ENVIRONMENTAL QUALITY MONITORING OF SELECTED WORK SITES OF

DOST-II REGIONAL STANDARDS AND TESTING LABORATORY

A special problem

Submitted to the Faculty of

College of Development Communication and Arts & Sciences
Department of Natural and Applied Sciences

Isabela State University – Cabagan Campus

In partial fulfillment of the requirement on

Bio 205 Advanced Microbiology

By

Jericho Duque Fronda

June 2022
 ABSTRACT

It is very important for laboratories to understand the levels of microorganisms within the
surrounding environment and identify them to reduce the risk of contamination. This work aimed
to assess air quality and critical surface area of the microbiology testing laboratory using Swab
Contact Method of (Moberg and Kornacki, 2015) in four different agars depending on the
organisms of interest. Standard count is 10 colonies per plate provided that no pathogenic
microorganism is detected. Confirmation and identification of isolated microorganism was based
on gram stain, and series of biochemical tests. Surface swab from the media preparation room
isolated Staphylococcus aureus which was confirmed with a positive catalase test. The possibility
of having a data on environmental quality could be a useful educational and training tool both for
those responsible for the sanitation procedures, since the results reflect the effectiveness and
suitability of the procedures adopted in order to increase awareness of the effects of a possible
non-adherence to behavioral standards.

I. INTRODUCTION II. SIGNIFICANCE OF THE STUDY

Environmental microbial monitoring plays a Environmental Monitoring (EVM) Testing is a

fundamental role in reducing the risk of
microbial contamination. Appropriate
microbial control requires an understanding
of abundance and community structures of
microbes in the target environment (Kawai,
2021).
Microbiological environmental monitoring is
a means of demonstrating an acceptable
microbiological quality in the controlled
environment and detecting changes in a
timely manner. It involves the collection of
data relating to microbial numbers recovered
from samples of air, surfaces, and people in
a clean area. Such data enable the
monitoring of trends over time, that is, the
detection of upward and downward changes
in that area. Besides running programs for
monitoring numbers and types of
microorganisms, particle counts (which may
represent viable organisms on carriers or
inert material) are also assessed as part of
the program.
This needs to come together into a program.
Hence the assessment of environments by
environmental monitoring is undertaken
through a defined environmental monitoring
program. The program should be
documented and detailed in a policy or
rationale together with accompanying
standard operating procedures (Sandle,
2019).
process which is conducted to monitor the
quality of the environment in areas where
microbial contamination is of concern. It is
very important for laboratories to understand
the levels of microorganisms within the
surrounding environment and identify them
to reduce the risk of contamination.
III. OBJECTIVES OF THE STUDY
The general objective of this study is to
assess air quality and critical surface area in
the Media Preparation Room, and Isolation
and Incubation Room of microbiology testing
laboratory.
Specifically, the study aims:
1. To isolate microorganisms using
Swab Contact Method in four
different agars:
- Plate Count Agar- Heterotrophic
Plate Count (HPC)
- DG18 Agar- Molds
- Baird Parker Agar-
Staphylococcus aureus
- Rambach Agar- Salmonella
2. To quantify the colonies appearing
per plate and report as the number of
colonies.
3. To confirm and identify isolated
microorganism based on:
- Gram stain
- Catalase test
 - Voges-Proskauer
- Methyl red-reactive
IV. SCOPE AND LIMITATION
Media Preparation Room, and Isolation and
Incubation Room will be subjected for
microbial monitoring using Swab Contact
Method of (Moberg and Kornacki, 2015).
Gram staining, and biochemical testing will
be conducted to confirm and identify isolated
microorganisms.
V. REVIEW OF RELATED LITERATURE
General Environment of the Laboratory
Conducive to Safety and Proper Practices
The laboratory should be air-conditioned and
well ventilated to minimize temperature
variations. The air-conditioning unit with
clean vent filters will reduce the number of
particulates in the air.
The laboratory should be designed with
worker safety in mind. It needs to be
spacious enough to include all necessary
equipment and have adequate workbench
space for the staff.
Adequate storage is needed to minimize
clutter, which allows for proper cleaning and
sanitization of surfaces. The laboratory
should be well lit, with a maintained light
intensity of approximately 50–1000 lumens.
A dependence on natural light is discouraged
during the day owing to high variability in
intensity. Direct sunlight should also be
avoided as it can negatively affect media,
reagents, and organisms.
Laboratory conditions should be comfortable
for workers. It is recommended that the
laboratory atmosphere be at an ambient
temperature between 21°C and 23°C, with a
relative humidity of 45%–50% (Brauniger
et.al, 2015).
Housekeeping and Environmental
Monitoring
A master cleaning schedule and appropriate
documentation should be established for the
laboratory to ensure that cleaning is
documented and can be monitored for
effectiveness. Laboratory surfaces should be
cleaned prior to sanitization with a cleaning
solution that contains surfactants to remove
dirt and organic materials. For sterilization
there are several disinfectant options, such
as iodophors, chlorine, quaternary
ammonium, or phenolic disinfectants. To
verify the effectiveness of the cleaning
schedule, a standard operating procedure for
environmental monitoring should be
established.
The operating procedure should describe the
sampling procedure, the locations to be
sampled, and the procedure for responding
to a positive result for qualitative analysis or
out-of-specification data for quantitative
analysis. Laboratory personnel should
perform the environmental sampling
technique consistently, and, although the
locations and frequency are detailed in the
operating procedure, the actual sites should
be chosen randomly (Brauniger et.al, 2015).
Principle of Monitoring the
Microbiological Flora
Natural selection is the underlying scientific
principle that applies to the need to monitor
the microbiological flora of a laboratory
environment. Based on the type of product
that is produced in the manufacturing
environment, microorganisms will be
selected that can best adapt and survive in
the environmental conditions encountered in
the laboratories, manufacturing equipment,
and residual food matrices. Failure to exert
control over the selection of these ‘‘normal
flora’’ will result in their proliferation with the
subsequent deterioration of product quality
and a potential increase in the safety hazard
(Moberg and Kornacki, 2015).
Enrichment Methods
Enrichment methods determine the
presence or absence of a target organism:
they are not conducted to determine the level
or quantity of that organism. Many direct
plating or quantitative methods exist for the
recovery and enumeration of target
microorganisms. However, quantitative
methods are not appropriate in several
situations: 1. The permissible level of the
organism is less than the maximum
sensitivity of the quantitative procedure. 2.
The organism is surrounded by large
numbers of competing microorganisms. 3.
The suspending food is inhibitory to the
target organism.
The goal of the enrichment method is to
permit the growth of the target
These pre-enrichment media are either
nonselective or are designed to be
moderately selective against competing
microorganisms. If the media contain any
selective components, these must be
balanced to permit the growth of the target
organism and repress the growth of
competing microorganisms in the food
sample. A pre-enrichment procedure may
not be necessary when the enrichment
medium has been validated to support the
microorganism, and if enrichment is
selective, to suppress or inhibit the growth of
competing microorganisms. There are
various types of enrichment protocols that,
either individually or combined, permit the
growth of the target organism to levels
necessary for detection or recovery by
diagnostic or selective plating procedures,
respectively.
Pre-Enrichment
The purpose of pre-enrichment is to allow the
stressed target microorganism to resuscitate
in either a non-selective or a moderately
selective environment. Although the
microorganism may resuscitate, very little
growth may occur during the pre-enrichment
step.
resuscitation and growth of the target
microorganism. The liquid version of Baird
Parker Agar (Liquid Baird Parker), without
agar, has been successfully used to recover
Staphylococcus aureus in foods.
The formulation of the pre-enrichment
medium will depend upon the level of
competing microorganisms and the ability of
the food to inhibit microorganisms. The US
Food and Drug Administration (FDA)
Bacteriological Analytical Manual describes
different pre-enrichment media for the
recovery of Salmonella in foods. These
range from sterile deionized water for
recovering Salmonella from non-fat dry milk,
to Brilliant Green-supplemented milk for the
recovery of Salmonella from chocolate.
These media use either the nutrients from
the food or contain ingredients (e.g., casein)
to neutralize the inhibitory nature of the food
(e.g., chocolate). Lactose is commonly used
as the pre-enrichment medium for
Salmonella, even though few species of this
genus are capable of metabolizing this
carbohydrate. The presence of lactose is not
directly essential for the recovery of
Salmonella. It has been suggested that some
selectivity of pre-enrichment media
containing lactose is generated by the
reduction in media pH when the lactose is
fermented by the mixed competitor flora.
Selective Enrichment
The pre-enrichment will result in
resuscitation of the target microorganism and
moderate levels of proliferation. Selective
enrichment furthers the growth of the target
microorganism while suppressing or
inhibiting that of competing microorganisms.
The selectivity of the medium is provided by
agents or conditions which are antagonistic
or inhibitory to competing microorganisms.
These selective agents include temperature,
antimicrobials, salts, acids, and metals. The
pre-enrichment step can also serve to dilute
or minimize interfering agents in the food
sample that can impair the selective
capability of the selective medium. Taylor
and Siliker determined that lactose pre-
enrichment prior to selective enrichment in
tetrathionate and selenite cystine increased
the recovery of Salmonella from albumin.
Furthermore, direct inoculation of food
samples with large numbers of competing
microorganisms into selective enrichments
may result in false negative results owing to
the reduction in the medium’s selectivity.
Conversely, food material in a pre-
enrichment transferred to a selective
enrichment will positively affect media
efficacy. (Abbiss, 1986) demonstrated that
the presence of food material in buffered
peptone water pre-enrichment enhanced the
recovery of Salmonella typhimurium from
selective enrichments. This improved
recovery was due to amelioration of the initial
inhibitory environment Salmonella
encounters when transferred to the selective
medium. In the case of Salmonella, neither
tetrathionate nor selenite cystine enrichment
broths alone will support the growth of all
strains of Salmonella. Therefore, to reduce
the risk of a false negative, many selective
enrichment protocols will employ more than
one medium following the pre-enrichment.
To ensure the availability of nutrients to the
target microorganism, enrichments may be
shaken during incubation. Facultatively
anaerobic microorganisms may experience
shortened lag phase and generation times
when oxygen is added during the
enrichment. The oxygen will support an
aerobic metabolism that yields higher energy
than anaerobic metabolism. (Duffy et. al,
1994) however, studied the growth kinetics of
L. monocytogenes and found that aeration of
the selective enrichment did not alter the
length of the lag phase or the growth rate of
L. monocytogenes.
Temperature Control or Management
Temperature control is a critical selective or
elective component of many microbiological
enrichment protocols. The Temperature
control is a critical selective or elective
component of many microbiological
enrichment protocols. The first step in the
quality assurance process of temperature
control is the purchase of the appropriate
equipment. Most inexpensive gravity flow
connection air incubators can maintain
incubation temperatures within about ± 3°C
of the desired temperature. For protocols
requiring more stringent control, forced air or
water-jacketed incubators, or shaking or
circulating water baths, should be used.
Incubators and water baths are usually
equipped with temperature controls and
indicators. These ‘‘built-in’’ devices should
never be relied upon as the sole means of
temperature verification. Incubators can
have hot and cold spots, so a calibration
should be performed annually to establish a
relationship between the temperature
reading of the internal device and the true
temperature at key locations within the
chamber. This can be done using a recorder
equipped with thermocouples that have been
calibrated against a reference thermometer.
Thermometers of the proper type (partial,
total or complete immersion) and of sufficient
accuracy and precision should be
permanently placed within incubators and
water baths. These thermometers should be
calibrated at least once per year against a
reference thermometer whose accuracy is
certified to be traceable to a National Institute
of Standards and Technology (NIST)
thermometer. Large incubators should have
at least two thermometers, one located
towards the top and one towards the bottom.
If calibration indicates a correction factor is
necessary, this correction factor must be
used each time the temperature is noted.
NIST-traceable temperature monitoring
devices are now available that monitor the
temperature continuously.
Laboratory Environment Management
Enrichment techniques are designed to be as
efficient as possible at detecting extremely
low levels of target microorganisms. This
means that they are extremely sensitive to
accidental contamination from the laboratory
environment. In addition to meticulous
aseptic technique, it is often desirable to
have a controlled ventilation system to
reduce the potential for contamination.
Laboratories should meet the general
requirements of at least a Biosafety Level 2.
Laboratories should be designed with
physical separation between critical areas
such as sample check-in, storage, pre-
enrichment set-up area, enriched culture
transfer areas, and media preparation and
sterilization areas. Hands-free wash stations
should be conveniently located and stocked
with soap. Disposable paper towels should
be available.
Analysts who work with enriched cultures or
highly contaminated samples should do this
only in designated areas that are separated
from other areas, and should wash their
hands and change their laboratory coats
before entering other areas of the laboratory.
In short, many procedures for limiting cross-
contamination that are considered good
manufacturing practices in the food
manufacturing plant should also be applied
to the food microbiology laboratory.
The air supply for laboratories conducting
enrichments and isolations should reduce
the levels of contamination, lower humidity,
and control temperature. Airborne
microbiological contamination should be
controlled by using filters, and the air quality
should be verified by microbiological
monitoring (air sampling devices, air settling
plates, surface swabs). Typically, total
bacteria or yeasts and molds are monitored,
but monitoring for specific target organisms
may be appropriate. Critical work surfaces
should be routinely monitored for the
presence of the target organisms being
enriched or isolated. Great care needs to be
taken when transferring pre-enrichment or
enrichment cultures. Analysts should be
trained in the elimination of aerosols and
microdroplets, and in all aspects of aseptic
technique. A useful technique for training
new analysts is to place brown paper towels
on the laboratory bench during transfer
practice exercises. After transfers are
complete, the towels can be checked for
droplets. Micropipettors should be used only
with extreme caution, and the use of
micropipette tips containing a filter should be
considered. Mixing test tube contents by
using a vortex mixer before transfer is not
necessary in most cases and can be a
source of cross-contamination. Receptacles
for contaminated pipettes and micropipettor
tips should be located as close as possible to
the operation being performed to reduce the
potential for dripping.
If testing indicates that results may have
been compromised by the laboratory
environment, policies and procedures must
be in place that allow for interpreting,
VI. METHODOLOGY
Procedural Details
1. Prepare appropriate agar plates for
the target organism.
2. Remove petri plate covers and
expose the plates in selected work
sites for 15 minutes. For the
biosafety cabinet expose the plates
for 1 hour duration. Replace covers.
Be sure to label plates with sample
site identification.
3. Incubate PCA plates at 35°C for 48
hours and DG18 plates at 25°C for
120 hours. Incubation time of other
media may vary.
4. Count the colonies appearing per
plate and report as the number of
colonies per 15 minutes.
Standard: Air control= 15 colonies
per plate per 15 minute exposure
provided that no pathogenic
microorganism is detected.
Swab Contact Method (Moberg and
Kornacki, 2015)
1. Prepare 0.1% Buffered Peptone
Water and salmocyst broth base (for
Salmonella), dispense 5 ml in a test
tube. Autoclave for 15 mins at 121ºC,
15 psi. Let it cool.
2. Label tubes with sampling sites for
identification.
3. Open the test tube containing the
0.1% Buffered Peptone Water and
salmocyst broth base (for
Salmonella), moisten the sterile swab
head, and press out the excess
solution against the interior wall of the
test tube with a rotating motion.
evaluating and reporting equivocal results.
Strict documentation at every step provides
valuable information for the investigation of
equivocal results. This documentation
information may include analyst, enrichment
time, transfer time, sample order, and rack
order.
4. Hold the swab handle to make a 30˚
angle contact with the surface. Rub
the swab head slowly and thoroughly
over a surface area of approximately
50cm2 three times, while reversing
direction between strokes. Move the
swab on a path 2cm wide by 25 cm
long or other dimensions to cover an
equivalent area.
5. Return the swab head to the solution
test tube, rinse briefly in the solution,
then press out the excess.
6. Swab four more 50cm areas of the
surface being sampled, as described
in item 3, and rinse the swab in the
solution after each swabbing.
Remove the excess.
7. After the areas have been swabbed,
position the swab head in the test
tube, and break or cut it with sterile
scissors or other device, leaving the
swab head in the test tube then
replace the screw cap.
8. Shake tube vigorously, making 50
complete cycles of 15 cm in 10
seconds.
9. Plate 1 ml and 0.1 ml portions of rinse
solution, plus additional solutions, if
deemed necessary.
10. Pour/Spread plates with appropriate
media, depending on the organisms
of interest:
Plate Count Agar- HPC
DG18 Agar- Molds
Baird Parker Agar- S. aureus
Rambach Agar- Salmonella
NOTE: Collected swab for
Salmonella detection should be
incubated for 6-8 hours at 35°C.
Then, add one tablet of salmocyst
(selective tablet) and stand for 30
 minutes. Shake vigorously until
dissolved. Incubate for 18-22 hours at
35°C. Then follow item 8-10.
11. Incubate PCA plates at 35°C for 48
hours, and DG18 Agar plates at 25°C
for 120 hours.
12. Count colonies and then calculate the
number of colonies recovered from
50cm2(equivalent to 1 ml of rinse).
Standard count = 10 colonies per
plate provided that no pathogenic
microorganism is detected.
Gram Staining
1. Prepare a light emulsion of the
bacterial growth from an agar slant in
Morphology of Microorganism after Gram Staining
Gram Positive
Blue to Violet colored
Catalase Reaction (Bennett et.al, 2015)
Emulsify growth from a TSA slant in 1
drop 3% hydrogen peroxide on a glass slide.
Immediate bubbling is a positive catalase
test. Cultures of S. aureus are catalase
positive.
Procedure for Biochemical Testing for
Escherichia coli
Voges-Proskauer (VP)
1. Inoculate tube of MR-VP broth and
inoculate 48 ± 2h at 35°C.
a drop of distilled water on a glass
slide.
2. Air-dry or fix by passing the slide
through a flame and stain for 1 minute
with the crystal violet solution. Rinse
the stained slide in tap water.
3. Apply Lugol’s iodine solution for 1
minute. Rinse the stained slide in tap
water.
4. Decolorize for approximately 15 to 30
seconds with acetone alcohol by
holding slide between the fingers and
letting acetone alcohol flow across
the stained smear until no more stain
is removed. Do not over-decolorize.
Rinse with tap water.
5. Counterstain with Safranin for 15
seconds, then rinse with tap water,
blot dry with absorbent paper or air
dry. Examine microscopically.
Gram Negative
Pinkish red colored
2. Transfer 1 mL to 13 x 100 mm tube.
Add 0.6 mL α-naphthol solution and
0.2 mL 40% KOH, and shake.
3. Add a few crystals of creatine. Shake
and let it stand for 2 hours.
4. Test is positive if eosin pink color
develops
Methyl red-reactive compounds
1. After VP test, incubate MR-VP tube
additional 48 ± 2h and 35°C.
2. Add 5 drops of methyl red solution to
each tube.
3. Distinct red color is positive test.
Yellow is negative reaction.
VII. RESULTS AND DISCUSSION
The data presented was based on the
microbiological work area monitoring record
of the RSTL-Microbiology Testing
Laboratory. Reporting of counted colonies
was every 24 hours intervals: 24-48 hours for
Total Plate Count and 24-120 hours for Total
Fungal Count. Microbial isolates from the 48
hour Total Plate Count were considered for
confirmation and identification. Standard
count is 10 colonies per plate provided that
no pathogenic microorganism is detected.

Table 1.a. Air sampling: 15 CFU/plate/15 mins. Exposure at Media Preparation Room

Date of Total Plate Total Fungal Count S. aureus Salmonella

Sampling Count
24H 48H 24H 48H 72H 96H 120H 24H 48H 24H 48H
06-06-2022 0 7 0 0 0 2 6 0 0 0 0
06-13-2022 0 2 0 0 0 1 3 0 0 0 0
06-20-2022 0 4 0 0 0 2 5 0 0 0 0

Table 2.a. Surface Swabbing at Media Preparation Room

Date of Total Plate Total Fungal Count S. aureus Salmonella
Sampling Count
24H 48H 24H 48H 72H 96H 120H 24H 48H 24H 48H
06-06-2022 0 3 0 0 1 3 8 0 1 0 0
06-13-2022 0 1 0 0 0 1 2 0 2 0 0
06-20-2022 0 3 0 0 1 3 4 0 1 0 0

Table 3.a. Confirmatory tests of isolated bacteria from Media Preparation Room at 48H Total
Plate Count

Sample Date of Confirmatory Test

sampling Catalase Voges- Methyl red-
Gram stain
test Proskauer reactive
Air sampling Gram-negative,
06-06-2022 Negative Copper Yellow
rod-shaped bacteria
Gram-negative,
06-13-2022 Negative Copper Yellow
rod-shaped bacteria
Gram-negative,
06-20-2022 Negative Copper Yellow
rod-shaped bacteria
Surface Gram-positive,
swabbing cocci, large round blue-
06-06-2022 Positive Copper Yellow
black colonies in
grape-like cluster
Gram-positive,
cocci, large round blue-
06-13-2022 Positive Copper Yellow
black colonies in
grape-like cluster
Gram-positive,
cocci, large round blue-
06-20-2022 Positive Copper Yellow
black colonies in
grape-like cluster
Table 3.a showed a negative result to Voges-
Proskauer and Methyl red-reactive with
copper and yellow color reaction
respectively. Isolated bacteria from air
sampling are rod-shaped gram-negative
bacteria while isolated bacteria from surface
swabbing were gram-positive with a cocci,
large round blue-black colonies in grape-like
cluster appearance. These samples were
subjected to catalase testing which confirms
all isolated bacteria from surface swabbing to
be Staphylococcus aureus with a positive
catalase test.

Table 1.b. Air sampling: 15 CFU/plate/15 mins. Exposure at Isolation and Incubation Room

Date of Total Plate Total Fungal Count S. aureus Salmonella

Sampling Count
24H 48H 24H 48H 72H 96H 120H 24H 48H 24H 48H
06-06-2022 0 0 0 0 0 0 0 0 0 0 0
06-13-2022 0 0 0 0 0 0 0 0 0 0 0
06-20-2022 0 0 0 0 0 0 0 0 0 0 0

Table 2.b. Surface Swabbing at Isolation and Incubation Room

Date of Total Plate Total Fungal Count S. aureus Salmonella
Sampling Count
24H 48H 24H 48H 72H 96H 120H 24H 48H 24H 48H
06-06-2022 0 1 0 0 0 0 1 0 0 0 0
06-13-2022 0 1 0 0 0 0 1 0 0 0 0
06-20-2022 0 1 0 0 0 0 1 0 0 0 0

Table 3.b. Confirmatory tests of isolated bacteria from Isolation and Incubation Room at 48H
Total Plate Count

Sample Date of Confirmatory Test

sampling Catalase Voges- Methyl red-
Gram stain
test Proskauer reactive
Air sampling Gram-negative,
06-06-2022 Negative Copper Yellow
rod-shaped bacteria
Gram-negative,
06-13-2022 Negative Copper Yellow
rod-shaped bacteria
Gram-negative,
06-20-2022 Negative Copper Yellow
rod-shaped bacteria
Surface Gram-negative,
06-06-2022 Negative Copper Yellow
swabbing rod-shaped bacteria
Gram-negative,
06-13-2022 Negative Copper Yellow
rod-shaped bacteria
Gram-negative,
06-20-2022 Negative Copper Yellow
rod-shaped bacteria

Table 3.b showed no pathogenic organism detected from all isolated bacteria with negative result
from catalase, voges-proskauer, and methyl red-reactive tests.
VIII. CONCLUSION AND
RECOMMENDATIONS
It is fundamental that the environment
complies with structural, organizational, and
managerial norms and adequate behavioral
models. The detection of microbial
contamination through environmental
monitoring may be the indirect expression of
shortcomings or failures in the above-
mentioned aspects. In terms of prevention,
environmental monitoring certainly
constitutes a secondary level intervention,
mainly aimed at an “early diagnosis” of
environmental unhealthiness, as
forewarning of sentinel events. Through
monitoring, indications are given of the
hygienic-sanitary quality not only of that
moment (the status quo) but also of the
quality that that environment will always have
under the same management and sanitation
conditions. Moreover, data on environmental
quality could be a useful educational and
training tool both for those responsible for the
sanitation procedures, since the results
reflect the effectiveness and suitability of the
procedures adopted in order to increase
awareness of the effects of a possible non-
adherence to behavioral standards.
IX. REFERENCES
Abbiss. (1986). A study of the dynamics of
selective enrichment of Salmonella.
The British Food Manufacturing, 1-
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Bennett et.al. (2015, June 8).
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