650mL in water.
Autoclave 15 min at 121°C.
Cool to room temperature.
 Materials for dilution:

or

Dilution bottles Screw cap Test tubes with cotton

plugs
Don’t forget your
Food Sampling
For Sampling and Wet Lab Reservation of Equipment:
Reservation: February 6, 2024 pH meter/pH pen (Group 4)
Materials (per group): Incubator (Group 2)
1 Thermometer (to be reserved)
1-2 Alcohol lamps (to be reserved)

1 Test tube rack

Beaker (waste container)
3 Glass pipette (1mL/5mL) or 1 Micropipette (1000uL) (sterilized)
Pipette tips; if micropipette (sterilized)
6 Petri dishes (sterilized)
Plate Count Agar (150mL)
Dilution Blanks; 99mL, 9mL, 9mL (sterilized)
Water bath
1 500mL Beaker
Icebox
Aluminum Foil (sterilized)
Ice
1 Glass Pipette (10mL)
Sampling/Lab Notebook
 Sampling Plan
1. Prepare materials needed for sampling.
 Sampling Plan
2. Assigned food sample per group.
o Group 1- Blue Lemonade
o Group 2- Melon Juice
o Group 3- Kalamansi Juice
o Group 4- Buko Juice
o Group 5- Pineapple Juice
 Sampling Plan
3. Collection of Samples.
Sample volumes should be sufficient to carry out all tests required.
Collect two 8-oz subsamples (236.59 ml) from any lot of food at
random. Place it inside a larger plastic cup to have a headspace for
your samples and to avoid spillage during transport. Seal it properly
using a sterile aluminum foil.
 Sampling Plan
4. Label your Samples.
Labelling of food samples is crucial for proper identification,
tracking, and analysis. The labels of your samples must contain the
following information:
o Unique sample identifier
o Date and time of sampling
o Product information
o Sampling location
o Analysis purpose
o Sampler name or initials:
 Sampling Plan
4. Label your Samples.

Code:
Date & Time of Sampling:
Product Info:
Sampling location:
Analysis Purpose:
Sampler Name or initials:
 Sampling Plan
5. Storage of Samples.
For the most accurate results, place your samples inside a cooler
with ice during transport to the laboratory. Keep samples cool but not
frozen (<10°C).
 Sampling Plan
6. Intrinsic/ Extrinsic Factors
Prepare data blanks and take note of other factors that could
possibly contaminate your samples during sampling.
Analysis (APC)
 Dilution
Dilutions:
10^-2
10^-3
10^-4

10^-2 10^-3 10^-4

 Dilution Blanks
Dilutions:
Dilution Blanks should contain 0.85% Saline Solution.

99 ml 9 ml 9 ml
 Dilution Scheme
99 ml 9 ml 9 ml
Dilutions:

Sample 10^-2 10^-3 10^-4

1 ml 1 ml 1 ml
 Dilution Blanks
Dilutions:
Shake dilutions vigorously 25 times in 30 cm (1 ft) arc in 7 s
after adding the 1 ml.
 Pour Plating
Why pour plating?
Allows microbial colonies either embedded within the agar
layer or formed on the agar surface.
The pour plate method is commonly used for the enumeration
of microorganisms with low microbiological limits.
 Pour Plating

WE MUST BE FAST!
 Pour Plating
Why should we be fast?
To avoid reshaking dilution bottle every 3 minutes.
To avoid the microbes to generate after 30 minutes.

Source: BAM Chapter 3: Aerobic Plate Count

 Pour Plating
How can we be fast?
Reshaking of dilution bottle if it stands more than 3 min is
necessary before it is pipetted into petri dish.
We need to add 12-15 ml plate count agar (cooled to 45 ± 1°C)
to each plate within 15 min of original dilution.
Maintain the sample’s temperature.
Not more than 15 min should elapse from the sampling time
until all dilutions are in appropriate media.
Source: BAM Chapter 3: Aerobic Plate Count
 Pour Plating
1 2
Pipette Diluted Sample onto Pour plate count agar (cooled
Petri dish to 45 ± 1°C)

3 Incubate 4
Swirl to mix Colonies grow on agar surface
and subsurface
 Before Incubation of Plates
Invert the plates after
the agar solidify to
prevent the
accumulation of water
condensation that
could disturb or
compromise a culture
Source: BAM Chapter 3: Aerobic Plate Count
 Incubation

Incubate the
plates for 48 ± 2
hours at 35 ± 1°C

Source: BAM Chapter 3: Aerobic Plate Count

Schedule of Activities
 Mark your Calendars...

Feb 6-Pre-lab Discussion

Feb 13-Media Preparation
Feb 14-Sampling and Wet Lab
Feb 16-Observation

**Feb 6-Reservation of Materials and Equipment

Time Allotment
To avoid delays, strictly follow the schedule...
Media Preparation (February 13, 2024) Sampling and Wet Lab (February 14, 2024)
15 mins. (Weighing of Media and 10-15 mins Sampling of assigned beverage
Preparation of Saline Solution) (measuring of temp and pH included)
20-25 mins. (Preparation of 20-25 mins Dilution and Pour Plating and
Materials) Preparing the incubator
Autoclaving 15-20 mins Cooling of petri dishes
Storing of media and sterilized Incubation (48 + 2 hrs incubation period)
materials

Observation of results (February 16, 2024)

Guidelines for calculating and reporting
APCs in uncommon cases
 Reporting Aerobic Plate
Count (APC)
Report all aerobic plate counts from duplicate plates.
For milk samples, report counts as follows:
1. Less than 25 colonies: Report as less than 25 estimated count.
2. More than 250 colonies: Report as estimated count.
3. Counts outside the 25-250 range may give inaccurate
bacterial composition indications.
 Reporting Aerobic Plate
Count (APC)
For all dilutions, record the counts as too numerous to count
(TNTC) for all but the plate closest to 250 when the number of
CFU per plate exceeds 250. Then, count CFU in the plate's areas
that represent the colony distribution.
 Aerobic Plate Counting
Normal plates (25-250)
Use spreader-free plates.
Count all CFUs, including pinpoint size.
Record dilutions and total colonies.
Plates with >250 Colonies
Record as "too numerous to count" (TNTC), except closest to 250.
Count CFUs in representative areas.
Mark calculated APC with EAPC to denote that it was estimated
from counts outside 25-250 per plate range
 Aerobic Plate Counting
Spreaders
Identify 3 spreader types:
1. a chain of colonies, not too distinctly separated, that appears to
be caused by disintegration of bacterial clump;
2. one that develops in film of water between agar and bottom of
dish; and
3. one that forms in film of water at edge or on surface of agar.
Report plates as spreaders if: a) Area covered exceeds 50% or (b)
Repressed growth exceeds 25%.
 Aerobic Plate Counting
When it is necessary to count plates containing spreaders not
eliminated by (a) or (b) above, count each of the 3 distinct spreader
types as one source.
For the first type, if only one chain exists, count it as a single colony.
If one or more chains appear to originate from separate sources,
count each source as one colony.
Do not count each individual growth in such chains as a separate
colony.
Combine the spreader count and the colony count to compute the
APC.
 Aerobic Plate Counting

Plates with No CFU

Report APC as <1 times lowest dilution.
Mark calculated APC with an asterisk for counts outside 25-250
range.
Record contaminated plates as laboratory accidents (LA).
COMPUTING AND RECORDING
OF COUNTS
Computing and recording counts
Report only the first two significant digits
Number of Third Digit Round off to

6, 7, 8, 9 Round Up

1, 2, 3, 4 Round Down

5, when second digit is ODD Round Up

5, when second digit is EVEN Round Down

fewer than 25 CFU Record as <25

 Number of
Round off to Example EAPC
Third Digit

6, 7, 8, 9 Round Up 12,700 13,000

1, 2, 3, 4 Round Down 12,400 12,000

5, when second
Round Up 15,500 16,000
digit is ODD

5, when second
Round Down 14,500 14,000
digit is EVEN

fewer than 25
Record as <25 12,700 <25 x 1/d
CFU
Calculating Aerobic Plate Count (APC)
Review:
Valid Plate Count
Microorganism Valid Count

Bacteria 25-250

Molds 10-150
 Traditional Method of Calculating CFU/mL
Used when valid counts come from only one dilution

Example:
1:10 1:100 1:1 000 1:10 000

R1 TNTC 237 18 5

R2 TNTC 243 22 11
 Traditional Method of Calculating CFU/mL
Used when valid counts come from only one dilution

Example:
1:10 1:100 1:1 000 1:10 000

R1 TNTC 237 18 5

R2 TNTC 243 22 11
Example: 1:10 1:100 1:1 000 1:10 000

R1 TNTC 237 18 5

R2 TNTC 243 22 11

Given: Solution:
Traditional Method of Calculating CFU/mL
Used when valid counts come from only two dilution
which are not consecutive in 10-folds

Example:
1:10 1:1 000 1:10 000

R1 TNTC 237 26

R2 TNTC 243 28
Traditional Method of Calculating CFU/mL
Used when valid counts come from only two dilution
which are not consecutive in 10-folds

Example:
1:10 1:1 000 1:10 000

R1 TNTC 237 26

R2 TNTC 243 28
Example: 1:10 1:1 000 1:10 000

R1 TNTC 237 26

R2 TNTC 243 28

For first dilution For second dilution

Given:
with valid plate count with valid plate count
Standard Method of Calculating CFU/mL
Used when valid counts come from only two
consecutive 10-folds dilution
Example: 1:10 1:100 1:1 000 1:10 000

R1 TNTC 237 18 5

R2 TNTC 243 22 11
Example: 1:10 1:100 1:1 000 1:10 000

R1 TNTC 237 26 5

R2 TNTC 243 29 11
Example: 1:10 1:100 1:1 000 1:10 000

R1 TNTC 237 26 5

R2 TNTC 243 29 11

Given: Solution:
 For plates in all dilutions with less than 25 CFU, record
actual plate count but record the count as less than 25 × 1/d
when d is the dilution factor for the dilution from which the first
counts were obtained.

Example:

1:100 1:1 000 EAPC/mL (g)

18 5 <2 500

0 0 <2 500
 All plates with more than 250 CFU. When plates from both 2
dilutions yield more than 250 CFU each (but fewer than
100/cm2), estimate the aerobic counts from the plates (EAPC)
nearest 250 and multiply by the dilution.

Example:

1:100 1:1 000 EAPC/mL (g)

TNTC 640 640 000

All plates with spreaders and/or laboratory accident.
Report respectively as Spreader (SPR),
or Laboratory Accident (LA).
 All plates with more than an average of 100 CFU per sq cm.
Estimate the APC as greater than 100 times the highest dilution
plated, times the area of the plate. The examples below have
an average count of 110 per sq cm.
Example:
1:100 1:1 000 EAPC/mL (g)

TNTC 7 150(a) >6 500 000

TNTC 6 490(c) >5 900 000

(a) Based on plate area of 65 cm2

(b) EAPC, estimated APC
(c) Based on plate area of 59 cm2
 ARE THERE ANY QUESTIONS?
CLARIFICATIONS? VIOLENT REACTIONS?
References
Food and Drug Administration (FDA). (n.d.). BAM Chapter 3: Aerobic Plate Count.
Retrieved February 3, 2024, from https://www.fda.gov/food/laboratory-methods-
food/bam-chapter-3-aerobic-plate-count

Park, J., & Kim, M. (2013). Comparison of dry medium culture plates for mesophilic
aerobic bacteria in milk, ice cream, ham, and codfish fillet products. Preventive
nutrition and food science, 18(4), 269.