Cosmetics Microbiology

D K Brannan, Abilene Christian University, Abilene, TX, USA

P A Geis, The Procter and Gamble Company, Cincinnati, OH, USA
ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Preservation

Background and Importance of Cosmetic Microbiology HACCP for Cosmetics
Sanitary Manufacture Test Methods
Sanitization Further Reading

Glossary threatening. An example is contamination of a cosmetic

adulteration The addition of any harmful substance
that may make a product harmful to users under usual
conditions of use. Adulterated products contain filthy,
putrid, or decomposed substances. Products are
adulterated if packed or held under insanitary conditions
or if the container is unsafe.
antimicrobial Compounds that kill or inhibit the growth
of microbes – used as disinfectants or preservatives.
Class II recall Recalls of products are classified from I
to III. Class I is an emergency situation where the
consequences are life threatening. Class II is a priority
situation where the consequences may be immediate or
long range and possibly hazardous to health or life
with a potential pathogen.
cosmetics Articles that someone applies to, sprinkles
on, or rubs into their body to cleanse, beautify, or
promote attractiveness or alter their appearance. The
product must not affect normal bodily function or
structure. This definition excludes ordinary soap.
disinfect Killing microbes on surfaces to levels that are
not harmful to health or the quality of the product.
preservative A chemical agent used to prevent
microbial growth in finished products. It prevents their
multiplication or kills them to prevent spoilage or
contamination of the product with pathogens.
sanitizer A chemical agent used to disinfect
equipment.

Abbreviations GMP Good Manufacturing Practice

ALS ammonium lauryl sulfate HACCP hazard analysis and critical control point
AOAC Association of Official Analytical Chemists HEPA high efficiency particulate air
BP British Pharmacopoeia MPN most probable number
CFUs colony forming units PCT preservative challenge test
CIP clean-in-place PVC polyvinyl chloride
CTFA Cosmetics, Toiletries and Fragrance QA quality assurance
Association Quats quaternary ammonium compounds
FDA Food and Drug Administration USP United States Pharmacopoeia

Defining Statement Background and Importance of Cosmetic

Cosmetic microbiology is a subdiscipline of microbiology.
Here, microbiologists produce cosmetics free of pathogens
and prevent their spoilage due to microorganisms. The goal
is to improve the safety and aesthetic quality of cosmetics. To
do this, one must understand microbial physiology, patho-
genic microbiology, and microbial ecology. In addition, one
must understand organic and physical chemistry, toxicology,
engineering, and regulatory/environmental laws.
Microbiology
Cosmetic manufacturers invest considerable effort to
reduce the risks of microbial contamination in their pro-
ducts since the economic effects are great. Contamination
requires having to scrap spoiled product, conduct Class II
recalls, and handle litigation from harmed consumers.
For those few, less reputable, cosmetics firms that are
unaware of the role of microorganisms in their products,

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 Applied Microbiology: Industrial | Cosmetics Microbiology
the discovery that their cosmetic contains contaminating
microorganisms creates a reactive, problem-solving flurry
of activity. As they become more aware of the microbial
world, this activity finally becomes proactive prevention.
Microbiologists can solve such preventable problems. In
fact, the joy of solving such problems provides a kinship
with the microbiological masters of old. Like Pasteur
solving the spoilage problems of French wines, the tech-
niques of microbiological sleuthing are still the same. Get
all information on the subject, formulate an hypothesis for
why the problem exists, test it, and then provide a prac-
tical solution for the problem.
Even the novice microbiologist can identify and quan-
tify microbial contaminants in a cosmetic. But it takes
considerable expertise and experience to eliminate the
contaminant and prevent it from occurring again. The
novice just adds more biocide, a guaranteed way to
adapt the contaminant to the biocide and compound the
problem. The experienced cosmetic microbiologist finds
the sources of the contamination, cleans them up, rede-
signs the product preservative system in case of tolerance,
and even helps determine how to reclaim or scrap the
affected product in an environmentally safe manner.
Regulations and History
In 1938, congress passed an administrative bill regulating
cosmetics – the Food, Drug, and Cosmetic Act of 1938.
This action was the culmination of a sequence of events.
The existing 1907 Drug Act was considered too weak to
ensure effective drug and food safety. A stronger bill was
proposed in 1933 but it did not pass both House and
Senate at that time. In 1937, a company marketed an
oral tonic made with a poisonous ingredient. The result-
ing deaths prompted congress to pass the Act in 1938. The
Act gave the Food and Drug Administration (FDA) a
means of regulating and defining cosmetics. The Act
also defined adulteration and allowed FDA to request
recalls. If a manufacturer fails to conduct the recall, the
Agency can seize the product. Finally, congress passed
tighter regulatory control on label claims, ingredient list-
ing, and product safety warnings – the Fair Packaging and
Labeling Act of 1973–75.
Cosmetics industries thrived in the 1930s. The major
microbial problems were preventing visible mold growth
with parabens. When high-volume manufacture of cos-
metics in the 1940s increased, so did bacterial and mold
spoilage. Companies began including bacteria in their
preservative challenge tests (PCTs) and using bacterici-
dal preservatives.
In 1943, the Toilet Goods Association (later called
the Cosmetics, Toiletries and Fragrance Association
(CTFA)) established its Scientific Section. Member com-
panies also founded The Society of Cosmetic Chemists to
discuss formulation and preservation on a scientific level.
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Physicians used antibiotics indiscriminately in the 1950s.
Industry also overused antimicrobials in cosmetics ranging
from deodorants and soaps to toothpastes and shampoos.
Our preoccupation with germs drove this market for pro-
ducts that killed germs. Often, there was an unclear
distinction between whether the biocide was used to provide
a functional antimicrobial in the cosmetic or used as a
preservative. Soon, Staphylococcus spp., Streptococcus spp.,
Pseudomonas spp., Serratia spp., Enterobacter spp., and Klebsiella
spp. caused contamination problems as they developed tol-
erance to biocides. Contaminated cosmetics were found at
higher rates than ever before on store shelves in the 1960s.
The FDA conducted 25 drug and cosmetic product
recalls during 1966–68. In a 1969 sampling, the FDA
found contamination in 20% of 169 cosmetics tested.
Thus, the least regulated of all consumer products came
under fire. Rather than face enforced and impractical
regulations, the industry launched a cooperative CTFA/
FDA relationship of self-regulation. In 1967, the CTFA
formed the Microbiology Committee from member com-
panies to address these contamination problems. The
committee conducted a survey of almost 4000 products
to show that cosmetics, at least from the reputable com-
panies, were free of objectionable microorganisms. This
committee developed test methods and conducted colla-
borative studies to improve manufacture of microbially
free cosmetics. They issued technical guidelines covering
good manufacturing and microbiological practices. With
microbiological test methods and good manufacturing
practices in place, the industry satisfied one of the
FDA’s chief concerns.
However, in 1974, instances of blindness occurred
from use of mascaras contaminated by the user with
Pseudomonas spp. The next concern of the FDA was if a
cosmetic product could withstand microbial insults added
during consumer use. In the 1980s, the FDA rephrased
this concern to ask if PCTs predicted the risks of in-use
contamination. PCTs are methods used to see how
well the biocide in a product kills microorganisms. The
CTFA Microbiology Committee in conjunction with the
FDA and the Association of Official Analytical Chemists
(AOAC) arranged for a variety of university and industry
collaboration to compare laboratory methods of assessing
the preservative efficacy of eye area cosmetics. They
found that a variety of laboratory methods for measuring
preservative adequacy in eye area cosmetics were satis-
factory. Although none of the tests were found to be
predictive of consumer contamination, they were shown
to be statistically reproducible and reliable. Nevertheless,
industry rose to the challenge without such extramural
support. Even to the present, there still remain only two
challenge tests that have published validation data in
peer-reviewed journals proving their abilities to predict
the in-use potential for consumer contamination; both are
modifications of the CTFA method. One of these, by
272 Applied Microbiology: Industrial | Cosmetics Microbiology
Lindstrom in 1986, used a retrospective validation proto-
col. The other, by Brannan et al. in 1987, used a
prospective validation protocol.
In the 1990s the focus was on harmonization of stan-
dards due to the effects of the European Community open
market in 1992. Uniformity of test methods, manufactur-
ing practices, and labeling requirements became more
important, once the US companies realized that poten-
tially global concerns (e.g., environmental and public
health) superseded regional ones. From a Darwinian per-
spective, these ‘altruistic’ actions were, nevertheless, still
self-serving since they permitted living within what was
increasingly becoming a global community.
Future Expectations
The twenty-first century finds us facing a sea change. No
longer is the 150-year-old agar plating hegemony of the
past necessarily affecting cosmetic microbiology as a
whole – we have begun to assimilate the molecular biol-
ogy revolution. Kuhn’s ‘paradigm shift’ is, once again,
potentially proving valid. Rapid methods (e.g., based on
ATP bioluminescence) have reduced confirmation of
plate counts from 5 days to 48 h or even possibly less,
depending on process control. Even our responses to the
threat of bioterrorism may become a boon to our industry
by permitting release standards which approach those of
‘real-time’. And our traditional 28-day PCT could be
drastically reduced in time, and far more insightful, by
advances using bioluminescent challenge organisms.
Sanitary Manufacture
Good Manufacturing Practices
Despite this major molecular shift, the principles of sani-
tary manufacture of cosmetics remain the same in
preventing microbial spoilage and protecting the consu-
mer from potential pathogens. These principles allow
cosmetic manufacturers to voluntarily meet the appropri-
ate sections of current food and drug regulations (21 CFR
x 110, 210, 211). Congress established the requirement for
Good Manufacturing Practices (GMPs) in 1969 and
revised them in 1980 for drugs (‘The Act’).
Even though the GMP rules define FDA’s expecta-
tions of manufacturers of food, drugs, and medical
devices, they provide guidelines for cosmetic manufac-
turers to voluntarily follow to produce ‘in control’
products. The Act states that such companies must pack,
hold, or prepare such products under conditions where
they cannot become contaminated. This requirement
typically implies suitable premises, equipment, raw mate-
rials, record keeping, sample retention, stability testing,
environmental control, and trained personnel who
operate using approved procedures. Companies who
manufacture their products under conditions whereby
they may have become contaminated or where potential
contamination was not prevented are subject to fines,
recalls, and even seizure. Note that it is only the potential
for contamination (or even just the perception of the
potential for contamination) that is required for legal
action to take place. Proof of contamination, or the pre-
sence of microorganisms, is not required – only the
perception of unsanitary manufacture is required. The
microbiological deficiencies most cited by the FDA
when carrying out a facility inspection are citations for
water contamination, inappropriate or absent testing of
final product, and lack of validated methods or validation
records.
Even though these regulations are voluntary for cos-
metics, most reputable companies follow them, especially
those companies who manufacture products that are
somewhere between cosmetics and drugs (e.g., antiper-
spirants, dandruff shampoos, and ‘cosmeceuticals’).
Water
Cosmetic plants classify water as ‘raw’ or processed. Raw
water comes from either the city or the plant’s own well
for use in personal hygiene, cooling, toilets, or drinking.
Processed water is water for making product. This water
is either softened, deionized, distilled, or reverse osmosis
treated.
A variety of organisms like Escherichia coli and
Pseudomonas spp. can contaminate raw water. In storage
tanks the numbers of bacteria may rapidly increase to
1  106 colony forming units per milliliter (CFU ml1).
This occurs due to chlorine depletion particularly during
warmer months. Softened and deionized water also con-
tain Gram-negative bacteria. In brine regenerated resins,
Bacillus and Staphylococcus spp. also may grow. These bac-
teria are in raw water at low levels, but then multiply
rapidly in the ion exchange resins that serve as fluidized
bed bioreactors. Distilled and reverse osmosis water is
free of microbes as it leaves the still or membranes. It
rapidly becomes contaminated with Gram-negatives in
storage and distribution. This contamination occurs
because of microorganisms that grow back from the var-
ious outlets to enter the storage tank and grow throughout
the water system.
Biofilms then form on the surfaces of the tanks, pumps,
and pipes of the water distribution system. This coloniza-
tion provides a microbial reservoir that contaminates
the water passing through. The shear force of the passing
water causes intermittent biofilm sloughing. Nonattached
microorganisms also may grow in unused sections of the
pipelines called dead legs. Turbulence of the water pas-
sing by carries the organisms into the main water line.
 Applied Microbiology: Industrial | Cosmetics Microbiology
Raw Materials
Most raw materials used in cosmetics are dry powders,
natural gels, or surfactants. A few examples of these are
talc and quaternized clay, aloe vera, and ammonium
lauryl sulfate (ALS). These illustrate the key concerns
and ways to handle raw materials. In dry powders and
natural gels like aloe vera, the primary contaminant is a
spore-forming Bacillus or Clostridium spp. In surfactants, a
wide variety of Gram-negative bacteria may grow.
In thickening agents and talc, the spore formers begin
growing during wet portions of their manufacture.
Fortunately, most of the spore-forming organisms involved
are nonpathogenic Bacillus spp. Washing the talc provides
an ideal setting for spore-forming bacteria to grow in the
moist powder. Drying of the talc preserves the spores. The
microbial content will be low if one reduces the process
time. One can cool the moist powder to below 25  C or heat
it above 45  C until the drying step, but the energy costs to
do so are high. One highly effective means of eliminating
spores is the use of gamma irradiation. Manufacturers refer
to irradiated quaternized clays and talc as ‘cosmetic grade’.
Although the only issues associated with use of irradiation
are for foods, one should be cautious of the regulatory
considerations surrounding the use of irradiated raw mate-
rials in cosmetics.
Aloe vera is also notorious for harboring spore formers.
Manufacturers harvest aloe in dry dusty areas. So it has a
high bioburden of spore-laden dust. Other organisms that
contaminate aloe are Erwinia and Pectobacterium spp.
Manufacturers pasteurize aloe once. This only destroys
vegetative cells. Since food manufacturers also use aloe,
they use food grade biocides. These are not sporicidal.
Therefore, it is common to receive aloe with counts as
high as 105–106 CFU ml1.
The solution is Tyndallization – double or even triple
pasteurization. However, slow cooling between each pas-
teurization provides a warm environment (25–45  C)
permitting growth for periods of 4–8 h. This can actually
compound the problem because the spores that survive
can germinate and grow forming even more spores.
Germination without growth is desirable. This allows
the next pasteurization to work against vegetative cells.
Thus, one should flash pasteurize the aloe, cool it rapidly,
and repeat within 24 h to get a microbially free product.
Surfactants are difficult to keep uncontaminated. The
exceptions are ammonium xylene sulfonate and sodium
lauryl sulfate. These are hostile due to pH extremes and
usually require no preservation. The only precautions are
keeping the domes of the storage tanks free of condensation
as this is where microbes grow. When the condensate drips
down onto the surfactant, it spreads microbes as a thin film.
Use of circulating fans on the tank domes controls this
condensation. The fans force air across dust filters and UV
lights and then circulate it across the top of the dome.
273
Other surfactants, like ALS, are highly susceptible to
microbial attack and require preservation. Some companies
use isothiazolinones (Kathon) at 5–10 ppm. Formaldehyde
at 100–150 ppm is also effective. This preservative can fail,
however, since many organisms easily develop formalde-
hyde tolerance. If the manufacturing stream is not
scrupulously clean, surfactants can become contaminated,
regardless of preservation.
Surfactants support a succession of microbes analogous
to the ecological succession of microbes in milk. Pseudomonas
spp. are the primary invaders. They have an inducible
formaldehyde dehydrogenase. However, Enterobacter or
Klebsiella spp. can also be primary invaders because of their
capsule-producing capacity. These organisms eliminate
formaldehyde either by enzymatic means or by nonspecific
reaction with polysaccharide capsular material. Several
organisms succeed Pseudomonas or Enterobacter. Serratia sp.
is the first. The surfactant may even turn pink. Proteus sp.
then follows. It reduces the sulfate portion of ALS to hydro-
gen sulfide.
Thus, one should know what the primary invader
species is to solve these problems. If it is Pseudomonas
spp., one will have to use a completely different preser-
vative in the surfactant. If it is Enterobacter, simple addition
of EDTA at 0.05–0.1% will be enough to destroy the
capsule. This allows formaldehyde to penetrate the cap-
sule and kill the organism. A simple capsule stain and
oxidase test can save countless hours of preservative
development.
Personal Hygiene
Topics to cover when training for hygienic manufacture
include personal hygiene, operator-borne contamination,
and ways of preventing cross-infection. Personal hygiene
includes washing hands, wearing clean clothing, and
keeping hair/beards covered. For aseptic manufacturing,
the manufacturer may use presterilized single-piece
suits, foot coverings, hair and beard covers, face masks,
and gloves.
Washing the hands often is the most important and
inexpensive way to prevent contamination. Employees
should not wear jewelry. They should wash their hands
every time they leave and return to the process area. Even
a brief 15-s handwashing period will cause significant
drops in microbial numbers compared to unwashed
hands. However, a thorough scrubbing with warm water
(32–43  C) and bactericidal soap for at least 1 min is best.
It is aesthetically displeasing to the consumer to find
human hair in a product. To prevent such contamination,
hair and beard coverings should be in all areas of manu-
facture. Employees should wear disposable hair covers
that completely block all hair from exposure. Personnel
should put on their hair cover first and then wash their
274 Applied Microbiology: Industrial | Cosmetics Microbiology
hands. The employee replaces the hair cover each time he
leaves and returns to the work area.
Clothing must be clean and without adornment.
Uniforms are good means of controlling compliance and
actually help promote good attitudes toward sanitation.
Light-colored uniforms or lab coats show a need for
cleaning much earlier than dark-colored ones. Pockets
on the uniforms should have button-down flaps. This
prevents materials in them from falling into the product
or process machinery.
Sanitary Design of Buildings
Roominess, simplicity, bright lighting, no clutter, and
even a fresh clean smelling environment will give a posi-
tive perception of cleanliness. This perception creates the
right attitude within the organization to promote compli-
ance with sanitation rules. The FDA regulations written
for food and drug plant design are often used by most
reputable cosmetic companies as a model since there are
no cosmetic GMPs. These regulations require adequate
and separate space for equipment and materials storage.
They require separate areas for operations that could
contaminate the cosmetic. Also required are adequate
lighting, ventilation, plumbing, and protection from pests.
The grounds surrounding a well-designed plant
should be neat. Keep decorative landscaping features –
such as ponds, fountains, and sites that provide nesting for
birds and rodents – at least 30 ft away from the building. A
3-foot perimeter of gravel should surround the building.
Trim all trees, shrubs, and lawns to avoid insects that may
find harborage in the long grass and unkempt shrubbery.
Keep the driveways, docks, refuse sites, and parking lots
free of debris. Also, keep these areas drained. Use a
perimeter fence around the plant grounds to filter paper
and debris.
The building should be a simple, box design with no
adornments, ledges, or architectural details to encourage
bird nesting. Instead, it should have coved or sloped
ledges. The exterior walls and foundations should have
no cracks or holes. Avoid porous and cracked walls. Avoid
or at least caulk baseboards so there are no spaces that
harbor insects. Roofs should have positive slopes of 1 inch
per 8 ft and have exterior drain spouts to take water away
from the perimeter of the building. Screen all vents on the
roof.
Ceilings should have limited overhead pipes. These
trap dust and provide pathways for insect movement.
Either cove, round, or slope overhead beams to ease
cleaning. Give inside and outside ledges on windows a
20 slope. The ceilings should be easy to clean, nonpor-
ous, and painted with epoxy paint. A plant may use
suspended ceilings if inspected regularly. Use load-
bearing walls instead of columns on the interior of the
building. If using columns, they should be cylindrical or at
least sloped at the floor to help cleaning.
Floors should be impervious to water, free of cracks
and crevices, and resistant to chemicals. Tiled floors are
desirable but expensive. Concrete is satisfactory but must
be water sealed. Surround edges of upper mezzanines and
wherever pipes pass through the floor with 4 inch curbs or
sleeves. This prevents water from passing to the processes
below. Keep floors dry. In areas where water is frequently
on the floors, use epoxy or urethane coatings on the
concrete to prevent water saturation. Provide adequate
drainage by using trench drains set in floors sloped at 1/8
to 1/4 inch per foot. When using circular floor drains,
place them every 400 ft2. However, it is typically difficult
to direct water toward these types of drains. Drains and
troughs should drain well since moist areas provide ideal
harborage for insects. Screen all floor drains to keep
rodents from entering via sewers.
Loading docks should be at least 3 ft above grade, and
entrances into the plant opened only when needed.
Screen all windows and doors with 16-mesh screen or
keep them closed. Doors should fit well, have automatic
closures, and be made of metal. Protect large openings
with air curtains. These should sweep air from the top to
the bottom of the opening at a rate of 4400 ft3 min1 and
at a 25–30 angle. Equip ventilation systems with high
efficiency particulate air (HEPA) filters capable of
removing 90% of particles that are 1–5 mm in diameter.
Provide handwashing facilities in the rest rooms and
near entrances to the manufacturing area itself. Surgical
style washing facilities are showy but superfluous and
expensive. Instead they should be easy to use, easy to
find, convenient, and accessible. They should be at least
8 ft away from any process stream to prevent contamina-
tion of the product.
Sanitary Design of Equipment
Manufacturers of cosmetics rely on sanitary design of
equipment developed by food sanitarians. Their standards
for sanitary equipment design apply to cosmetic equip-
ment design. Engineers should design equipment with
sanitation in mind.
The material used for pipelines should be smooth to
prevent biofilm formation. Stainless steel such as AISI 302
or 316 with sanitary pipe junctions (dairy fittings) should
be used. Pipe interiors should be smooth without rough
seams. Keep bends smooth and rounded without sharp
right angles. The center-line radius of pipe bends should
never be less than the outside diameter of the pipe. Slope
all pipes 1/8 inch per foot away from tanks to permit
proper drainage. Avoid sags or depressions that will trap
stagnant fluids. Plastic or polyvinyl chloride (PVC) pip-
ing is undesirable.
 Applied Microbiology: Industrial | Cosmetics Microbiology
Cosmetic plants should use only diaphragm, plug
cock, or butterfly valves. Each of these has advantages
and disadvantages. The key concern is that disassembly is
easy to permit cleaning. Washers, O-rings, and diaphragms
provide microenvironments particularly susceptible to
microbial colonization. Ones made of silicone rather than
natural rubber are more resistant to microbial growth.
Use tanks made of stainless steel. Interiors should have
no sharp corners to complicate cleaning. Welds should be
flush and ground to 120 grit compatible with the surface
finish of the rest of the tank. Tank roofs should be domed
and their bottoms rounded. Openings into tanks should
have protective lips surrounding them. Hatches, covers,
and lids should be overlapping to prevent debris from
entering into the tank. Protrusions into the tank such as
thermometers, pressure sensors, and spray balls should
form smooth welded junctions. Viewing ports are flush
with the tank interior. Never place a well or depression
anywhere in a tank. These trap stagnant product.
Seal all bearings on stirring devices and keep them oil-
free. Drains should be flush with the tank wall and ground
smooth. Design inlet pipes to the tank with air gaps to
prevent back siphonage. Fit them with flared protective
shields directly above the gap to prevent contamination
from the environment. Never seal pipes into the top of the
tank. They will serve as reflux columns to permit microbial
growth in the condensate that drips back into the tank.
Peristaltic and diaphragm pumps are more sanitary than
rotary positive displacement or reciprocating pumps.
Pumps should be self-draining and free of pockets or
crevices that trap product. The most important criterion
for pumps is that they are easy to disassemble and clean.
Pumps bolted together require tools to take them apart, so
personnel will clean them less. Clamped pumps are more
amenable to cleaning. Product contact parts should be
stainless steel and without bearings, which directly contact
the product stream. Rotors attached to their shafts – only
by pressure contact – are superior to those that use bolts,
cotter pins, or hexagonal screws. Pipes entering and leaving
pumps should be in smooth curves. Allow plenty of space
around the pump to help cleaning and maintenance.
Gaining Employee Commitment
The only way to get employees involved in sanitary man-
ufacture is to get the managers involved. If the employees
see the managers wearing hair coverings and washing their
hands, then the employees also will obey the rules. People’s
communal instincts will compel them to cooperate when
their superiors set the pace. One achieves cooperation
through education, training, and dedication to a sanitary
program. Achieving this dedication is sometimes not possi-
ble until people experience the negative impacts of ignoring
sanitary manufacture. Unfortunately, management views
the microbiologist as a roadblock to profitable manufacture.
275
This attitude changes rapidly when consumer complaints,
regulatory action, or the expense of scrapping product
occurs. Sometimes, this is the only way to gain the attention
and cooperation of an uneducated management.
The ideal situation is to have everyone cooperate at the
start. This occurs best with an organization scheme where
the microbiologist reports to the quality assurance (QA)
manager. The QA manager reports directly to a vice pre-
sident. The production manager reports to a manufacturing
manager who also reports to a vice president. It is unwise to
have the production manager and QA manager report to the
manufacturing manager. The QA manager has the respon-
sibility for good quality product; the production manager for
producing it profitably. Short-sighted individuals view these
two goals as conflicting. Some cannot see that long-term
insanitary manufacture is typically far more expensive than
maintaining a quality product.
The ongoing commitment of managers, and their
employees, to proper sanitation is facilitated by an active
and knowledgeable QA manager. Typically, the most suc-
cessful QA functions are those where the QA manager’s
reporting relationships are independent of day-to-day
manufacturing needs. This allows the QA manager to
direct appropriate activities such as employee practices
and training and product testing with the primary objective
of maintaining product quality.
A corporate microbiologist is also needed to enforce
QA and to audit a plant’s conformance to GMPs. The
corporate microbiologist is responsible for problem-
solving support, developing test methods, and preserva-
tion systems for products. A company concerned with
total quality allows the plant microbiologist and the
corporate microbiologist to communicate directly with
each other. Plant management may think this communi-
cation leads to whistle-blowing about sanitation problems.
As a result, the microbiologist frequently is accused of
‘not being a team player’. Actually, this is one of the finest
accolades for a microbiologist. One knows he/she is doing
the job right when he/she is not popular.
The corporate microbiologist is also responsible for
training and education. This can be through classes with
outlines and books, or clever use of visual aids such as
posters and films. Management and employees should
attend these training sessions. Training should cover the
regulatory requirements for clean manufacture and the
illnesses that can result from products contaminated with
microorganisms. This training includes personal hygiene,
good housekeeping/sanitation, and sanitary equipment
design. Training is effective only if the employee is
given valid reasons for the hygienic principles taught.
This means he/she will need a basic understanding of
microbiology without a scientific, esoteric approach. The
rapid nature of microbial growth, their ability to adapt,
their small size, and their ubiquity should be taught in
easy-to-understand terms.
276 Applied Microbiology: Industrial | Cosmetics Microbiology
Sanitization
Several elements of effective cleaning and sanitizing are
important in cosmetic microbiology. These include the
types of cleaning and sanitizing agents used, the kinds of
equipment, the type of soil to be removed, when to clean,
and how frequently to clean. Typically, cleaning and
sanitizing are done at the end of a production run. Other
times for cleaning and sanitizing may be after a set num-
ber of manufacturing hours or between shift changes.
Base the choice of when to clean and sanitize on facts
rather than convenience. Such facts are gained by mon-
itoring the system for microbial contamination during
production. The first detection of microbial counts is
the maximum length of time to go between sanitizations.
Cleaning
Immediately before washout, one should pump the system
free of all product. Sometimes a ‘pig’ (a foam rubber bullet)
is air blown through long lines to remove traces of product.
Detergent-based cosmetics, like shampoos, rarely use
cleaning agents. Typically, the system is only washed out
with hot water followed by sanitization. Use detergents or
caustic materials for cosmetics with an oily nature, emul-
sions, and powders. These lift and suspend the oily portions
by reducing interfacial and surface tension.
Factors that govern the choice of cleaning agent are
whether it is safe and effective, nondamaging, and com-
patible with the formulation. Alkaline cleaning agents
remove the lipid portions of cosmetics. Other cleaning
agents for mineral scale or very fatty materials may be
acid or solvent based, respectively. For heavy-duty clean-
ing, use concentrations as high as 2000–3000 ppm.
Concentrations of solutions for clean-in-place (CIP)
detergents range from 1000 to 1500 ppm. Chlorinated
cleaners enhance cleaning but are not sanitizers. Many
companies sell what they call chlorinated sanitizing clea-
ners. Cleaning agents work well at a pH >9. Chlorine
works well at a pH range of 4–7. Mix them and you have
either an ineffective cleaner or an ineffective sanitizer.
The water used for cleaning is as critical as the cleaner
used. It should be potable, low in hardness, and hot (70  C)
for fatty residues or at least warm (43–54  C) for most other
cleaning operations. Control of hardness can be done
using softeners or ion exchange columns. Check and ser-
vice these routinely so that high bacterial counts
(>100 CFU ml1) are avoided. Alternatively, one can
use detergents with organic chelators or phosphates.
Regardless of cleaning agent used, make routine checks
for microbial growth. Quaternary detergents are especially
susceptible to pseudomonads, but all detergents can sup-
port growth if not frequently changed.
Use physical methods to clean – especially hard to
clean systems. Probably the most effective physical
method is scrubbing by hand with a brush and detergent
in water solution. Use plastic brushes with synthetic
fibers, never use sponges and rags. Dry using air rather
than with drying cloths. Unfortunately, this is labor inten-
sive, costly, and dependent on the attitude of the one
doing the scrubbing.
Alternatives to hand scrubbing are use of high pressure
cleaning and CIP methods. High pressure spray systems
can deliver from 500 to 1000 psi. CIP systems are by far
the most popular methods. These are line loops connect-
ing the various tanks and equipped with a CIP pump that
can deliver 2–3 gallons min1 at 400–800 psi. Cleaning
agent, sanitizer, or rinse water should be pumped through
the system. High flow rates (four to five times the product
flow rate) provide shear that will strip biofilms from the
inner pipe surfaces. Locate spray balls on the top of tank.
Assure that they rotate with the pressure of the solution so
the spray reaches all points inside the vessel. The critical
points to check are the shear flows and that the cleaning
agent and the sanitizer contacts every surface.
Sanitizing
For some cosmetics, a thorough cleaning of the manufac-
turing equipment will be adequate to provide microbial
control. Microorganisms are controlled by physical
removal or by removing the nutrients required for growth.
Some cosmetics require that the manufacturing equipment
is sterile.
The perfect sanitizing agent should be safe for use by
the plant personnel and act rapidly under conditions of
use. It should not interact with the cosmetic or leave a
residue that could interact with it. The agent should be
easy to use and inexpensive. Of the various types of
chemical sanitizers, none meet all aspects. The major
types of chemical sanitizers available for use in cosmetics
are halogens, quaternary ammonium compounds (quats),
phenolics, aldehydes, and alcohols.
The most commonly used halogen in the cosmetics
industry is chlorine in the form of hypochlorous acid
(HOCl). A variety of chlorine sources exist including
sodium hypochlorite, calcium hypochlorite, chloramines,
chlorocyanurates, and gaseous chlorine. Use these at con-
centrations that will provide from 100 to 200 ppm
available chlorine. The pH of the chlorine solution should
be at or just slightly below 6.5. A pH below 3.5 may
corrode the metal or even give off chlorine gas. One
should assay used solutions after a sanitization for free
chlorine. Significant drops to <20 ppm show the system
was improperly cleaned and contained materials that
provided a chlorine demand. In this case, repeat the
cleaning and sanitization before production begins.
 Applied Microbiology: Industrial | Cosmetics Microbiology
Cosmetic plants use glutaraldehyde as a disinfectant
rather than formaldehyde due to the potential carcino-
genicity of formaldehyde vapors. Glutaraldehyde used at
2% and buffered to pH 7.5–8.5 is active against Gram-
positive and Gram-negative bacteria, fungi, viruses, and
spores. It acts within 10 min. Formaldehyde used as a
preservative is not carcinogenic, but some consumers
have a negative perception of its use.
Quats are cationic surface active agents that are parti-
cularly effective against Gram-positive bacteria but
ineffective against Gram-negatives. In fact, dilute in-use
solutions serve as selective media for Pseudomonas spp.
Plants use quats to sanitize small coupling pieces, asso-
ciated piping, and exterior surfaces of tanks. Make and use
the solutions daily to avoid adaptation problems. One
advantage to quats is that they are substantive and so
provide residual antibacterial activity on treated surfaces.
Apply quats at 500–1000 ppm without rinsing. They are
most effective at higher temperatures and at pH 10.
The cosmetics industry uses phenolics only for floor,
wall, and ceiling disinfection. The activity of phenolics
diminishes markedly in the presence of organic material
on surfaces. Therefore, clean the floors with an alkaline
detergent and rinse before sanitizing. Phenolics are most
effective at acid pH. They are effective against vegetative
bacteria and molds.
Use alcohols as surface disinfectants for tank tops and
other working surfaces around packing lines. The most
commonly used alcohols are ethanol (70% aqueous) and
isopropanol (50% aqueous). Due to volatility, their action
is brief and limited to vegetative cells.
Disinfectant suppliers have tried developing detergent–
sanitizer combinations. Mixtures of quats and nonionic
detergents, or solutions of anionic detergents plus chlor-
ine-releasing compounds are most common. More often
than not, the combination results in either an ineffective
cleaner or an ineffective sanitizer. Rarely are the two com-
pletely compatible with each other. The better approach is
to apply a cleaning compound to the system followed by a
rinse and then the sanitizing agent.
The most effective sanitizing agent is heat. Exposure of
the making system to 180  F (82  C) for 15–30 min is
effective. The advantage in using heat over chemical
sanitizers is that heat penetrates the biofilm where che-
mical sanitizers do not.
It is critical to design water systems that permit effec-
tive sanitization. Holding tanks and associated piping
should be stainless steel. Avoid using pipes made of cop-
per or galvanized piping. Never use PVC or black iron
pipe. Water lines entering tanks should not permit back
siphonage of the tank contents into the water system. This
is easily done by providing a shielded air gap. Prevent
back siphonage in the system when pressure drops in the
water line by avoiding cross-connections. Construct
277
filters in the water system in parallel. This allows a filter
needing service to be isolated from the rest of the system.
Supply water treatment systems with chlorinated city
water. This treated water should go directly into the
stainless steel storage tanks and be periodically heated
to 180  F. Circulate the heated water through the system
and into the storage tank. An alternative to heat sanitation
of water is ozonation. Use ultraviolet lights at the point of
use to remove the ozone and further sanitize the water.
Preservation
Both clean manufacture and preservation should compli-
ment each other. One should never use preservation to
mask unsanitary manufacture. Instead, preservation is
primarily a way to protect the consumer during use of
the cosmetic product. In addition, preservation is an aid to
extend the shelf life of the product.
Cosmetics are such complex products that their pre-
servation is more of a subjective art than an objective
science. There is such a complexity of interacting factors
in the cosmetic that using ones intuition to select a pre-
servative is often more successful than relying on isolated
facts. The formulation factors to consider when choosing
a preservative include pH, oil–water partitioning, and
interaction with raw ingredients. In addition, preserva-
tives may localize in an emulsion, and even react with the
container.
Some of the more common and effective preservatives
in use today are isothiazolinones (Kathon), hydantoins
(Glydant), imidazolidinyl or diazolidinyl urea (Germall),
oxazolidines (Nuosept C), formalin, and parabens. One
chooses a preservative based on effectiveness as deter-
mined in a PCT (described below). Other considerations
should be safety, compatibility with the aesthetics of the
product, cost, ease of formulation, and availability. Several
choices of preservative systems for a cosmetic should be
available in case tolerance to the preservative develops.
Some microbiologists like to use combinations of several
different preservatives; the logic is to prevent microbial
tolerance development to a preservative. This logic is
based on an assumption that preservative tolerance devel-
ops similarly to antibiotic resistance. This assumption is
false since preservatives often act on multiple nonspecific
targets as opposed to antibiotics that act on specific mole-
cular targets. This practice is also unnecessary in plants
where sanitation standards are high. It also can create more
safety risks and be expensive. If the microbiologist insists
on using multiple preservative systems, he/she would at
least be sure that the combination acts on different targets
entirely. For example, it makes no sense to use two differ-
ent formaldehyde donors since they both ultimately
work in the same way despite having two different chemi-
cal formulae.
278 Applied Microbiology: Industrial | Cosmetics Microbiology
HACCP for Cosmetics
Hazard analysis and critical control point (HACCP)
Analysis was established for regulated consumer products
(e.g., foods and drugs), not for cosmetics. Nevertheless,
many principles of HACCP may be voluntarily adopted
for cosmetics in order to maximize profits. HACCP,
whether it is adapted to foods, drugs, cosmetics, or any
consumer product is simply a formalized set of principles
that permit products to be manufactured ‘in control’.
Human nature desires predictability, reliability, and con-
sistency; HACCP allows these desires to be realized in a
rational and clearly understood format. Regardless of
product form, seven basic steps for conducting HACCP
are (1) determine hazards and preventative measures that
may eliminate or control that hazard; (2) determine cri-
tical control points for each hazard; (3) establish critical
limits for each control point; (4) establish an ongoing
monitoring program; (5) know what actions you will
take when each critical control point is beyond predeter-
mined limits; (6) establish documentation procedures; and
(7) monitor and reevaluate the program systematically to
ensure validity.
In order to prevent costly product contamination by
microbes (in some cases, $10MM) and ensure consumer
safety, HACCP can be used to ensure – via QA – that only
low levels of transiently surviving microbes exist in the
manufacturing environment. Sterility is not cost-effective
in a cosmetic plant. Consequently, the goal should be no
more than these low levels of transient microbes. However,
without HACCP, one can expect either high-level popula-
tions of nonsurviving microbes or low levels of survivors.
These two situations are rife for the development of pre-
servative-tolerant microbes; the latter situation is typical of
quality control rather than assurance. In such situations,
process control and product preservation are relied upon to
control the high levels of nonsurviving microbes. But this
situation will rapidly turn into the third possibility of
preservative-tolerant microbes at low levels of survival
and then progress into high levels of the same. At this
point, the company is faced with product recalls. The key
difference between the QA, that HACCP offers, and qual-
ity control is that test sensitivity, sampling frequency, and a
variety of control parameters have simply not been well
thought out.
The idea that quality control, as contrasted with QA,
can be effective ignores the reality that microbes are
incredibly adaptive – they evolve rapidly. Microbes are
well known for their ability to grow in the presence of
preservatives if sufficient numbers are allowed to be con-
stantly exposed to any selective environment, like a
preserved product. Once manufacturing has reached this
situation, the best it can hope to achieve is finished
product quality by testing which units are contaminated
and which are not. Considering the thousands of units
produced per hour, the statistical validity of detecting
contaminated units is nil, until a consumer is adversely
affected. By then, one is faced with at least a Class II
recall.
Alternatively, a complete HACCP program may be
established by evaluating even systems peripheral to pro-
duction: water systems, environmental and compressed
air systems, steam and waste water removal systems.
Other areas to be assessed are those associated with for-
mulation: raw materials, premix/intermediate, and
finished product. Analysis of the production system
includes hold and processing tanks, delivery and transfer
lines, bulk storage and filling/packing transfer, and pro-
cess systems. Some of the key hazards to look for are: areas
where liquids may remain stagnant, equipment that traps
product within it, wherever sites cannot be drained com-
pletely, areas where heat sanitization cannot penetrate,
areas that cannot be cleaned and sanitized with chemicals,
wherever human intervention is required to accomplish
sanitization, and any process that leaves pathways idle
where product can remain stagnant. As applied to both
active and dormant manufacturing systems, HAACP
serves as the most effective means of quality assessment.
Test Methods
There are two major test methods for the cosmetic micro-
biologist: the PCT and the Microbial Content Test
(MCT). All the other test methods are variations of
these two basic tests. In addition, the microbiologist
needs to know the basic microbiological techniques that
support the conduct of these two tests.
Preservative Challenge Tests
The basic way to conduct a PCT is simple. Inoculate a
product and see how long it takes to eliminate or reduce
the inoculum to an acceptable level. Variations of the
PCT include its adaptation for use in water miscible
and immiscible products, and eye area products.
Additional variations are those designed by several
groups such as the United States Pharmacopoeia (USP).
The AOAC is developing a standardized PCT in colla-
boration with CTFA and the FDA. As opposed to the
USP, who set PCT methods based on collective anecdotal
experiences, the collaborative work by the AOAC/
CTFA/FDA based the test on rigorous scientific testing.
Unfortunately, the test will still not predict consumer
contamination. No test can do this since there are too
many variables to predict.
There are many variations to this simple technique.
Some of these variations are: which organisms to use,
concentration of the organisms, single versus multiple
 Applied Microbiology: Industrial | Cosmetics Microbiology
inoculation, and how often or how long to follow the
elimination process. Other variations include if the pro-
duct should be diluted, if the challenge organisms should
be a mixed or pure inoculum, and what diluents/plating
media are best.
The most used PCT tests are summarized here. For
additional information on these tests the reader should
refer to the section ‘Further reading’. The most used PCT
methods are those of the CTFA, and the Society of
Cosmetic Chemists of Great Britain methods for cos-
metics. For pharmaceuticals, the USP and the British
Pharmacopoeia (BP) have developed PCT methods.
Other PCT methods are the ones being developed by
the AOAC, and a variety of rapid test methods that are
simple adaptations of D-value methods to cosmetics.
Nearly all the methods include Pseudomonas aeruginosa,
Staphylococcus aureus, Candida albicans, and Aspergillus niger.
In addition to these, USP includes Escherichia coli. CTFA
includes E. coli, Bacillus subtilis, Penicillium luteum, and
spoilage isolates. Only the Society of Cosmetic Chemists
of Great Britain (UK test) makes specific inoculum
recommendations for each type of cosmetic product.
The levels of inoculum range from 105 to
6
10 CFU per ml or gram and a single challenge for the
USP test to 106–107 CFU ml1 with several challenges for
the UK test. Interpretations of the tests also vary. The
CTFA test makes no formal recommendations but
requires that the test continue for at least 28 days. The
USP test requires a 3-log reduction of bacteria within 2
weeks. Yeasts and molds should remain at or below the
initial count. Over the 28-day period, all organisms are to
remain at these levels.
The BP test requires that a 3-log reduction of the
bacterial load occurs. It requires a 2-log reduction of
yeasts and molds within 7–14 days. The BP test expects
shampoos to be ‘self-sterilizing’ in 7 days, creams and
lotions to ‘show drastically reduced counts’, and eye cos-
metics to be ‘bactericidal to P. aeruginosa’.
Criticisms of these tests are improper selection of
challenge organism, microbial load of the challenge, num-
ber of challenges, and the end points stipulated. The
FDA’s chief criticism is that PCT methods do not predict
if consumers can contaminate the product during use. As a
result, Brannan in 1987 and Lindstrom in 1986 developed
methods that were predictive of consumer contamination
potential. These are the only methods with published data
to validate their ability to predict the potential for con-
sumer contamination.
Microbial Content Tests
These tests detect the numbers and sometimes the types
of organisms present in the cosmetic. Most tests for
microbial content are plate counting methods. However,
one can use other techniques as well.
279
Plate counting methods are also known as aerobic
plate counts (APC) or total plate counts (TPC). In plate
count methods, use an appropriate diluent to neutralize
the preservative and any other antimicrobial ingredient in
the cosmetic. A variety of neutralization agents are avail-
able. Not all are effective or nontoxic to the organism
needing detection. If neutralization via some agent is not
possible, then one should use physical dilution or mem-
brane filtration. Similarly, the medium for plating the
product is critical for accurate recovery of the microor-
ganisms potentially present in the product.
Use either spread plates or pour plates. Use as low a
dilution as possible. For example, a 1:10 dilution of the
product will yield a better detection limit than a 1:100
dilution. Once inoculated into the agar plate, incubate for
1–5 days. Count the viable colonies that form and record the
number as CFUs. There are several sources of error using
this method. The biggest error is that no single medium will
detect all types of microbes in all types of products.
Other methods for enumerating microbes in cosmetics
are the direct microscopic count, the most probable number
(MPN), radiometric methods, and impedance measure-
ments. In the direct microscopic count, one spreads a
known amount of cosmetic on a slide and counts it. The
advantage of this technique is that it is fast. It counts both
living and dead cells, and is only good for heavily contami-
nated products. Also, the products interfere with seeing
the microbes and the technician will be highly susceptible
to fatigue.
In the MPN technique, the technician places various
dilutions of the product in replicate tubes of nutrient
medium. He then compares the number of replicates
showing growth to a standard MPN table. From this, he
estimates the number of bacteria in the sample. This
method is useful when the organisms do not grow well
using standard plating techniques.
Radiometric methods rely on the organisms present in
the product to degrade a 14C-labeled substrate into
14
CO2. The 14CO2 evolved is related to total microbial
levels in the product. The radiolabeled substrate used is
typically glucose. Use 14C-glutamate or 14C-formate
instead of 14C-glucose to detect Pseudomonas. The major
drawback of this technology is the expense; however, it
offers rapid results (6–8 h) compared to traditional plating
methods.
Impedance measurement is a widely used method for
detecting microorganisms in cosmetics. It is especially
useful for following microbial activity during preservative
challenge testing. One also can use impedance methods to
approximate microbial numbers in finished product. The
method uses the ability of the growing microbial culture
to produce changes in impedance of electrical current as
it metabolizes the nutrients in the medium. This method
estimates microbial activity within 5 h. The major
280 Applied Microbiology: Industrial | Cosmetics Microbiology
drawback of this method is the cost and poor detection
limits: 10 000 CFU g1 of product.
Plate counts will always be the benchmark of quality
control in the cosmetic microbiology laboratory. They are
easy to perform, inexpensive, and result in number of
microbes to which most people can easily relate.
However, even plate counts do not accurately quantify
the numbers of microbes present. First, microbes typically
clump together. A CFU is likely not a result of a single
microbe but, instead, anywhere from one to several thou-
sand microbes.
Reliance on plate count methods should be reevalu-
ated. Regardless of method used, the key for microbial
monitoring is to show relative microbial levels and trends
during the process. One should base such monitoring on a
system in control. The data should be used to keep the
system within total quality limits. Since total quality
concepts and just-in-time production require rapid turn-
around, the use of rapid methods may one day replace the
time-consuming plate counting methods.
See also: Biofilms, Microbial; Food Spoilage,
Preservation and Quality Control; Sterilization and
Disinfection; Water Treatment, Industrial; Water
Treatment, Municipal
Further Reading
Ayliff GAJ, Bagg J, Baird R, and Fraise AP (2004) Russell, Hugo and
Ayliffe’s Principles and Practice of Disinfection, Preservation, and
Sterilization, 4th edn. Cambridge, MA: Blackwell Scientific
Publications.
Block SS (ed.) (2000) Disinfection, Sterilization, and Preservation,
5th edn. Media, PA: Lippincott Williams & Wilkins.
Brannan DK, Dille JC, and Kaufman DJ (1987) Correlation of in vitro
challenge testing with consumer-use testing for cosmetic products.
Applied and Environmental Microbiology 53: 1827.
Denyer SP, Hodges NA, and Gorman SP (eds.) (2004) Hugo and
Russell’s Pharmaceutical Microbiology, 7th edn. Oxford: Blackwell
Publishing Ltd.
Geis PA (ed.) (2006) Cosmetic Microbiology: A Practical Handbook,
2nd edn. New York, NY: Taylor & Francis.
Lindstrom SM (1986) Consumer use testing: Assurance of
microbiological product safety. Cosmetics and Toiletries 101: 71.
O’May GA, Allison DG, and Gilbert P (2004) A rapid method for
evaluation of both extrinsic and intrinsic contamination and resulting
spoilage of water-in-oil emulsions. Journal of Applied Microbiology
96: 1124.
Sapsford KE, Shubin YS, Delehanty JB, et al. (2004) Fluorescence-
based array biosensors for detection of biohazards. Journal of
Applied Microbiology 96: 47.
Troller J (1983) Sanitation in Food Processing. Orlando: Academic
Press.