PZ Cussons – Industrial Hygiene

Training

Module One

Microbiology & Contamination

Overview

Phil Greaves
BiotiQ Consulting
 Slide 2

Microbiology and Contamination Control

CONTAMINATION

Why does it matter?

 Slide 3

Products at Risk of Microbial Contamination

Medicines Foods Cosmetics

Contaminated Counterfeit
Chinese milk Heparin
blamed for
300,000 victims 200 deaths
Disinfectants Paint Pesticides Aviation Fuel
 Slide 4

Contamination Matters Because:

• Customers may get infected from using our products

• Some customers may be more vulnerable than the general population

• Contamination can cause spoilage of products during storage, distribution

and use
• Discolouration, odours, cloudiness

• It damages business reputation and costs a lot of management time!

• Product recalls are highly damaging

• Adverse publicity
• Very costly
 Slide 5

Consequences of Microbial Contamination

Multiple Deaths

Infections / Death of
High Risk Individuals
Contaminated
Chinese milk
300,000 victims
Commercial &
Reputational
Damage
 Slide 6

Contamination is Expensive!

Some of Phil’s recent work:

• Public recall of skin cleanser due to bacterial contamination. Lost batches

valued at £500,000

• Contamination and recall of 130 batches of new cosmetic and personal

care brand launch in Germany = €2 Million

• Contamination of sanitizer chemicals used to sanitize hospitals and

personal care plants! Factory shut down for over 6 months with lost sales
of €58 Million
 Slide 7

Microbiology and Contamination Control

TO PREVENT CONTAMINATION

WE NEED TO UNDERSTAND OUR

ENEMY!
 Slide 8

Understanding Micro-Organisms

Micro-Organisms:
•4 billion years of evolution = ability to grow on diverse carbon source
•Can exist in very extreme environments:
• Hyperthermophiles
• Anaerobes
• Acidophiles
• Methyotrophs
• Lithotrophs
• Grow in aviation fuel
• Spore formers
• Biofilm
 Slide 9

What are Micro-Organisms?

Understanding Micro-Organisms

Organisms that require a microscope to be seen – typically less than 0.1 mm (but
may grow to visible size)

True Bacteria Fungi Protozoa

Archae Bacteria Algae Viruses

 Slide 10

Understanding Micro-Organisms

Viruses:

• Particulate entities – ‘non-living’

• Typically 30 to 250 nanometers long
• Can only reproduce inside living cells
• Takes over cell function
• Important diseases of animals and plants
• Not significant to personal care products

• But can be highly disruptive to our

businesses and way of life!
 Slide 11

Understanding Micro-Organisms

Fungi (moulds):

• Non-photosynthetic, single or multi-celled

• Several micrometers to many metres long
• Live on organic matter, dead or living
• Tiny amount of water required for growth
• Produce large numbers of spores – often found in
wood, cardboard, dust etc.
• Important causes of disease in plants and animals

• Frequent cause of contamination of personal care

products
 Slide 12

Understanding Micro-Organisms

Bacteria

• Single celled, typically 1 to 5 µm long.

• Widespread on, under and above the earth
• Cause disease but most are non-pathogenic

• Different shapes
– Rods (bacilli)
– Spheres (cocci)
– Spirals (corkscrews)
 Slide 13

Understanding Micro-Organisms

Bacterial Spores

• Formed by Gram positive rods

– Bacillus, Clostridium
• Very resistant to drought
• Also very resistant to chemicals and heat
• Germinate in suitable conditions
 Slide 14

Understanding Micro-Organisms

‘Good’ Micro-Organisms:

• Most micro-organisms are harmless

• Commensals protect us from pathogens
• Break down waste & Sewerage
• Food Processing
• Genetic Engineering
• Our microbiome
 Slide 15

Understanding Micro-Organisms

‘Bad’ Micro-organisms:

• Spoilage
– Food
– Materials
• Contamination
– Pharmaceuticals
– Cosmetics & personal care products
• Toxins
 Slide 16

Understanding Micro-Organisms

‘Ugly’ Micro-organisms: • Fungal infections

• Viral Disease - Candida
– Hepatitis - Aspergillus
– Coronavirus - Cryptococcus
– Influenza • Protozoa
• Bacterial - Malaria
– Gangrene - Cryptosporidium
– Meningitis - Toxoplasmosis
– Whooping cough
 Slide 17

Conditions for Growth

• Many micro-organisms have very basic requirements for growth:

• Water – even low levels of moisture can be enough to support growth
• Carbon source – may be as simple as methane but more typically
complex molecules containing carbon
• Often the carbon source is biological – derived from plants or
animals
• Heat – typically in the range 15° to 35° C
• Growth will occur at lower temperatures – only slower
• Some species can grow at much higher temperatures
• Absence of growth inhibitors – chemicals that prevent growth e.g.
preservatives
 Slide 18

Conditions for Growth – Personal Care Products

Growth Requirement Product Manufacture

Water

Carbon source

Room temperature

Absence of growth
inhibitors
 Slide 19

Preservatives

• Many products ‘protected’ by a preservative

• Best preservatives (that are ‘guaranteed’ lethal to micro-organisms) tend to
be very toxic to people e.g. formaldehyde
• Very restricted use in EU, US and Japan
• Increasing trend to lower toxicity and ‘preservative free’ formulations

• Even good preservative systems can fail:

• Resistant bacteria or fungi
• High numbers of micro-organisms will ‘over-power’ the preservative

• Preservative may allow ‘survival’ but not kill micro-organisms

• Beware products that ‘kill 99.9% of all germs’. That still leaves 1,000 to
grow from an initial population of 1,000,000!

Common Personal Care Preservative Systems
Preservative System
Parabens (methyl-, propyl-,
butyl-parabens)
Formaldehyde donors
Phenol derivatives
Quaternary ammonium
compounds (QACs / Quats) e.g.
Benzalkonium chloride
Alcohols (phenoxyethanol,
benzyl, ethyl alcohol)
Isothiazolones
Organic acids (e.g. benzoic
acid, Triclosan)
Slide 20
Optimum pH range Comments
pH 3.5 – 6.5 Anti-fungal, high toxicity (?), volatile (loss
during manufacture)
pH 3 – 9 Anti-bacterial, less effective against fungi
(Bronopl 5 – 8) at use levels; potential carcinogenicity
pH 4 - 9 Anti-bacterial; potential odour
pH 4 - 10 Broad spectrum anti-bacterial and anti-
fungal; activity affected by water
hardness and anionic detergents
pH 3 - 10 Anti-bacterial, weakly effective against
fungi. Volatile and some safety concerns
with phenoxyethanol
pH 2 - 8 Broad spectrum against bacteria and
fungi; not accepted in Scandinavia
< ph 4.5 Anti-fungal; very pH dependant
 Slide 21
Bacteriostasis – Survival Without Growth
(until conditions are right!)

• Preservatives may kill micro-organisms (bacteriocidal) or prevent their

growth (bacteriostasis)
• Even bacteriocidal preservatives may allow survival if:
• Formulated incorrectly
• pH is outside optimum range
• Resistant organism or high population

• Bacteriostatic organisms may grow very slowly (e.g. doubling every 24 –

48 hours) or return to normal growth when conditions are favourable

• Bacteriostasis may result in number being below limit when product is

tested immediately after manufacture, but can result in unacceptable
contamination after several weeks slow growth
 Slide 22

How They Grow

• They don’t need sex!

• Simply divide into two – binary division
 Slide 23

Growth Rates

• Some species an replicate (double) every 6 – 8 minutes under optimum

conditions
• More normal rates are doubling every 20 – 60 minutes

If just 10 bacteria survive disinfection, and assuming a doubling rate of every

20 minutes, how many will there be after 8 hours?
 Slide 24

Growth Rates – Our 8 Hour Example

Time (Minutes) Number of Bacteria

0 10
20 20
40 40
60 80
80 160
100 320
120 640
140 1,280
160 2,560
180 5,120
After 8 Hours 83,886,080
 Slide 25

How They Grow

 Slide 26

Rapid Evolution

• Micro-organisms have enormous adaptive capacity

• Rapid exchange of genes
• High Mutation rates

• Highly capable of adapting and

developing resistance
e.g. MRSA
 Slide 27

Biofilm

• In natural environments, relatively few

bacteria (and fungi) occur as free
swimming (planktonic) organisms
• Micro-organisms prefer to attach to
substrates – offers protection from being
swept away
• Colonies can develop that are complex
communities of several interacting species
– a natural phenomenon called biofilm
• Secrete extracellular lipopolysaccharide
(slime) which protects the colony
 Slide 28

Biofilm in Manufacturing Environments

• Bacteria in production vessels, pipework or water systems are unlikely to

be planktonic (free swimming)
• Bacteria attach very rapidly to surfaces – by hydrogen bonding and with
attachment structures called pili
• Form thin surface biofilm on pipework, on filters, valve seats….anywhere
with a surface

• Difficult to detect – attached so not usually sampled (random high counts)

• Large numbers of organisms detached sporadically – ‘chunks’ of biofilm
slough off (sometimes as a result of a mechanical knock)
• Protected from many chemical disinfectants
 Slide 29

Bioflim in Manufacturing Environments

Even antimicrobial products can be overwhelmed!

Biofilm growth is favored by:
Plastic over stainless steel
Rough over smooth surfaces
Nutrient rich over nutrient poor conditions
(left-over product, raw materials (specific –
pH)
Sites where flow is slow or stopped:
Dead legs
Undrained hoses
Depleted tanks and totes that are not
fully emptied and dried
You don’t always see it … but sometimes you do!
We fight biofilm at home too:
Dental plaque
Dog dish
Humidifier
Shower
 Slide 30

Microbiology and Contamination Control

Microbiological Testing

and

Quality Control
 Slide 31

Microbiological Quality Control

• Testing usually performed on (some) raw materials, process water,

finished product
• Sometimes testing extended to vessels after cleaning, and people (finger
dabs)

• Testing performed using culture media – nutrients, growth factors and agar
(a gel)
• Solid culture media allows visible colonies to develop
• Shape, colour and smell of each colony helps in identification of the
species
 Slide 32

Microbiological Quality Control

• One cell produces one colony…..in theory!

• In practice, cells are rarely ‘free individuals’ but occur in clumps, either
attached to a substrate (skin flake, hair) or sloughed from a biofilm

• Therefore one colony may represent a relatively unknown number of

bacterial cells or fungal spores

• Microbiologists report results as ‘colony forming units’ or CFU

• Microbiology is not an exact science!
 Slide 33

Identifying Bacteria and Fungi

• Microbiologists need to know not only how many micro-organisms are

present, but also what species
• Some species are more likely to contaminate product, and some may
be harmful to customers

• Identification based on
• Gram’s stain – a staining process that results in violet (Gram positive)
or red (Gram negative) cells
• Use of selective culture media – allow only the problem species to
grow, or produce a colour reaction specific to selected species
• Biochemical tests
• DNA sequence
 Slide 34

Rapid Test Methods

• Rapid methods for identification (speciation)

• Genomic methods (DNA fingerprinting)
• MALDI-TOF (Matrix assisted laser desorption – time of flight) = protein
analysis

• Rapid methods for detection and enumeration (number)

• Early detection of micro-colony growth
• Detection of ATP (metabolic molecule only found in live cells) by
luciferase test
• Detection of whole living cells by UV-fluorescence of biomolecules
using flow cytometry
 Slide 35

Rapid Detection and Enumeration Methods

• Results are not reported as ‘colony forming units’ - CFU

• ATP test reported as ‘Relative Light Units’ - RLU
• UV-fluorescence as a ‘Relative Fluorescence Units’ – RFU

• RLU and FLU are NOT directly comparable to CFU

• Measuring quantity of biomolecules within cells
• Bigger bacterial cells – more biomolecule
• Different species of bacteria – different levels of biomolecules
• Different phase of growth – different levels of biomolecules
 Slide 36

Why We Cannot Rely on Testing

• We cannot test everything – number of samples tested is a very low

fraction of the total batch
• Sampling requires contamination to be uniformly present across batch
• Contamination of one bottle in 1,000 is 98% likely to be missed on
testing
• Sampling plans aimed to focus on parts of the batch that may be more
contaminated
• We cannot grow everything
• Some cells may be ‘stressed’ and difficult to grow…but can slowly
recover in product
• We may not be able to detect biofilm
• ‘Acceptable’ numbers may slowly grow to unacceptable numbers
 Slide 37

So Why Bother Testing?

• Trends are often more important than absolute numbers, especially in

water systems
• Rising trend may indicate that a failure condition is going to occur

• Provides data from which we derive knowledge about manufacturing

quality

• Final ‘link’ in our chain of microbiological quality assurance

• Design
• Procedure
• Monitoring
 Slide 38

Some Typical ‘Manufacturing Environment’ Bacteria

Organism Sources

Staphylococcus People – carried on skin flakes. Very

Micrococcus
Pseudomonas and Burkholderia
Bacillus
Fungi (Aspergillus, Penicillium etc)
common
People – carried on skin flakes. Very
common
Water, or any aqueous environment.
Chemically-resistant bacteria that can grow
in disinfectants
Spore-forming bacteria present in soils, and
plant-derived materials. Can be left over
from building work or carried in on footwear
Building works , cardboard

E. coli & ‘Coliforms’ Gram negative bacteria indicative of faecal

contamination e.g. bird droppings
 Slide 39

Microbiology and Contamination Control

How Does Contamination Happen?

 Slide 40

How Does Contamination Occur

• Remember the conditions for growth:

• Water
• Carbon source (product / product residues)
• Heat (room temperature)
• Absence of growth inhibitors
• Time

• Contamination occurs when one or more of these is increased more than

normal
 Slide 41

Contamination of Personal Care Products

Fortunately, very few micro-organisms are able to grow in personal care products:

Organism Sources
Burkholderia cepacia Raw materials, water, poor plant hygiene

Psuedomonas aeruginosa Raw materials, water, drains, poor plant

hygiene
Pluralibacter gergoviae Raw materials of plant origin, drains, poor
plant hygiene
Klebsiella species (e.g. oxytoca) Raw materials of plant origin

Fungi (Aspergillus, Penicillium etc) Environment (air) building works ,

cardboard
 Slide 42

Sources of Contamination – Raw Materials

• Not all raw materials are a contamination risk

• High risk materials:
• High water content
• Carbon source
• pH between 5 and 8
• Biological / organic origin
• Site of manufacture

• Risk ranking of raw materials

• Accreditation programme for new suppliers
• Very high risk materials tested routinely upon receipt
• High risk materials – supplier audit
 Slide 43

Source of Contamination - Water

• Process water must be treated to kill bacteria

• Contamination can occur when this treatment loses efficiency

• Low or very low levels of bacteria may survive the water treatment

• These bacteria will grow where water is stagnant – pipework deadlegs or when the system is
shut-down for maintenance
• Growth will be rapid and reach high concentrations of bacteria

• Contamination enters manufacturing lines when the deadleg is opened

 Slide 44

Source of Contamination - People

• People are the greatest source of micro-organisms in manufacturing

• 100,000,000,000,000 bacteria per person!
• 10,000,000,000 per gram of human poo
• 20% of women & 40% of men do not wash their hands after using the
toilet
• The number of bacteria on your hands doubles after using the toilet
• These bacteria survive for up to three hours
• We shed about 1,000,000 skin cells per day – 3.5kg per year
• Between 4 and 100 bacteria per skin cell

• Hygiene procedures (hand washing, protective clothing) should prevent

this contamination entering manufacturing lines….when we do it properly
 Slide 45

Source of Contamination – Poor Plant Design

• Some bacteria will always be present in manufacturing lines

• From raw materials, water and from the environment
• Problems occur when poor plant design allows these bacteria to grow

Common issues:
• Vessels and pipework that cannot be fully drained
• Pipework deadlegs that trap water or product
• Surfaces that allow bacteria to attach e.g. flexible
tubing
• Valves that cannot be fully exposed for cleaning
and disinfection e.g. ball valves
• Inadequate ventilation
 Slide 46
Source of Contamination – Failure of Cleaning &
Sanitization

• Cleaning & sanitization may not adequately remove or kill these bacteria:
• Small areas of plant or pipework that are not fully treated e.g. ball
valves, deadlegs, mixer blades, mixer headplates
• Product residues allow re-growth
• Chemical treatment that loses efficiency as it progresses down the
plant
• Residual warm water allows re-growth

• Cleaning & sanitization may actually introduce bacteria

• Stagnant water in the cleaning & disinfection plant
• Water / chemicals used for cleaning and sanitization
 Slide 47

Source of Contamination - People

People are the greatest RISK of causing

contamination if they are not trained and
supervised!
 Slide 48

Source of Contamination - People

Personal Hygiene
 Slide 49

Personal Hygiene

Remember! People are the greatest source of micro-organisms in

manufacturing
• 100,000,000,000,000 bacteria per person!
• 10,000,000,000 per gram of human poo
• 20% of women & 40% of men do not wash their hands after using the
toilet
• The number of bacteria on your hands doubles after using the toilet
• These bacteria survive for up to three hours
• We shed about 1,000,000 skin cells per day – 3.5kg per year
• Between 4 and 100 bacteria per skin cell
• Hygiene procedures (hand washing, protective clothing) should prevent
this contamination entering manufacturing lines….when we do it properly
 Slide 50

Clean Hands and Common Sense

How many of you would be prepared to…

•Eat animal faeces?
•Eat soil?
•Lick the floor/carpet?
•Pick food up off the floor and eat it?
•Drink water from the toilet bowl?
•Lick the hands of a microbiologist?

Why not?
•Common sense tells you that these actions may make
you ill
•Your body and your brain have a way of telling you what
is safe and what is dangerous
 Slide 51

Personal Hygiene

• Fun facts:
Human skin and
• The human body contains more microbial cells than
hair are colonized human cells (90:10)
by
• Babies are born with no microorganisms but begin
MICROORGANISMS acquiring them soon after birth
• We NEED bacteria to keep our skin and gut
healthy!
 Slide 52
Personal hygiene procedures PROTECT YOU AND
PRODUCT

• Wear gloves whenever handling chemicals or packaging

– Protect your skin from harmful materials
– Protect our products from contamination
• Hairnets and beardnets are required in production – change out daily
– Hair carries a LOT of bacteria!
• WASH/SANITIZE YOUR HANDS!!!
 Slide 53

Personal Hygiene

• Jewellery can be vulnerable to product contamination, and has been

shown to be a harbor for microorganisms
• Any piercing that is not covered by clothing could be considered a
harborage
• Artificial fingernails and nail lacquer should not be allowed in
manufacturing areas due to contamination risk
• Even cosmetics, hair and lash extensions could adulterate products if they
are shed in the manufacturing environment
 Slide 54

When Should You Wash Your Hands?

• AFTER USING THE TOILET!!!

• Before eating or drinking
• After sneezing or blowing your nose
• Before handling contacts or touching your face
• Before putting on gloves

• AFTER REMOVING GLOVES!!!

– Your skin bacteria are multiplying like crazy in
that nice, warm environment!
 Slide 55

How Do You Wash Your Hands?

• Wet hands with warm water

• At least 40˚C
– Apply soap to hands

• Rub hands together vigorously

• 15-20 seconds (sing Happy
Birthday)
• Cover all surfaces of hands
and fingers
• Rinse hands with warm water
• Dry hands with disposable
towel or air blower
• Use towel to turn off tap
Take care of your skin:
•Use warm, not hot water
•Thoroughly dry hands – air
drying is hard on the skin
•Use lotion often and cover
your hands in cold weather
•Use sanitizer instead of
soap washing - sometimes
 Slide 56

WHO Causes Contamination?

• The person who thinks preservative challenge testing means product is

safe form contamination

• The engineer who designs and installs pipework … and has not been
trained

• The production supervisor who designs production procedures … and has

not been trained

• The production technician who performs cleaning & sanitization … and

has not been trained
 Slide 57

Conclusions

• Micro-organisms are far cleverer than us – they’ve been doing it for 3.5 billion
years!
• Contamination of products is a real risk with major consequences
• Bacteria can grow very fast – a few residual organisms can rapidly multiply into
millions of cells
• Products are less protected from contamination due to the reduction in
preservatives
• The main risk of contamination arises from a mix of water and product residues –
failure to clean and drain adequately
• Microbiological QC provides a limited view of what is happening in our plant – we
must rely on quality by design
• Properly applied, hygienic controls are effective at preventing contamination
• YOU have a role to play in helping ensure products are free from contamination
Any Questions?