Introduction to Pharmaceutical Microbiology

Introduction to
Pharmaceutical Microbiology
Mohamed Abo-Elgheit
Pharmaceutical Microbiology Expert
Quality System Supervisor in Egypt Otsuka Pharmaceuticals
Microbiology Supervisor in Egypt Otsuka Pharmaceuticals
BS. Microbiology, 2010 - TQM Diploma, 2018
Email: mhmd.aboelgheit@gmail.com

Aim of this lecture
• To understand the role of microbiology lab in the pharmaceutical
industry.
• To get an overview on what is conducted in the pharmaceutical
microbiology lab
• To be familiar with microbiological tests.
• To get to know career path and qualifications of the
pharmaceutical microbiologist.
Mohamed Aboelgheit mhmd.aboelgheit@gmail.com 2

Content
• PART 1: Microbiology Lab
• PART 2: Activities in Microbiology Lab
• PART 3: To be a Microbiologist.

PART 1: Microbiology Lab
• What is Microbiology?
• What is Pharmaceutical Microbiology?
• Pharmaceutical Microbiology Lab
• Aseptic Technique

(1) What is Microbiology?
• Microbiology is the study of
microscopic microorganisms:
• Bacteriology (the study of bacteria)
• Mycology (the study of fungi)
• Phycology (the study of algae)
• Virology (the study of viruses)
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mhmd.aboelgheit@gmail.com
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Microbiology in Tree of
Life
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Bacteria
• Bacteria are
prokaryotic single cell
organisms
• Prokaryote == lack
nucleus, no nuclear
membrane around the
chromosomal DNA.
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Bacteria: Gram Stain and
Cell Wall
• Bacteria are 2 groups:
• Gram +ve Bacteria:
• High peptidoglycan in cell
wall
• Retain the stain
• Gram –ve Bacteria:
• Low peptidoglycan
• High lipopolysaccharide
in cell wall
• Don’t retain the stain
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Bacteria: Gram Stain and
Cell Wall
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Staphylococcus
auresus
E. coli

Bacteria: Aerobic vs
Anaerobic
• Aerobic bacteria utilize oxygen to make energy.
• Anaerobic bacteria don’t require oxygen to make their
energy so they can survive without oxgen.
• Some anaerobic bacteria cannot survive in presence of
oxygen, also called “obligate anaerobic bacteria”
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Fungi
• Fungi are a group of eukaryotic living organisms that
include, but not limited to, yeasts and molds.
• Eukaryote == has a true nucleus
• Yeasts: single cell eukaryotic organisms
• Molds: multicellular filamentous eukaryotic organism.
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Fungi: yeast and molds
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Candidia albicans
Rhizopus sp.

Normal Flora
• Normal Flora is present in:
• Skin e.g. Staph. aureus
• Upper respiratory tract e.g. Staph. aureus
• Oral Cavity e.g. Viridans streptococcus
• Intestine e.g E. coli
• Vaginal Tract e.g. Lactobacillus spp
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(2) What is Pharmaceutical
Microbiology?
• Microbiology is the biology branch
involved with the study of microscopic
organisms e.g. bacteria, fungi, algae,
viruses.
• Pharmaceutical Microbiology is an
applied sub-branch of microbiology
concerned with the study of
microorganisms associated with the
manufacture of pharmaceuticals.
Biology
Microbiology
Pharmaceutical
microbiology

Pharmaceutical
Microbiology Scope
• Study of microorganisms associated with the
industry:
• control and monitor microorganism through the process to
ensure the safe manufacturing of pharmaceutical products:
• Microbiological Analysis Tests
• Environmental monitoring

Flowchart of Pharmaceutical
Manufacturing
Aseptic
Environment
control
Environment
control

(3) Microbiology Lab
Microbiology lab
Clean Areas
Washing Media
Preparation
Sterilization
Testing
Areas
MLT area
Sterility Area
BET Area
Live Culture Areas
Growth
Promotion
Strain maintenance
Microbial
Identification
Decontamination
Area

Microbiology lab Equipment
• Glassware
• Weighing
balance
• pH meter
• Filtration System
• Oven
• Autoclave
• Laminar Air Flow
• Safety Cabinet
• Incubator

Autoclave (steam
sterilization)
Steam Sterilization
• Media
• Glassware
• Gowns
Decontamination
• Incubated Cultures
• Contaminated items

Autoclave: mechanism
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Laminar Air Flow (LAF)

Laminar Air Flow: air
direction
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Microbiological Incubators
Bench Incubator Walk-in Incubator

Good Manufacturing
Practices (GMP)
• Good Manufacturing Practices (GMP) are a set of
practices which ensures that pharmaceutical products
are consistently produced and controlled according to
the quality standards (WHO).
• Areas under GMP are: facilities, equipment,
production, process control, etc.
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Good Laboratory Practices
(GLP)
• Good Laboratory Practices (GMP) are a set of
practices which ensures reliability and reproducibility in
the lab.
• Areas under GLP: testing facilities, personnel training,
equipment, records, reports, etc.
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GLP in Microbiology Lab
• Personnel: trained,
qualified:
• Aseptic technique
• colony counting
• plate pouring
• media preparation, etc.
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GLP in Microbiology Lab
• Lab Environment:
• Separation of clean and dirty activities
• Dedicated spaces for samples, media, reference
microorganisms.
• Disinfection and sterilization can be effectively done
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(4) Aseptic Technique
• Bacteria are everywhere. It is important to take
measures to avoid contaminating your work with
undesired bacteria.
Disinfection of working areas, and hand sanitation
Use of flames to kill bacteria that may enter vessels
openings.

Aseptic Technique

PART 2: Activities in
Microbiology lab
• Media Preparation
• Media Growth Promotion
• Microbiological Analysis Tests:
• Microbial Limit Tests
• Sterility Test
• Bacterial Endotoxin Test
• Viable Environmental Monitoring
• Rapid Microbiological Methods

(1) Media Preparation
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Media Preparation

Media Preparation
Weighing
Adding
water
Dissolving
SterilizationDispensingStorage

Media Preparation
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Agar Media
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• Solid media usually poured in plates
• Used for: enumeration, isolation and purification
• e.g. TSA, Mac. Agar, SDA, R2A, etc

Broth Media
• Liquid media usually dispensed in tubes, beakers, etc.
• Used for: enrichment
• e.g. TSB, FTM, Mac. broth,..etc.
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(2) Growth Promotion Test
• Growth promotion test is a QC test of prepared media to
ensure the media are able to enhance the microbial
growth as intended.
Inoculation
(> 100 CFU)
Incubation Inspection
Growth 50-
200%
Growth <50%
or >200%
PASS
FAIL

Reference microorganism
in GPT for TSA
Reference Microorganism Incubation period and conditions
Staphylococcus aureus ATCC 6538 30°–35° ≤3 days
Pseudomonas aeruginosa such ATCC 9027 30°–35° ≤3 days
Bacillus subtilis ATCC 6633 30°–35° ≤3 days
Candida albicans ATCC 10231 30°–35° ≤5 days
Aspergillus brasiliensis ATCC 16404 30°–35° ≤5 days
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(3) Microbiological Tests
• Microbial Limit Test
• Enumeration test
• Test for specified microorganisms
• Sterility Test
• Bacterial Endotoxin Test
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(3.1) Microbial Limit Test
MLT
Microbial
Enumeration Test
Membrane
Filtration
Plate Count
Method
Test for specified
microorganism
Determination for
presence of absence
of objectionable
microorganism
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(3.1.1) Microbial Enumeration
Test (bioburden)
• In this test, a general solid (agar) media is used to
enhance the growth of all microorganisms in a sample and
hence these microorganisms can be enumerated.
• This test is conducted for:
• Non-sterile finished product
• Water Samples
• Intermediate stages of sterile products

Microbial Enumeration Test:
Membrane Filtration

Microbial Enumeration Test:
Plate Count Methods
Sample

Membrane Filtration VS Pour
plate: think microbiologically!
Membrane Filtration Pour Plate
Sample size Whole sample 0.1 – 1.0 mL
Accuracy Higher Lower
Sample
form
Soluble samples
Samples with low
bioburden
Soluble or insoluble
Samples with high
bioburden
Tools Membrane filtration
system
MFS is not required

Microbial Enumeration
Test: Media
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Purpose Media
Total Aerobic
Microbial
Count (TAMC)
TSA (tryptic soy
Agar)
R2A
Total Yeast
and Mold
Count (TYMC)
SDA (Sabouraud
Dextrose Agar)

(3.1.2)Tests for Specified
Microorganisms
• In such test, a solid media is used to enhance the
growth of a specified microorganism e.g. test for E. coli
• This test is conducted for:
• Non-sterile finished product
• Water Samples
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Example: Tests for E. coli
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90 ml diluent
+ 10 gram
sample
1
TSB
10 ml
(= 1 g
sample)
Incubation
2
30 – 35ᴼC
18- 24
hrs.
4
1 ml
(Mac.Broth)
Incubation
42 – 44ᴼC
24- 48 hrs.
5
Streak
Incubation
30 – 35ᴼC
18- 24 hrs.
6
7
3

Test for specified
microorganism
Test Media used
Escherichia coli MacConkey Agar (MCA) 42-44 ⁰C
MacConkey Broth (MCB) 30-35⁰C
Salmonella spp. Rappaport (RVB) 30-35⁰C
Pseudomonas aeruginosa Cetrimide Agar 30-35⁰C
Staphylococcus aureus Mannitol Salt Agar 30-35⁰C
Clostridia spp. Reinforced Medium for Clostridia 30-35⁰C
Columbia Agar 30-35⁰C
Candida ablicans Sabouraud Dextrose Broth (SDB) 30-35⁰C
Sabouraud Dextrose Agar (SDA) 30-35⁰CMohamed Aboelgheit mhmd.aboelgheit@gmail.com 48

MLT Acceptance Criteria
(USP, ch. 1111)
Dosage form TAMC TYMC Specified microorganism
Nonaqueous oral use 103 102 Absence of E. coli
Aqueous oral use 102 101 Absence of E. coli
Rectal Use 103 102 NA
Cutaneous use 102 101 Absence of S. aureus
Absence of P. aeruginosa
Vaginal use 102 101
Absence of S. aureus
Absence of P. aeruginosa
Absence of C. albicans
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(3.2) Sterility Test
• Sterility test: is a qualitative test for presence or absence of
microorganisms. (sterile == no microbial presence detected)
• In this test: a broth (liquid) media is used to enhance the
growth of any microorganism in the sample and hence these
microorganisms can be detected as a turbidity in the culture
media.

Sterility Test
• Aseptic technique is very critical to
this test, as a single bacterial cell
growth fails the test.
• The Test is done in Class A with
background Class B.
• This test is conducted for: Sterile
finished product.

Sterility Test: media
Media Used
Incubation
Conditions
Targeted microorganisms
Tryptic Soy Broth
(TSB)
20 – 25 ⁰C Aerobic bacteria and fungi
Fluid Thiglycolate
Medium
30 -35 ⁰C Anaerobic bacteria (mainly)
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Sterility Test
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Filter the
sample
solution
Incubate
for 14 days
Inspect
Clear Turbidity
PASS FAIL

Sterility Test: closed
system

Rapid Microbiological
Methods
Rapid Microbiological method are microbiological
testing method alternative to the traditional ones where
microorganism detection requires days or weeks.
Traditional methods Rapid methods
Upfront cost Low Very high
Speed
Very low (days or
weeks)
Real time / minutes
or hours
Sensitivity Low High

(3.3)Bacterial Endotoxin
Test (BET)
• When gram negative bacteria are killed, the
disintegration of cell wall results in
lipopolysaccharides which cause fever if introduced
into the blood. This group of lipopolysaccharides
produced as byproduct of cell wall disintegration are
known as bacterial endotoxins.

Bacterial Endotoxin Test
(BET)
• In this test, a specific reagent called Limulus
Amebocyte Lysate (LAL) is mixed with the
sample to detect or quantify the endotoxin
content.
• Methods: Gel clot BET & Quantitative BET
• This test is conducted for sterile injectable
solutions and for water feeds.

BET : Gel Clot Method
incubation at 37◦ C
for 60 min
sample + LAL reagent (Gel) Firm Gel (+) i.e. Endotoxin detected
then inverting 180◦C
No firm gel (-) i.e. No endotoxin detected

BET: Quantitative Methods
sample + LAL reagent (Turb)
incubation at 37⁰C
Measuring the turbidity
Calculation of endotoxin amount using
the standard curve

(4) Viable Environmental
Monitoring

Viable Environmental
Monitoring
• Pharmaceutical manufacturing must be operated in air-
controlled room, so called “cleanrooms”.
• EM is concerned with:
• Nonviable particles e.g. dust and fine particles (0.5 – 5 µm)
• Viable particles e.g. bacteria and fungi.
• Viable EM role is monitoring number and types of
microorganisms in the air, on the surfaces and within the
personnel in the cleanrooms.

Clean Room Classes (WHO)
Grade 0.5 µm 5 µm Operation
A 3520 20 Filling of product
B 3520 29 ----
C 352000 2900 Preparation of solution
D 3520000 29000
Handling of components
after washing
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Monitoring
Viable EM
Air samples
Personnel samplesSurface
samples

Monitoring Techniques
Category Sample title Description Photo
Air
samples
Passive
Air
Sample
Settle Agar plates left
open for a specific
period in specific
locations
Active
Air
Sample
Using a pump to
collect a specific
amount of air.

Surface
samples
Surface
swab
Using a cotton swab
to collect the
microbes from a
surface
Contact
plate
sample
plates filled with
agar to the highest
level contacting the
surface of
examination.

Personnel
samples
Finger
plates
Agar plates contacting the
fingers of the personnel
hand
Gown
swab
Using a cotton swab to
collect the microbes from
the personnel gown

PART 3: To be a
Microbiologist or Not to be
• to be a pharmaceutical microbiologist
• Pharmaceutical Microbiologist Career Path

To be a pharmaceutical
microbiologist
• You must have BSc. in Microbiology or in Pharmaceutical
Science or any relevant field.
• Love to work with microbes
• Detail-oriented
• Logical thinking
• Problem solving

Pharmaceutical
Microbiologist Career Path
• Lab Microbiologist
• Lab Supervisor/ Section Head/ Manager
• Microbiology Consultant
• Quality Control Manager

References
•WHO Technical report no. 961, 2011:
• Annex 2: WHO good practices for
pharmaceutical microbiology laboratories
• Annex 6: WHO good manufacturing
practices for sterile pharmaceutical
products
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References
• United States Pharmacopoeia USP :
• <1117> Microbiological Best Laboratory Practices
• <61> Microbiological Examination of Nonsterile
Products: Microbial Enumeration Tests
Products: Tests For Specified Microorganisms
• <71> Sterility Tests
Products: Acceptance Criteria For Pharmaceutical
Preparations And Substances For Pharmaceutical Use
• <85> Bacterial Endotoxins Test
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Other references
• British Pharmacopoeia (BP)
• Japanese Pharmacopoeia (JP)
• European Pharmacopoeia (Ph.Eur.)
• ISO Standards
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Introduction to Pharmaceutical Microbiology

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