Unit4 Microbiology

S.Y.B.Pharm Sem 3
- Dhruvi Jain
 Microbiological assay

Measuring the
Determine relative On a test
amount required
activity of organism under
to produce the
compound standard condition
effect

Principle: when compounds are present in limited amounts the

microbial growth corresponds to the amount of the compound
 Microbiological assay
Liquid or gel medium

Test compound Test organism

• Antibiotic • bacteria
• Vitamins • yeast
• Amino acids • mold

Response
• antibiotics - negative
• vitamins and a.a.- positive
Standardization of Antibiotic
 Microbiological assay
Merits Demerits
• Suitable for compounds which • Requirement of specific test
cannot be assayed by physical and organism for assay of specific
chemical methods compound
• Minimize the mortality rate of • Requires sterile conditions in
animals laboratory
• Simple and rapid than bioassay • Invalid results due to slight
• Standardization of medicine variations in incubation
• Concentration and activity can be • Time consuming
determined • Requires well trained and expert
• No requirement of large amount individuals
of sample and instruments
• Can be automated
 Standardization of Antibiotics
• Comparison of inhibition of growth by test vs standard
- Test : Antibiotic under examination
- Standard : Known concentration of antibiotic with known activity

A. Cylinder Plate Method / B. Turbidimetric method /

Cup plate method Tube Assay Method
- Depends on diffusion of antibiotic - Depends on inhibition of growth in
from cylinder fluid
- Examine zone of inhibition - Examine turbidity
 Standardization of Antibiotics
• Media
- support the growth of test organism
- ingredients dissolved in 1000mL water and ph adjusted by 1M NaOH or
1MHCl
 Standardization of Antibiotics
• Buffer Solution:
- Dissolve following quantities of KH2PO4 and K2HPO4 - total volume
1000mL
- Adjusting the pH with 8M phosphoric acid or 10M potassium hydroxide.
 Standardization of Antibiotics
• Preparation of Standard Solution:

- Standard Preparation: authentic sample of antibiotic for which potency is

determined by International standards

- Potency: ability of antibiotic to kill target microorganism

concentration at which effective killing is achieved
- expressed as International units or mg/mg of antibiotic
- Certified by Indian Pharmacopoeia Commission or other notified laboratories
- Replaced by working standard prepared in laboratory
- Stock is prepared by dissolving specific amount of antibiotic in diluent.
 Standardization of Antibiotics
• Preparation of Sample Solution:
- Unknown sample of assumed potency
- Prepare stock and test dilution as standard

• Test Organism: Identification number - American Type Culture Collection

 Standardization of Antibiotics
• Apparatus:

1) Temperature control – Incubator or water bath

2) Spectrophotometer – device for measuring transmittance

3) Plate Assay – Petri dishes, sterile cylindrical borer

4) Turbidimetric assay – Glass or plastic tubes

 Standardization of Antibiotics
• Potency:
- ability of antibiotic to kill target microorganism
- concentration at which effective killing is achieved

Estimation of Potency

One Level Assay Two Level Factorial Assay

 A. Cup Plate Method

Cork Borer

Inoculated with
Uninoculated Microorganism
Media Seed layer - 4ml
Base layer – 21mL
 One Level Assay
• Standard Solution: from stock prepare 5 dilutions
Eg: S1 - 2mg/ml, S2 - 4mg/ml, S3 - 6mg/ml, S4 - 8mg/ml, S5 - 10mg/ml

• Sample Solution: assuming its initial concentration adjust to its median

Eg: U1 - 6mg/ml

• Method :
S3
Any 1 of the
4 dilutions
 One Level Assay
• Method:
- incubate the plates for 18hrs at specific temperature
- measure the diameter or zone of inhibition

• Total standard
plates = 4*3=12
• Total unknown
plates = 3
 One Level Assay
• Estimation of potency:

a) Calculate the average zone diameters:

- Average of S1, S2, S3, S4 and S5
- Average of all 36 S3

b) Correction
- Eg:

Average of 36 Average of one Difference

S3 set of S3
c 6 5.8 +0.2
 One Level Assay
Sample Conc. Average Corrected
mg/ml value value
a S1 2 2 2.2
b S2 4 3.8 4
d S4 8 8.1 8.3
e S5 10 10.3 10.5

12

L = calculated 10zone diameter H = calculated zone diameter

Zone diameter

for lowest concentration

8 for highest concentration
L = 2.02
6

4
H= 10.38
2

0
0 2 4 6 8 10 12
L = 2.02 Concentration H= 10.38
 Two Level Factorial Assay
• Standard and sample solution:
- Parallel dilutions of two levels : highest and lowest (S1, S2 and U1, U2)

• Estimation of Potency:

Percent Potency = Antilog (2.0 + a log I)

𝐚 = (𝐔𝟏 + 𝐔𝟐 ) − (𝐒𝟏 + 𝐒𝟐 )
(𝐔𝟏 − 𝐔𝟐 ) + (𝐒𝟏 − 𝐒𝟐 )

U1, U2 = sum of zone diameters of unknown solutions of high and low

concentration
S1, S2 = sum of zone diameters of standard solutions of high and low
concentration
I = Ratio of dilutions
 Two Level Factorial Assay
U1 (20mg/mL) = 10 S1 (20mg/mL) = 10
U2 (5mg/mL) = 2 S2 (5mg/mL) = 2

𝐚 = (𝐔𝟏 + 𝐔𝟐 ) − (𝐒𝟏 + 𝐒𝟐 )
(𝐔𝟏 − 𝐔𝟐 ) + (𝐒𝟏 − 𝐒𝟐 )

𝐚=0

Percent Potency = Antilog (2.0 + a log I)

Percent Potency = 100%

 B. Turbidimetric Assay
Prepare dilutions of standard and sample antibiotic

Concentration adjusted
Stock
to median

Control Tubes

1 ml of antibiotic solution +
9ml of media inoculated with microorganism

Media Culture
Blank
control control

Incubate the tubes for 3-4 hrs Measure growth by taking absorbance
at 540nm
 B. Turbidimetric Assay
• Estimation of Potency:
- Calculate the average for each dilution and plot standard Absorbance vs
concentration
 B. Turbidimetric Assay

Advantages Disadvantages

• Not recommended for

• Shorter incubation period cloudy or turbid solutions
• Solvent residues interfere
with the assay
Standardization of Vitamins
 Standardization of Vitamins
• Principle:
- Vitamins: essential for growth of microorganisms
- the ability of test organisms to use the substance being standardized under a
proper nutritional condition is measured.
- Growth response will be proportional to amount of vitamin added

Turbidimetric Titrimetric

• Prerequisites:
1. Basal Medium stock solution: medium containing limited nutrients but will
be free from the vitamin to be tested
2. Standard Vitamin stock solution
3. Organism
Standardization of Vitamins

Vitamin Organism
Vitamin B12 (Cobalamin) Lactobacillus leichmanii
Vitamin B6 (Pyridoxine) Saccharomyces uvarum
Vitamin B2 (Riboflavin) Lactobacillus casei
Vitamin B1 (Thiamine) Lactobacillus viridescens
Vitamin B3 (Niacin)
Vitamin B5 (Pantothenate)
Lactobacillus plantarum
Vitamin B7 (Biotin)
 Standardization of Vitamin B12
(Cyanocobalamin)
1. Standard Cyanocobalamin (0.01-0.04mg/mL) and unknown sample is added to
clean test tube.

1mL 1.5mL 2mL 3mL 4mL 5mL 1mL 1.5mL 2mL 3mL 4mL 5mL
Standard Unknown

2. Add 5 ml of Basal medium and adjust the volume to 10mL with water
3. The tubes are covered to prevent bacterial contamination and autoclaved for 5
minutes at 121°C
4. Tubes are inoculated with 0.5mL organism Lactobacillus leichmanii
5. Tubes are incubated for 24hrs.
 Standardization of Vitamin B12
(Cyanocobalamin)
1) Turbidimetric:
- After incubation record transmittance of each tube at 530nm
- High vitamin conc. – high growth – low transmittance
- Low vitamin conc. – low growth – high transmittance

2) Titrimetric:
- After incubation contents of tube are titrated with 0.05N NaOH using
bromothymol blue indicator
- Yellow – Green – Blue (Acidic – neutral – basic)
- More vitamin will cause more growth
and more acid production thus higher titration value
- Less vitamin will cause less growth
and less acid production thus lesser titration value
• The titration values of standard and test solutions are determined and plotted
on a graph considered titration values (in ml) of 0.05 N NaOH against the
concentration of standard cyanocobalamin solution.
• A linear graph is obtained by interpolating the standard curve to determine the
concentration as activity per ml of vitamin B12. From the graph the
concentration of the test solution of cyanocobalamin is calculated
Standardization of Amino Acids
 Standardization of Amino Acids
• Amino acids – proteins : essential for growth and replication of
microorganisms

• Utilization of amino acids result in growth which is measured by

turbid metric or titration (pH change) method

• Guthrie and Susie – modified the assay by using diffusion in gels

• Phenylalanine: amino acid responsible for production of

neurotransmitters (dopamine), melanin

• Phenylketonuria
- genetic inherited disease
- Cause: mutation in gene responsible for the utilization of
phenylalanine therefore results in its buildup
- Symptoms: neuronal disorders, eczema
 Standardization of Amino Acids
• Guthrie Test: Determines the levels of phenylalanine in blood sample
• Principle:
- phenylalanine : helps in growth of Bacillus subtilis
- b-2-Thienylalanine: competitive metabolic antagonist i.e. inhibits Bacillus
subtilis
- competition between the two
 Standardization of Amino Acids
• Method:
1. Plate with low
nutrients and limited
amounts of
b-2-Thienylalanine
2. Inoculate plate with
Bacillus subtilis

3. Incubate overnight

• Result: No bacterial growth because b-2-Thienylalanine inhibits Bacillus

subtilis
 Standardization of Amino Acids

4. Dip a filter paper (4mm) in blood

A
sample and place it on the plate

5. Incubate overnight

A- High Phenylalanine
B- Low Phenylalanine
B

• Result:
- Sample A will show zone of growth because high levels of
phenylalanine competes with antagonist b-2-thienylalanine
- Sample B will show no growth because there is no phenylalanine to
compete with b-2-Thienylalanine
 Standardization of Amino Acids
• METHOD: Guthrie Test

1. Filter paper discs of 4 mm diameter are soaked in blood and placed on the agar
surface along with the blood soaked discs of phenylalanine standards.

2. These agar plates are incubated at 37°C overnight.

3. Bacterial growth is observed only when phenylalanine concentration in the

blood discs is sufficient to overcome the effects of β-2 thienylalanine.

4. Growth is observed in zones of growth around each disc.

5. The next day diameter of each zone of growth is measured and related to
phenylalanine concentration.
Designing of Aseptic Area
 Designing of Aseptic Area
• Aseptic Area: any area which is sterile and provides a controlled
environment such that entrance of viable (microorganisms) and non-viable
(particle dust) contaminant is minimized.

• Requirement:
- Production of sterile products
- Products not sterilized after production are filled in this area
- eg: ophthalmic or parenteral preparations, sterile medicinal products, vaccines,
microbial extracts
- Products that can be sterilized after production are filled in clean area and not in
aseptic area

• Example: Laminar Air Flow Hood (LAFH),

Biological Safety Cabinet (BSC)
 Designing of Aseptic Area
• Design and Layout:
- Located separately
- Should be buffered i.e., unauthorized personnel should not gain access to this
unit
- Safe and organized workflow
- Risk of cross-contamination is reduced
- Cleaning is easy and dust accumulation is reduced
 Designing of Aseptic Area

Air Lock
office Preparation/
Aseptic Filling Quarantine
Compounding
Area Area Storage/
Area
Shipping
Area
Stock/
store Packaging/
Area Sterilization
Clean-up Area Labeling
Area
Area Quality
Control
 Designing of Aseptic Area
• Design and Layout:
1) Stock Area:
✓ Area in which raw materials / active pharmaceutical ingredients needed for
manufacturing are kept
✓ Excipients like buffering agents, solvents, stabilizers, etc
✓ In order to maintain stability of raw materials stock room has a suitable
temperature and humidity
✓ Area should be treated with disinfectant to prevent contamination

2) Clean-up Area:
✓ Area for cleaning bottles, vials, ampoules and glass things used for parenteral
preparation.
✓ The room should be free from dust, fibers , microorganisms
✓ It should withstand moisture, steam and detergent.

3) Preparation/Compound Area:
✓ Area for mixing of ingredients and preparation for filling operation
✓ Not necessary for it to be aseptic but contamination should be avoided
 Designing of Aseptic Area
4) Sterilization:
✓ Some parenteral preparations are directly prepared and sterilized in this
room
✓ Alternatively formulations which cannot be sterilized are transferred to
aseptic filling area

5) Aseptic Filling Area:

✓ After preparation, the formulation is transferred from preparation area to
aseptic filling area
✓ In this area the parenteral preparation is filled into containers and finally
sealed after filtering it
✓ Only trained person should be eligible for entry
✓ For the air to be free from microorganisms and dust particle High Efficiency
Particulate Air (HEPA) filters are used

6) Quarantine Area:
✓ This area consists of a store where the in process batches as well as approved
batches are stored separately.
✓ This area has limited access and is under the control of a responsible person.
 Designing of Aseptic Area
6) Packaging and Labeling area:
✓ In this area, the batches are packed and labelled
✓ Packing is carried out by packaging machines, while labels are obtained by
over printing devices.
✓ At a time, only one product labels are printed
✓ Packing should be carried out in such a manner that the sterility of the
product is maintained.

8) Quality Control:
✓ The packaged products undergo quality checks and are then transferred to the
storage room

9) Storage and shipping area:

✓ The finished, approved and packaged products are held in this room until
shipment is confirmed.
 Designing of Aseptic Area
• Construction:
1) Surfacing Materials: (Floors, walls and ceiling)
✓ smooth, impervious, and unbroken to reduce the release and accumulation
of contaminating particles and organisms
✓ can withstand the effects of cleaning agents and disinfectants.
✓ Floor – Polyvinylchloride (PVC) : cheap, simple to clean and easy to repair
✓ Walls – non-inflammable or fire resistant
1% of 8- hydroxyquinolone, pentachlorophenol added to paint to reduce
fungal growth
Epoxy resin paints and polyurethane paints are also used to avoid cracking
and peeling
✓ Ceiling – sealed to prevent entry of microorganisms
✓ Minimum shelves, ledges, cupboards, and equipment should be present
 Designing of Aseptic Area
2) Doors:
✓ Doors and windows should fit flush with the walls.
✓ Limited number of doors for entry
✓ Self-closing doors to allow easy movement
✓ Double doors/ Air lock doors
✓ All the doors should have an alarm system which should ring when more
than one door are being opened.

3) Windows:
✓ Non opening and sealed windows
✓ solely to provide illumination and are not for ventilation.

4) Light:
✓ Should be fitted with the ceiling to prevent accumulation of dust
✓ Also airflow of the room is not disturbed
 Designing of Aseptic Area
5) Services:
✓ Piped liquids and gases must be filtered first
✓ Position of pipes and ducts should be such that it can be easily cleaned
✓ Sinks and drains should not be present anywhere in the clean area
✓ The areas having sinks and drains should be designed, positioned, and
maintained such that the risk of microbial contamination is reduced
✓ they are fitted with easily cleanable traps, installed with electrically heated
disinfection devices.
 Laminar Air Flow Hood

• Provides:
1) Clean air to the critical sites (immediate aseptic compounding area),
2) Constant flow of air out of the work area to prevent the entry of room
air, and
3) Outward flow of air from the hood that suspends and removes
contaminants which have been introduced in the work area by
personnel.

• HEPA Filter:
 Laminar Air Flow Hood
Horizontal LAFH Vertical LAFH
• Sweeps the filtered air from back to • Sweeps the filtered air from top to
the front down
 Laminar Air Flow Hood
• HEPA Filter (High Efficiency Particulate Air):
✓ The air within the room is taken into this filter and passed through a pre-filter
- removes the gross contaminants (lint, dust, etc.).
✓ The air is then blown at a uniform velocity through the hood and HEPA filter
in a unidirectional (laminar flow) manner
✓ HEPA filter is a particulate filter which traps the airborne particles and
microbes; but allows the gases to pass through.
✓ A pre-filter is fitted upstream of the HEPA filter, thus extending the final
filter's life.
✓ A fan is also fitted which pumps the air through the filter.
✓ Provides:
1) A high air flow rate,
2) A high particulate holding capacity, and
3) A low-pressure drop across the filter.
Sources of Contamination

Personnel

Manufacturing Building and

Process Facility

HVAC system Raw material

Equipment and
utensil
 Sources and Prevention of
Contamination
1) Personnel:
✓ Skin scales released by the operator
✓ Inadequate training of GMP and aseptic techniques
✓ Improper hygiene
✓ Direct contact between hands and starting material, intermediate and final
product
✓ Improper hygiene
✓ Insufficient gowning and protective equipment

• Prevention:
➢ Adequate training
➢ Restricting access to unauthorized individual
➢ Protective clothing: PPE, gloves, head gears, masks, etc
➢ Maintain good personal hygiene
 Sources and Prevention of
Contamination
2) Buildings and Facilities:
✓ Inadequate size and organization of the space,
✓ Poor filth and pest controls,
✓ Rough floors, walls, and ceilings,
✓ Absence of air filtration systems,
✓ Improper washing, cleaning activities

• Prevention:
➢ Smooth, crack-free, and easily cleanable floors, walls, and ceilings should be
used as they facilitate easy and effective cleaning.
➢ Windows should be sealed
➢ The design of pipe work, ventilation should be free from recesses
➢ Sinks should be of stainless steel
 Sources and Prevention of
Contamination
3) Equipment and Utensils:
✓ Unsuitable design, size, corrosion-causing materials
✓ adulteration with lubricants, coolants, dirt, and sanitizing agents,
✓ Inadequate cleaning and sanitization,
✓ Inappropriate calibration and irregular service

• Prevention:
➢ Time-to-time cleanliness and disinfection of the areas should be done
➢ Cleaning agents of proper grades should be used
➢ Contact time, application, temperature, mechanical action, and the chemistry
of cleaning agents should be considered during the cleaning process.
 Sources and Prevention of
Contamination
4) Raw Materials:
✓ Improper storage and handling, which leads to mix-ups or selection errors,
✓ Microbial or chemical contamination,
✓ Wrong labeling.
✓ Degradation due to extreme environmental conditions
✓ Using materials not meeting the acceptance criteria.

• Prevention:
➢ Water used should be of pharmaceutical grade, microbiologically controlled
and monitored
➢ Clean and additive-free steam should be used for cleaning and sanitization of
production tools and equipment, and to supply for autoclaves and
humidification.
 Sources and Prevention of
Contamination
5) Manufacturing Processes:
✓ Improper cleaning between batches for minimizing the amount of product
changeovers
✓ Use of an open manufacturing system for exposing the product to the room
environment
✓ Lacking an area line clearance (as per the approved procedures) after each
cleaning process and between each batch,
✓ Lack of cleaning status labeling on all equipment and materials used within
the manufacturing facility

• Prevention:
➢ Proper cleaning
➢ Closed manufacturing system, open-container processing should be
performed within the aseptic area
➢ area line clearance
➢ cleaning status labeling
 Sources and Prevention of
Contamination
6) HVAC System (Heating, Ventilation and air conditioning):
✓ Organic materials accumulate in or near HVAC air intakes,
✓ Inadequate air filtration system,
✓ Inadequate magnitude of pressure differentials, which causes flow of
reversal

• Prevention:
➢ The differential air pressures should be higher than adjacent area