The document provides an overview of aseptic practices and microbiology basics. It discusses: - Definitions of aseptic, sterile, and related terms. - A brief history of aseptic techniques and milestones like the development of the microscope, iodine, and HEPA filters. - The importance of microbiology in contamination control and why microbes are studied. It describes common types of microbes like bacteria, fungi, and viruses. - Sources of contamination and methods for contamination control including cleaning/disinfection, hygiene practices, facilities design with HEPA filters and air flow, and the four pillars of aseptic techniques. - Personal responsibility in contamination prevention through practices like hand

1. Jim Hardy
GHGBioSciences, Inc.
fredcobio@gmail.com
gahaga@gmail.com

2. Agenda
 Background
 Microbiology Basics
 Contamination Control
 Sources of
Contamination
 Cleaning & Disinfection
 4 Pillars of Aseptic
Techniques
2

3. What are Aseptic Practices?
Definitions
Aseptic = without microorganisms
 A methodology that prevents the
introduction of unwanted organisms into
an environment.
 A practice that removes or kills
microorganisms from hands and objects.
Sterile = the complete destruction of all forms
of microbial life, including bacterial spores.
 The meaning of this word is absolute; there
is no such thing as "partially sterile“
 Something is either sterile or non-sterile
3

4. Aseptic Techniques
History & Milestones
4
1600 1700 1800 1850 1900 2000
Microscope
Iodine
Carbolic Acid
HEPA
19th Century SurgeryIgnaz Semmelweis

5. Useful:
-E.coli for production of rDNA/insulin
-Aspergillus for production of penicillin
-Production of foods, for example:
-Dairy industry: Streptococcus
thermophilus to make cheese
-Yeast in beer / wine making
-Bakers yeast
Harmful:
-contamination
-food decay
-contamination of medication
-germs

6. When are Aseptic Practices Used?
 Sampling Raw Materials for Qualification Release
 Sampling of Utilities (WFI, pure steam,
compressed air, and specialty gases)
 Bioburden & Sterility Testing
 Environmental Monitoring
 Manufacturing in classified areas
(i.e. cleanrooms)
 Compounding/Formulation
 Fill/Finish Operations
6

7. Good Aseptic Practices
Regulatory Aspects
 21 CFR 211
 211.28 (b) Personnel shall practice good sanitation and health habits.
 211.113 (b) Appropriate written procedures, designed to prevent microbiological
contamination of drug products purporting to be sterile shall be established and
followed.
 211.84 (c) Sterile equipment and aseptic sampling techniques shall be used when
necessary.
7
To ensure our drug products are safe, pure, and effective
 Eudralex Vol. 4 Part 1, Ch. 2
 Personnel working in areas where contamination is a hazard, e.g. clean areas or areas
where highly active, toxic, infectious or sensitizing materials are handled, should be
given specific training. (2.10)
 EC Guide to Good Manufacturing Practice Revision to Annex 1: Manufacture of
Sterile Medicinal Products
 The manufacture of sterile products should be carried out in clean areas entry to which
should be through airlocks for personnel and/or equipment and materials. Clean areas
should be maintained to an appropriate cleanliness standard and supplied with air
which is passed through filters of an appropriate efficiency.

8. Microbiology and Microorganisms
8

9. Why is Microbiology Important?
 Defined as “the study and science of microorganisms”
 Most microbial contaminants are not pathogens; however,
their presence in drug product and raw materials can affect
the safety, purity, and efficacy by:
 Causing turbidity
 Causing product degradation
 Shifting the pH
 Introducing endotoxins and other toxins
 Microbial testing (bioburden) is performed to ensure that an
adulterated product does not reach the patient
9

10. Types of Microorganisms
 Bacteria
 Fungi
 Yeast
 Mold
 Mycoplasma
 Microscopic algae
 Viruses
10
T4 Virus
Mycoplasma Bacteria
Saprolegnia Mold E Coli Bacteria

11. Bacteria
 Prokaryotes: Unicellular microorganisms with no
nucleus & rarely have membrane-bound organelles
 Ubiquitous to every habitat on Earth
 0.5 -1.0 microns in length : 3 million can fit
into an area the size of a pin head
 Wide range of shapes, spheres, rods, spiral
 Approximately 10X as many bacterial cells as human
cells in the human body
 Large populations on the
skin and in digestive tract
11

12. Bacterial Reproduction
Binary Fission
 One cell divides into
two identical cells
 A single bacterial cell can
multiply to 9 x1030 in a
24-hour period in ideal
conditions
 Refrigeration slows
bacterial growth, but
does not stop it
completely
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Bacterial Binary Fission
E.coli can double their number every 20 minutes!!

13. Fungi
 Eukaryotic organism
 A cell that has a complex
structure enclosed within a
membrane
 Contains a nucleus
 Ubiquitous to every habitat on
Earth
 Types
 Yeast
 Mold
 Can be multi-cellular (mold)
or single-celled (yeast)
 Reproduction is sexual or
asexual
 Commonly via spores 13
Mold
Yeast

14. Yeast
 Unicellular (single-celled) fungi
 Average size = 3 - 4 µm in diameter
 Both aerobic and anaerobic respiration
 Ideal growth conditions:
 Neutral pH
 Temperature 10˚ – 37˚ C
 Part of normal flora of human body
 Reproduce asexually by budding or
sexually by spores like other fungi
 Converts sugar into alcohol = alcohol
tolerant
14
Budding Yeast Cells
Yeast Cells with Spores

15. Molds
 Multi-cellular filaments called “hyphae”
 A colony of hyphae is called a mycelium
 Functions as a decomposer
 Ability to survive extreme temperature and
pH
 Visible as a downy or furry coating on food
or surfaces
 Reproduce through small spores
 Spores can be asexual (mitosis) or sexual
(meiosis)
15
Aspergillus niger
Penicillium

16. Microbes and the Human Body
Where are microbes
found on humans?
 Skin
 Eyes
 Nose
 Mouth
 Upper throat
 Intestines
16
Hands
10,000 -100,000 cm2
Groin
1-20 million / cm2
Feet
1 million / cm2
Scalp
~ 1 million / cm2
Forehead
100 -1,000 / cm
Saliva
~ 10 million / gm
Nasal Fluid
~ 10 million / gm
Armpit
1-10 million / cm2
Feces
>100 million / gm

17. Identification of Microorganisms
 Why?
 Identify sources of contamination
 Identify trends in the environment
 Monitor disinfectant effectiveness
 How?
1. Appearance or morphology
2. Gram staining to identify Gram-negative or Gram-positive bacteria
3. Secondary tests to identify families
4. Biochemical tests to further speciate (Vitek and API)
5. Genetic sequencing analysis (MicroSeq)
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Fewer than 1% of the world’s microbes have been identified!!
Morphology: Size, Shape
Texture, Color

18. Bacterial Identification
Gram Positive Microorganisms
18
Gram Positive Cocci
Staphylococcus aureus
Gram Positive Rods
Bacillus sp.

19. Bacterial Identification
Gram Negative Microorganisms
A source of endotoxin
A single E Coli can contribute 2 million
Endotoxin Molecules!
19
Gram Negative Rods
Escherichia coli
Gram Negative Cocci
Neisseria gonorrhoeae
Endotoxin
Cell Wall

20. Contamination Control
20

21. What is Contamination?
The introduction of undesirable impurities
into an environment
 Viable (living) Contamination:
 Examples: bacteria, yeasts, and molds
 Non-Viable Contamination:
 Examples: lint, dust particles, skin flakes, hair, pollen, smoke, chemical
substances, etc.
21

22. Contaminants
Common Particulates
22
Water Vapor
Bacteria
Skin Flakes
Influenza Virus

23. Contamination Control
 Contamination – any effect or action that has a
negative impact on a product's integrity making it
unfit for use
 Chemical composition
 pH
 Sterility & Pyrogenicity
 Biological or Therapeutic Potency
 Physical appearance
 Particulate Matter (e.g. dust, glass or precipitation)
 Importance of Aseptic Technique
 Parenteral (needle injection) administration bypasses
the skin and gastrointestinal tract, the body’s natural
barriers to infection
 Giving a patient a contaminated product can cause
serious adverse events including DEATH
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24. Contamination
Sources
 Personnel (most
common)
○ Touch Contamination
○ Skin / Hair / Mucous
Membranes
○ Clothing
 Equipment
 Improper Cleaning
/Contaminated Supplies
 Air
○ HVAC / HEPA Failure
○ Infiltration
○ Internal Generation
24

25. Particle Generation
Personnel
25
NOTE: Men shed more particles
than women
Activity Particles
Generated
Standing or Sitting 100,000
Slight head/hand
movement
500,000
Body, arm
movement w/ toe
tapping
1 million
Changing from
sitting to standing
2.5 million
Slow walk 5 million
Running 30 million

26. Contamination Control
Preventative Practices
 Gowning
 Labcoats in the labs
 Scrubs/Cleanroom suits for manufacturing areas
 Face masks, beard covers, hair nets, etc.
 Good hygiene practices
 Personnel/material/waste flow in critical areas
 HEPA filters
 Sanitization of equipment/materials
 Slow, deliberate movements in controlled areas
 Cleaning techniques
26

27. The HEPA Filter
High Efficiency Particulate
Attenuation

28. Unidirectional Air Flow
Air Flow Patterns
•Air moving devices displace the air
in a unidirectional (one direction)
pattern to reduce turbulence of the air
and displace contaminants.
•Air is directed using walls, curtains,
or panels.
•Smoke studies are done to allow the
airflow to be visualized.
•Machinery and large objects can
affect airflow by creating local zones
of air turbulence

29. Room Air Changes
 Air is circulated through the
Cleanroom and carries
contaminants as air passes
through in unidirectional flow.
 Recirculated air passes through
HEPA filters.
 To maintain ISO 5 (Grade A,
Class 100) conditions requires
the air volume to be replaced a
minimum of 25 air changes per
hour (ACH) ; air velocity should
at least be 0.45 m/s.
 20 ACH are typically required for
ISO 7 areas (Grade C areas;
Class 10,000) and ISO 8 (Grades
D areas; Class 100,000.
29
AHU1:
Recirculated Air
AHU2:
Outdoor Air
HEPA Filters

30. Types of
Cleaners/Disinfectants
30
Spor Klenz
Active Ingredient
22% Acetic Acid
4.5% Peracetic Acid
10% Hydrogen Peroxide
70% IPA
LpHse
Phenolic Disinfectant
Acidic, (pH 2.6 – 3.0)
Vesphene IIse
Alkaline pH 10.4-10.6
Bleach
Sodium Hypochlorite

31. Disinfectant Action
31
UV Light
Surface
Proteins
Protein Denaturation by Base
Disruption of
Cell Membrane
Protein Hydrolysis by Acid

32. Microbial Killing Efficiency
Antimicrobial Agents Compared
32

33. Contamination Control
Personal Resposibility
Personal Hygiene
 Regular Bathing/showering (includes washing
hair)
 Practice good oral hygiene
 Clean clothes and shoes
Illness
 Notify your management of illness or open
wounds
 Restrict access to controlled areas
When working in Controlled Areas
 No make-up, jewelry, or cologne
 No food, drinks, or gum/candy
 Avoid getting sunburned (flaking skin)
33

34. Contamination Control
Personal Responsibility
 Frequent hand washing
 If you smoke, drink
water after to reduce
the introduction of
smoke particles into
controlled areas
 Maintain a clean,
organized workspace
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35. 4 Pillars of Aseptic Techniques
Personnel training & monitoring
Environmental monitoring
Facilities design & HVAC validation
Process simulation (media fills)

36. Let’s Review!
 What does “aseptic” mean?
 Name 3 scenarios that require aseptic technique
 Name 3 types of microorganisms
 List the different techniques that QC employs for microbial
identification
 What are the two categories of particulate contamination?
 Name 3 sources of contamination
 List 3 ways to control contamination
 Describe YOUR personal responsibilities toward contamination
control
36

37. Q & A
37