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Sanitation Effectiveness of 3D-printed Parts for Food and Medical Applications
Matthew Thomas1, Abdennour Seibi 2, Israd Jaafar3 Abolfazl Amin4
1College of Engineering, Orem, 84058, United States
2Department of Engineering, Orem, 84058, United States
3Department of Engineering, Orem, 84058, United States
4Department of Engineering, Orem, 84058, United States
A R T I C L E I N F O
A B S T R A C T
Article history:
Received:
Accepted:
Online:
Sanitation effectiveness of 3D-printed parts for food and medical applications has been
established in a 4-month lab study and controlled tests. The present study examined the
continued use of sanitation techniques across 3 more months of testing and experimentation
in household kitchens. Multiple specimens of the most common thermoplastics for Fused
Filament Fabrication (FFF), were printed with a range of settings to test for pathogen
contamination, biofilm production, bacteria, or other pathogens masking (hiding) in the
layer lines, gaps, and other imperfections of said prints. This study investigates methods of
sanitation and cleaning to reduce or eliminate pathogens along with its biofilms from the
defects and interstitial spaces that naturally occur in FFF printing. Results from various
testing methods used in hospitals and FDA approved microbial surface testing, indicate
that 3D printed parts of PLA/PLA+ (Polylactic Acid), and PETG (Polyethylene
terephthalate glycol) can be cleaned to safe levels using warm water (120 °F), and non-
concentrated dish soap. Examination and verification of cleanliness were completed via
Petri dish preparations, and protein residue testing. It was found that Colony Forming
Units (CFU) and Plaque Forming Units (PFU) had been reduced by 90%. Experimental
results indicate that using 2g of baking soda, when used with soapy water, eliminates
biofilms by chemical and physical action, neutralizes acidic bacteria, and removes mucus.
It is recommended and tested by surgical technicians, that a 2-minute room temperature
bleach water soak (200ppm), after washing and rinsing should be done to ensure pathogens
are at safe levels. Acetic acid from vinegar was tested as well via petri dish for CFU
reduction and can effectively eradicate biofilms due to the ability to penetrate the biofilm
matrix and the cell membrane. It is noted to the reader that sanitation in this context refers
to the method of bringing a surface or object to safe levels of cleanliness for food or medical
preparation and storage. Furthermore, mass spectrometry readings indicate that no
contamination from heavy metals, or other toxins are present in PLA+, and PETG before
and after printing. Lastly, filaments made from a pull-trusion method from recycled soda
or water bottles has been tested as well. When using 3D-printed items for liquids, it is highly
recommended to coat the 3D-printed parts in resin. This is a simple, fast, safe, and very
effective way to smooth parts to ensure easy cleaning.
Keywords:
Food safe 3d-printing
3d-printing
Biomedical Engineering
Food safety
ASTES
AJSP15-2308-
03357
Special Issue On Innovation In Computing, Engineering Science & Technology Vol:15
www.astesj.com
H. David et al. / Advances in Science, Technology and Engineering Systems Journal Vol. 15, 08-31 (2023)
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1. Introduction
This journal is an extension of work originally presented in the
IETC conference hosted by IEEE at the Brigham Young
University in Provo Utah United States on May 12th 2023 [1].
Please see reference at the end of this journal for more details,
including the DOI reference number.
With the rise of Covid and mass shutdowns during early 2020,
effecting the production of Personal Protection Equipment (PPE),
frontline workers found themselves in a big mess and used
whatever they could get their hands on. Concerned makers decided
to take matters into their own hands and 3D printed respirators,
connectors, valves for ventilators, and even test swabs. This
brought many questions to the table such as Can 3D printed parts
be cleaned, sanitized and reused for food or medical applications?
Common sense and instinct say yes, however, it must be
documented, tested and approved for hospitals, where lives are at
stake, to use 3D printed items.
Due to its increasingly widespread use in the medical field,
there are growing concerns regarding risks associated with
contamination. Prior to the pandemic, federal agencies have
implemented regulations and guidelines for compliance of
3Dprinted medical devices [2]. However, regulations take time to
be drawn up and implemented, and typically lag innovation and
use. This may be the reason why, during the COVID-19 pandemic,
the United States Food and Drug Administration (USFDA)
provided emergency use authorizations, waiving the strict
requirements for good manufacturing practices. This may have
been done to address the urgent need for this equipment and
supplies. Hence, cleaning and decontamination of 3D-printed parts
is vital in ensuring its safe use.
One area of interest for 3D-prinitng, that has exponentially
gained popularity, is the manufacturing of hands and limbs for
children or those who have lost limbs due to poor living conditions,
birth defects and other various reasons, and can not afford or have
insurance to cover the costs of prosthetics. These 3D-printed hands
and limbs are not a one-time use device and creating a set of
standards for cleaning and sanitizing will be very beneficial in
keeping those recipients healthy and safe. Some users have even
tried to use acetone vapor smoothing which can be toxic and does
not always yield the best results.
2. Preliminary Information
In this current study, the scope however, is to provide an
understanding and a methodology of how 3D-printed objects from
a variety of common thermoplastics, can be feasibly cleaned to
levels that are safe for food-related and medical applications. In
this regard, testing methods, findings and all procedures are
documented in this journal.
A common idea among users, groups, and medical personnel,
is the notion that the gaps in the layer lines of 3D-printed objects
or dimples, pose the largest threat to making 3D-prints food-safe
or cleanable. The argument has been that bacteria could grow and
reproduce in tiny spaces causing difficulty in cleaning due to the
small space size. The average bacterium from food poisoning is 2-
5 microns in length and 0.5-1.5 microns in diameter. Even bacteria
need room to grow, like any other specimen. Think of the goldfish
and small fishbowl analogy.
When studying 3D-printed items under a VEGA3 Tescan SEM
imaging machine, it is found that the smallest dimple is 0.5
microns in size. These dimples make up between 1-2% of all
imperfections, meaning the bacterium that can cause food
poisoning, will have a difficult time fitting in those 0.5 micron
spaces. The figure below (Fig.1) shows the machine as well as the
scanned image of the 3D printed surface of a respirator.
Figure 1: VEGA3 SEM Imaging Machine and Scanned Surface.
The other 98-99% of flaws are larger than 0.5 microns, with an
average size of 1 micron. To the user, this seems small, but to
bacteria and pathogens, its fairly large. Much of the 3D-printing
community feels that this is the biggest concern and poses a major
problem, but we will soon find out that it does not cause as many
issues as one thinks. Larger spaces may allow for bacteria growth,
but it’s easier to clean.
Lead contamination has been another concern for the safety of
food applications. The amount of lead present in a 3 gram, standard
0.4mm 3D-printer brass nozzle is 1.5%. It is less for larger bore
sizes since more material is removed during its manufacturing
process. 1.5% of 3 grams amounts to 0.045 grams of lead. 21%
(0.63 grams) of a 3-gram standard 0.4mm nozzle is removed for
the filament to flow into as indicated in Fig. 2 below.
Figure 2: Cross Section of a Standard Brass Nozzle.
H. David et al. / Advances in Science, Technology and Engineering Systems Journal Vol. 15, 08-31 (2023)
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This means that 21% of the 0.045 grams of lead were removed
as well, leaving a total number of 0.035 grams of lead remaining
in the nozzle. Even less is in contact with the filament as it passes
through the bored nozzle. The filament is only in contact with 21%
of the TOTAL amount of lead, which means a total possible
contact with 0.007 grams of lead.
Lead leaches out in very small amounts, and usually from
handling, or from water sitting/flowing through a brass fixture. The
possible amount of lead being leached, or friction rubbed onto the
filament is hence considered to be negligible. Masses of new
nozzles compared to used nozzles (1000 hours of use) show that
either no mass was lost due to printing, or the amount lost due to
friction was so small, that the scale did not pick it up, meaning,
such an extremely small amount of lead had been friction rubbed
onto the thousands of meters of filament over 1000 hours of use. It
is noted that studies have shown, consumers who use the standard
brass key are exposed to 19 times the safe amount of lead [3].
To push the study further and gain more accurate results, the
VEGA3 Mass Spectrometer was used to view all the molecules
and elements that make up thermoplastics. PLA/PLA+ as well as
PETG were tested. No contamination from lead or other heavy
metals and toxins were present. Figure 3 shows the output from
scanning PLA+. Its 99% Calcium Carbonate with traces of Silica.
Figure 3: Mass Spectrometry Reading of PLA+
The last concern to address is the manufacturing process of the
filament itself. The color additives may leach out of the filament
when placed in a liquid. These colors that are added during the
melting stage may not be food safe unless otherwise noted. During
the manufacturing process, pigments are added to the melted
plastic. Companies, to keep their color a trade secret, will avoid
using any type of pigment that requires a listing on the MSDS
therefore, pigments used in filaments are usually non-toxic and
inert to most treatments and foods. However, to avoid any possible
risk, coating in resin adds an extra step of safety. In most cases, the
color of plastic does not bring any danger or changes the properties
more significant than changing its melting point. As the chosen
pigments are inert to most treatments, they do not need to be listed
on the MSDS, and thus omitted, allowing the companies to keep
them a trade secret that helps them compete against other
companies for only they have this one specific color. To mitigate
these concerns, coating a 3D-printed item in resin is safe according
to title 21 volume 3 of the FDA [4]
Three types of testing were done: reduction percentage count
of CFU, Adenosine Triphosphate (ATP) monitoring, and protein
residue testing. The latter is the most accurate of the testing
methods. Visual inspection is not nearly good enough for hospitals
or food establishments. So, we must use other testing metrics to
determine how clean surfaces are. Using an ATP monitor (very
expensive) you can test how many light units reflect to the monitor.
A statement from Bio Med Central reports “Measurement of ATP
is nowadays frequently used to measure cleanliness of surfaces in
hospitals. By measuring ATP, the presence of all kinds of organic
material is measured; e.g. microbial contamination and organic
contamination (skin flakes, bodily fluids, food scraps, etc.). As the
amount of ATP is quantified, ATP measurements give insight into
the level of environmental contamination within the healthcare
setting. The ATP results are available within seconds which enable
immediate feedback.”
ATP is present in every living cell. The cells use it as an energy
source, and they leave it behind as they move about. With dead or
non-living cells and contamination from microbial organisms,
ATP monitoring will not be sufficient. Some disinfectants can
interfere with ATP monitoring as well. Syscehm, a UK company
states the following: “ATP Hygiene monitoring does not represent
contamination itself. Therefore, microbial, or organic
contamination will always be an indirect measurement when
measured by ATP. On the other hand, however, Protein Residue
tests directly measure organic/microbial contamination.”
3. Experimental Investigation
To ensure proper testing, a set of print metrics and a list of
equipment was constructed. Following these guidelines and
procedures ensure that a proper conclusion can be made.
According to the FDA guidelines for microbial surface testing,
each lab must come up with their own set of metrics and
methodologies to prove that a surface or object is safe to use for
food or medical applications.
3.1. Printer Settings and Metrics
Three different printers were used for the experiments;
1. Tevo Tornado
2. Anet A8
3. Custom built Prusa
The printer settings used were;
• 0.16-0.24mm layer height in increments of 0.02mm and speed
increments of 5mm/s between 40-70mm/s
• Nozzle temperature 205-220°C in 5°C increments
• Bed temperature: 60°
Using these printers and settings will provide a wide range of
surface qualities. The Anet A8 is a printer known for having issues
with surface quality. However, the hypothesis is that parts with a
higher quality surface will be the most difficult to clean, and that
those with larger gaps will be the easiest.
3.2. Materials and Equipment
*Corresponding Author Name, Address, Contact No & Email
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•Bunsen burner was used to clean tools and keep a positive
convection updraft, to keep contamination from falling into the
petri dishes or cultured broths.
• Petri dishes 100mm x 15mm (quantity 50 in total)
• 3 different plastic filaments were used in the study: Polylactic
acid (PLA/ PLA+) and polyethylene terephthalate glycol
(PETG).
• Cultured broth containing pathogens
• Autoclave • Inoculation loop
• Scale and or balance
• Culture tubes
• Incubator set to 37C (98.6F)
• Erlenmeyer flasks
• Single channel precision pipettor
• Yeast extract, peptone, beef extract, salt, glucose, and agar are
all used to make nutrient rich broth and gel for petri dishes
• PH stabilizer
• Tryptic Soy Agar (TSA)
• Tevo Tornado 2019 Gold Edition 3D printer,1 custom-built
Prusa and one Anet a8
• VEGA3 TESCAN Scanning Electron Microscope and Mass
Spectrometer
• Gold and Carbon powder for SEM and Mass Spectrometer
preparation
• Pathogens for testing
• Dawn dish soap
• 200 ppm molarity bleach water at room temperature for
sanitation
• 70% IPA (Isopropyl Alcohol) Some people referrer to this as
“rubbing alcohol” they are similar. Rubbing alcohol is IPA
diluted with water.
• PRO-Clean Hygenia protein residue swabs
3.3. Pathogens For Culture on Specimen
The pathogens cultured on the printed specimen include the
following: Klebsiella pneumoniae, Acinetobacter baumanii,
Pseudomonas aeruginosa, Escherichia coli, Shigella sonnei ,
Salmonella typhimurium. Proteus mirabilis, Citrobacter freundii,
Bacillus cereus, Streptococcus pyogenes.
For best testing results for normal household conditions, 3D-
printed parts were soaked in chicken blood or livers for 24 hours,
then allowed to dry another 24 hours once removed from the liquid.
4. Results and Discussion
When water is placed on a plastic, it will bead up and roll off.
This is due to the surface tension of water as well as the typical
hydrophobic nature of plastic surfaces. This is known as
Flowability. Higher surface tension fluids do not flow well into
small spaces unless they get some help from some sort of additive
that loosens the tension of the hydrogen bonds. Think of a ball of
elastic bands tightly bond together. If you stretch the bands, they
will return to their normal position, but if you apply a small amount
of heat, they will loosen and hold to a new shape. This idea and
concept of loosening the bands is what we will take advantage of.
To accomplish this an additive must be added to the water to loosen
said bonds, this is where dish soap comes into play. Basic soap and
detergents will dramatically weaken the hydrogen bonds of water,
making it flow into very small spaces. Table 1 below shows some
common surface tensions of liquids.
Table 1: Surface Tension Forces of Common Liquids.
IPA and soapy water have very low surface tension and almost
0-degree wetting angle. Using a 0.1-micron filter paper, 70% IPA
has complete flow into the filter medium instantaneously, and
shows the grid pattern, indicating that it has flown into the 0.1-
micron sized pores. Soapy water was then tested on the same
medium yielding the same results as shown in figure 4. This
indicates that soapy water can flow into spaces much smaller than
the smallest bacteria that can cause food poisoning or the smallest
imperfection of a 3D-printed item. For comparison, the size of a
Corona virus is 0.125 microns. To prove the results, a single drop
of water placed on the same filter material never showed any signs
of flow. Then water was placed on a 0.45 micron filter paper, it
took 5 minutes for half of the water to flow through the paper.
Figure 4: Flowability of Water, IPA, and Soapy Water.
Preliminary testing and culture growth is step 1 of the process.
A 3D-printed respirator mask that had not been sanitized for 90
days, with constant wearing during the COVID 19 pandemic, was
used for the first preliminary test. The mask was swabbed with a
clean, moist sterile swab and then spread onto a 100mm x 15mm
Petri dish and allowed to incubate for 4 days in a room set at 83
°F. Clean glass tiles that were handled with “clean” (soap washed)
and “dirty” (un-washed) hands were also swabbed and cultured for
comparisons and controls. Lastly, “clean” fingernails and hands
were swabbed and then spread onto a dish as well. This preliminary
study was conducted to obtain an initial idea of how many Culture
Forming Units (CFU) grow between what is “clean” relative to
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“dirty” conditions. Once the Petri dishes were incubating, the mask
and glass tiles were then washed with basic soapy water using
nonconcentrated soap. After the washing, each item was then
swabbed and cultured again to determine a percent reduction in
CFUs. It was found that a 90% reduction of growth occurred after
washing with basic soap on the 3d printed mask contaminated with
bacteria and virions (Figures 5, 6, and 7). This leads to the possible
conclusion that the 3D-printed object is safe for food use. From
visual inspection, the growth had been reduced, but it is noted by
the authors that further testing must be done.
Figure 5: 90% CFU reduction of pathogens on 3d printed respirator.
Figure 6-7: Glass Tiles Used as Control; Before and After Washing.
After the first set of preliminary tests, 3 groups of 3D-printed
cubes of various wall thicknesses and layer heights were
inoculated with different pathogens found in common household
settings, such as E. coli or salmonella (chicken blood). The goal
was to allow pathogen growth. These pathogens were then stressed
by applying heat so that they would create biofilms, a protective
blanket. These biofilms are what need to be removed during
cleaning which allows cleansing agents to clear away the bacteria
that were veiled by the biofilm. Groups 1, 2 and 3, all consisted of
4 single-walled prints, 4 double-walled prints, and 4 triple-walled
prints. Each print varied from 0.16 to 0.24 mm layer height in
0.02mm increments, printed with different speeds and
temperatures. All groups consisted of PLA, PLA+, and PETG as
the choice of materials used. Each cube was inoculated and
allowed to culture. Once cultured, each cube was then swabbed
and spread onto a Petri dish. Each part in Group 1 was washed with
warm 120 °F water, and non-concentrated dish soap for 30
seconds. Group 1 was then re-swabbed and tested on a Petri dish
for visual comparison and percentage reduction in CFUs to
determine if the number lies below the Infectious Dose (ID)
number. To further prove the results, Group 1 underwent further
testing with PRO-Clean Hygenia protein residue testing. Group 1
were re-inoculated, with a soak in chicken blood and other
common bacteria found in food establishments. After the allotted
time to grow and produce biofilms, group 1 was rewashed and
allowed to air dry for 2 minutes. One printed object was left out of
the cleansing group to be used as a control. This contaminated cube
was tested to show a failure result as indicated in figure 8.
Figure 8: Control Swab for Protein Residues
The rest of the cubes from group 1 were tested using the residue
method after being cleaned with basic soap and water, and each
cube indicated a pass. Refer to figure 9 below.
Figure 9: Three Green Passes and 1 Purple Fail for The Control Group
To further test and study, each cube from group 1 was left out
for 48 hours to see if any contamination would occur. They were
re-tested and passed, however, after 3 hours, the catalyst in the vial
had turned light purple, indicating contamination of samples.
Groups 2 and 3 underwent the same inoculation and culturing. The
cleaning process for Group 2 was the same method as Group 1, but
with an added 2-minute soak in bleach water. This method has
been known to help reduce and dissolve E-coli (table 2). It is
required by the FDA that for all food establishments to be deemed
food safe, dishes and surfaces must be soaked or wiped in a
molarity of 200ppm (1 tablespoon bleach per gallon of H2O) of
cold bleach water for at least 1 minute.
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Table 2: BLEACH VS E. COLI. TNTC IS TOO NUMEROUS TO COUNT.
Since warm water deactivates bleach, this is typically
performed using room temperature water. Group 3 used a small
amount of baking soda on the 3D-printed part during washing. 2g
of baking soda was placed on the part and was scrubbed on using
fingertips [5]. The part was then washed with soapy water.
Diamond Back Plumbing stated: “Baking soda makes an excellent
cleaning agent when you need to get rid of the biofilm. This is on
account of the fact that it works by both chemical and physical
means. The rough particles in the baking soda will dislodge any
biofilm from the plumbing surfaces, while the basic pH will also
help in chemically removing most of the waste. It’s very simple to
use baking soda for this indication; you only need to wet a rag, put
a bit of the baking soda in it and then scrub away. In no time, you
will have cleared most of the slime.”
Once dry, the parts from Groups 2 and 3 were tested for residue
and again these indicated a pass result. The 3D-printed parts were
left out for 48 hours and then re-tested. Tests came back green,
indicating a pass, and after 3 hours, had not turned light purple
meaning, no re-contamination occurred. A 5th group was used for
clarity to help determine the best methods for cleaning.
70% IPA was introduced to the list. It was found that only with
physical agitation, such as rubbing, that IPA cleaned the parts to
safe levels. However, due to recent studies [6], IPA could
potentially increase biofilms with specific pathogens such as
Staph. Therefore, 70% or higher IPA should be used AFTER
cleaning as a precautionary sanitization step, as you would with
bleach water. Figure 10 shows this possible increase of staph.
Figure 10: Possible Increase of Staph
For the next set of tests, a 3-walled cube was filled with chicken
blood and water, then put under pressure to allow the fluids to seep
into the print. After washing the part with soapy water and baking
soda technique, sterile, distilled water was added to the cube and
put under pressure to allow the water to flow into the gaps and
layer lines, collecting any type of contamination that could be
present. The water was then tested with the protein residue method.
The catalyst in the vial turned green indicating a pass for safety
(Figure 11). Waiting 3 hours, the liquid in the vial remained green.
Further testing should be done with the more hazardous and
uncommon pathogens such as Pseudomonas or others listed in our
group.
Figure 11: Pressure Test Contamination Results Indicate a Pass.
Before concluding the experiments and study, the 3D-printed
parts that were cultured for a percent reduction were compared
with standard household items that had been washed with soapy
water and swabbed for petri dish culturing. A plastic cutting board
(80% reduction), a glass plate (99%), a standard spoon (91%) that
when examined under a digital scope, had many holes, cracks, and
scratches the same size range as 3D printed parts, and lastly,
fingernails (40%) that had been recently washed. (Figure 12 and
Chart 1).
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Figure 12: Scratches and Holes in a Spoon Roughly the Same Size Range
as Imperfections In 3D-Printed Parts.
Chart 1: Displays the CFU Reduction of Common Kitchen Equipment vs
3D Printed Items.
Plastic cutting boards seem to have more contamination than
3D-printed parts. More CFU’s grew on the petri dishes from a
plastic cutting board that had been washed with soapy water, than
3D-printed parts that were washed with the same method.
Surgical technicians were contacted at two different hospitals
(choose to remain anonymous) to test specimens for safe levels.
Each specimen underwent a wash, rinse, and sanitize in bleach
water for 2 minutes. ATP and residue testing was performed on the
parts and were found to be within specifications safe for food and
medical establishments.
To enhance the study and extend the testing results, a testing
metric was formed using acetic acid from vinegar to cleanse 3D-
printed parts. In order for acetic acid to be effective against
biofilms, the pH of the weak acid in question must be below its
pKa value. Vinegar has a pH of 2.5 and a pKa value of 4.6. This
makes vinegar a decent choice for testing [7]. Vinegar, in certain
situations is a good disinfectant, but for the majority of cleansing,
it is not recommended for sanitizing or disinfecting. Use vinegar
to help remove biofilms before washing with soapy water.
Spritzing the 3D-printed part with full strength white vinegar, and
leaving for 60 seconds, will help dissolve the biofilms covering the
bacteria. Then proceed to use the baking soda and soapy water
method to clean the prints.
In this last figure, you will see a petri dish that was divided into
3 equal parts: before washing, after washing and control. A swab
test for culture growth was done on a standard home kitchen sink
before washing and cleaning. Vinegar was then spritzed onto the
surface of the sink, allowed to sit for 60 seconds, and then scrubbed
with a paper towel. A final swab of the cleansed area was taken
and spread onto the petri dish. The last section of the dish is the
control, and nothing is to be done to this area. If nothing grows in
the control area, then the petri dish is of sound construction.
Figure 13: Disinfectant Efficacy of Vinegar is Low but Can Still Eradicate
or Weaken Biofilms. [7-8]
5. Conclusion
Experimental results proved that baking soda, when used
with soapy water, does eliminate biofilms by chemical and
physical action. Further testing helped authors conclude that a 2-
minute room temperature bleach water soak (200ppm) without
baking soda scrub, helped dissolve biofilms and pathogens to
safe levels when tested by surgical technicians. It has been
concluded from the results that the best method of cleaning 3D
printed parts is to use warm soapy water with a small (1/8th
teaspoon or 2g) amount of baking soda on a dish rag. Spend
about 15 seconds washing and scrubbing with a small amount of
force, as to not damage the part. Once clean, either soak in cool
bleach water for 1-2 minutes of a 200ppm molarity, 1 tablespoon
bleach per gallon of water or apply 70% or greater IPA by
soaking for 1 minute. It is highly recommended, when using
filaments that have color or non-food safe additives, to coat the
3D-printed part in resin and allow to cure fully, if said 3D-
printed part will be in contact with liquids.
Conflict of Interest
The authors declare no conflict of interest.
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Study on the Sanitization Efficacy for Safe Use of 3D-Printed Parts for Food and
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