INDUSTRIAL TRAINING IN

PHARMACEUTICAL
MICROBIOLOGY TECHNIQUES FOR
QUALITY CONTROL

A Report submitted to Manipal Academy of Higher Education in partial

fulfilment of the requirement for the award of the degree of

BACHELOR OF Technology
in
Biotechnology

Submitted by

Ronak Singh
(Reg. No. 190924008)

DEPARTMENT OF BIOTECHNOLOGY

MANIPAL INSTITUTE OF TECHNOLOGY

(A Constituent Unit of MAHE)

MANIPAL – 576104, KARNATAKA, INDIA

June 2022
 DEPARTMENT OF BIOTECHNOLOGY

MANIPAL INSTITUTE OF TECHNOLOGY

(A Constituent Unit of MAHE)

MANIPAL – 576 104 (KARNATAKA), INDIA

Date: 05/06/22

CERTIFICATE

This is to certify that Ms. Ronak Singh (Reg. No. 190924008) has successfully

completed INDUSTRIAL TRAINING in partial fulfillment for the award of the Degree

of Bachelor of Technology (B.Tech.) in BIOTECHNOLOGY of Manipal Institute of

Technology Manipal, Karnataka, (A Constituent Unit of MAHE), during the academic

year 2022-23.

Dr. Balaji S

HOD, Dept. of Biotechnology

M.I.T, MANIPAL

1
2
 ACKNOWLEDGEMENTS

I would like to take the opportunity to thank Dr. Prasad M. P, Director of the
organization for the opportunity to undergo the Advanced Diploma Training at the
esteemed offices of Sangene Biotech. Having been my mentor throughout the training
process, he has been an invaluable asset towards my development of knowledge of the
industrial standards enshrined in the pharmaceuticals pipeline.

I would also like to thank my co-mentors, Ms. Lata and Ms. Haripriya for their constant
guidance with the conduction of experiments at the facilities. Without their help, a lot of
experiments I had undertaken would have gone unvalidated. They filled any potential
gaps in knowledge that I had with regards to important theoretical and practical concepts,
without which my experience at the lab would have been erroneous.

The awareness of the opportunity and the subsequent completion of this industry training
would have been possible without the assistance, motivation and appreciation of my
friends and family, for which I am truly indebted to them.

Last but not the least I wish to show my heartiest appreciation to the Biotechnology
department at Manipal Institute of Technology for giving me the avenue to undertake the
aforementioned training towards not only the completion of my degree, but also the
provision of experience and clarity towards my career goals.

3
 TABLE OF CONTENTS

CHAPTER 1: INTRODUCTION Page No.

1.1 Organization information 6
1.2 Abstract 6
1.3 Motivation for Training 7
1.4 Objectives 8
CHAPTER 2: EXPERIMENTAL SET-UP
2.1 Laboratory Organization 9
2.2 Laboratory Protocol 9
2.3 Documentation Resources 10
CHAPTER 3: TESTS AND PROCEDURES
3.1 Culture of Media and Sterilization Techniques 11
Preparation of LB Broth and LB agar 12
Agar plate/slant preparation 13
Aseptic Culturing of LB broth and LB agar media 14
Microscopy of cultured media 15

3.2 Microbiological Quality Control Procedures 15

Growth Promotion Testing (GPT) 15

Pathogen Testing/Microbial Limit Test (MLT) 17

Water Analysis 20

Bioburden Monitoring 21

Lactobacillus (probiotic) assay 23

Antibiotic sensitivity testing 25

Minimum Inhibitory Concentration (MIC) 27

3.3 Equipment and Work Environment Validation Tests 29

Disinfectant and preservative efficacy testing 29

Personal hygiene monitoring 32

Lab environment monitoring 33

Autoclave Validation and Thermal Death Time (TDT) 35

4
 Effect of growth parameters on microbial cultures 3

CHAPTER 4: CONCLUSION 41
CHAPTER 5: REFERENCES 42
LIST OF FIGURES

1.1.1 Logo of the organization 6

2.1.1 Layout of the experimental facilities 9
3.1.1 LB broth tubes, LB agar and autoclave 12
3.1.2 LB agar slants with B. subtilis colonies 13
3.1.3 Gram-negative E. coli colonies under a microscope 15
3.2.1 MacConkey’s Agar plate for GPT 17
3.2.2 Mannitol salt Agar, EMB Agar, Cetrimide Agar, Bismuth Sulphite Agar 19
3.2.3 B. subtilis colonies as observed after Gram staining 20
3.2.4 SCDA assays of starch and Paracetamol 22
3.2.5 Lactobacillus assay duplicates for Yakult. 24
3.2.6 AP, S, IPM, TE, AMC and CC discs on E. coli lawn culture. 27
3.3.1 Disinfectant efficacy test tubes of varying concentrations with control (L) 30
3.3.2 Preservative efficacy test tubes of varying concentrations with control (L) 31
3.3.3 Hand, ears and elbow samples on SCDA plates. 33
3.3.4 Environment monitoring plates in LAF rooms. 34
3.3.5 Control; periods of 5, 10 and 15 minutes respectively. 36
3.3.6 Autoclave test tubes from top and bottom of chamber. 36
3.3.7 Broth cultures incubated at room temperature, and from 20°C to 50°C 38
3.3.8 Broth cultures incubated at salt concentrations between 100 mg to 2.5 g. 39
3.3.9 Broth cultures of pH 4, 5, 6, 7 and 8. 40
LIST OF TABLES

3.2.1 Media preparation criteria for pathogen testing 18

3.2.2 Antibiotic sensitivities against E. coli in terms of zones of inhibition 25
3.2.3 Result of antibiotic sensitivity testing assay 27

CHAPTER 1
5
 INTRODUCTION

1.1 Organization Information

Sangene Biotech is a centre for advanced research training and education in a Contract
Research Organization (CRO) setting for industry participants and students in a vast
plethora of fields related to biotechnology, such as Protein Chemistry, Molecular
Biology, Microbiology and the like. Their mission is to foster the growth of the R&D
sector as well as its impending demand for skilled professionals in the healthcare
technology landscape.

Established in 2004, the laboratories at Sangene have catered to thousands of students

over the past 19 years in the attainment of practical knowledge as well as the completion
of their envisioned thesis ideas. Under the guidance of mentors, every apprentice is
taught to use the facilities to their maximum potential while ensuring coherence with the
selected training module opted for by the student.

Figure 1.1.1: Logo of the organization

1.2 Abstract

Pharmaceutical microbiology in its essence is the usage of microbiology concepts in

application to validate, monitor and control the presence, growth and contamination of
pharmaceutical ingredients, materials or products by microbial entities. The subject is
intended to ensure the overall safety and efficacy of every aspect of the drug
manufacturing process, ranging from validating disinfectants to evaluating the sterility of

6
surfaces. Assuring the existence of clean-rooms, aseptic conditions and extremely well
controlled environments according to set standards or protocols for their qualification as
well as mitigating or controlling any existing risks is at the heart of pharmaceutical
microbiology [1].

The presence of microbes like E. coli, Salmonella spp. and Staphylococcus aureus in raw
materials, water, equipment, environment or even final products have a high chance of
degrading the potency and efficiency of any drug. To combat and create a baseline
against which the viability of any aspect of pharmaceutical production can be measured,
countries like the United States, Japan, Europe and Britain have developed
Pharmacopoeias [2] that are intended to establish thresholds to judge the risk posed to
patients with formulations, such as tablets, creams, ointments, nasal sprays etc. These
thresholds are crucial not only for compliance purposes, but also to maintain the
consistency of aseptic environments by monitoring the bioburden that is involved in the
manufacturing process. Total Viable Counts (TVC) of microbes, identification of species,
efficacy testing of chemicals used to maintain sterility and maintenance of equipment and
cultures through the application of microbiology techniques for validation and
characterizing the conditions that must be fixed to prevent contamination, which shall be
discussed in further detail through this report.

1.3 Motivation for training

While collegiate and academic channels for education can offer hands-on learning and
conceptual understanding of the important aspects of the pharmaceutical industry, very
little implementation can be translated into on-field expertise of the tools of the trade.
Having taken up a specialization in Pharmaceutical Biotechnology, I found it imperative
that I understand the pipeline of drug development as best as I could.

Quality Control (QC) is a pivotal aspect of this process that involves the measurement of
safety and hygienic practices throughout the manufacturing process of pharmaceuticals.
Being a microbiology enthusiast, I found my curiosity willing to explore the application
of the subject to testing ingredients as well as laboratory conditions in a CRO setting,

7
which is the predominant mode of manufacture of drugs and cosmetics in the Indian
landscape.

Sangene offers a one-month Advanced Diploma to help one pick up the techniques and
best practices of the QC evaluation process, ranging from the fundamentals of cell culture
techniques to the process of ingredient, environmental and personnel monitoring that
certifies a well-rounded training in the Good Manufacturing Practices (GMP) of the
pharmaceutical industry.

1.4 Objectives

Over the course of the one-month module, trainees are prepared to meet the following
objectives:

1. Exposure to preparatory measures involved in participating in drug manufacturing

process
2. Performance of experiments according to industrial best practices (GMP)
3. Understanding of the need for different protocols involved in the monitoring
process
4. Theoretical basis for measures taken and materials and tools used in the
evaluation process
5. Contextual awareness of the standards and guidelines followed according to
established benchmarks i.e., the US Pharmacopoeia
6. Knowledge of procedure in chronological order and awareness of the potential
points for error-making in the QC process
7. Qualitative and quantitative interpretation of results obtained through inference or
evaluation against industry standards
8. Thorough documentation of all tests and procedures and maintenance of work log
books to be replicated in a professional setting.

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 CHAPTER 2

EXPERIMENTAL SET-UP

2.1 Laboratory Organization

Figure 2.1.1: Layout of the experimental facilities

Given above is a rudimentary blueprint for the laboratories at Sangene, which is equipped
to provide all necessary technological capabilities required to carry out the experiments.
The environments most closely used for the course of my training revolved around the
reagent room, the preparation room, the experiment room, the culturing room and the
front office for lab record-related work. Resources used for the purposes of the module
included the incubators, microwave oven, autoclave, weighing scale, LAF bench,
refrigerators, water bath and Ph meter probe.

2.2 Laboratory Protocol

All attending laboratory users were required to enter the laboratory and immediately
sanitize before proceeding into the facilities. Wearing a lab coat is mandatory for the

9
conduction of any experiments, and carrying a pair of gloves is required to perform any
procedures that involved the use of chemicals or handling of biological specimens. Every
equipment used is to be washed and disinfected after every use, and any breakage or
damage resulting from use or recklessness has to be reported to the Director or the Staff.

In the case of a lack of materials/damage to apparatus for experiment, the shortage is to

be reported to the Director who would arrange for the reagents/equipment to be
refilled/replaced. If there is a paucity of time, the necessary training would be provided
through demonstrative means in video or through theoretical explanation by the Director
or the Staff.

2.3 Documentation resources

Every step of experiment that involves the utility of a resource was to be recorded in the
log-book provided next to each material or equipment being used. Reported results were
to be documented in a notebook and presented to the director for validation, and the use
of Internet resources for retrieving protocols related to known procedures was to be kept
track of for generating any subsequent reports. Pictures were to be taken of the results of
any experiments for documenting purposes in order to curate any final reports, which
were to be validated by the Director of the Staff for accuracy.

Apart from the above, laboratory conditions were to be maintained at all times and
timings had to be adhered to as prescribed by the Director. In the case of any
misdemeanors or damages posted, a record of the same was to be made in a registry,
which was to be compensated for by the student on completion of the duration of the
industry training. The certificate and report for the training was to be vetted by the
Director before submission to the University/concerned authority and has to be accurate
and in accordance with the prescribed methods and procedures as were communicated at
the time of training.

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 CHAPTER 3

TESTS AND PROCEDURES

3.1 Culture of Media and Sterilization Techniques

The first practice to master for the purposes of the subsequent experiments involves the
knowledge of proper media preparation procedure as well as culture introduction
techniques. For this purpose, we learnt the process behind the preparation of the basic
lysogeny broth, also known as Luria-Bertani (LB) broth and LB agar [3]. The intended
purpose of these media is for growing bacterial colonies, especially those of Escherichia
coli (E. coli) colonies as a standard for the past 70 years. Outlined below is the typical
method for preparing this media.

3.1.1 Preparation of LB broth and LB agar

Reagents required

Mentioned are the quantities of reagents required for preparing 100 mL of LB agar and
LB broth:

LB Agar: Tryptone: 1 g, Yeast extract: 0.5 g, NaCl: 1 g, Agar: 1.6 g, Distilled or

deionized water: 100 mL

LB Broth: Tryptone: 1 g, Yeast extract: 0.5 g, NaCl: 1 g, Distilled or deionized water:

100 mL

Procedure

Given below are the procedures for preparing 100 mL of LB agar and LB broth:

1. Weigh out and add 1 g of tryptone, 0.5 g of yeast extract, 1 g of NaCl, and 1.6 g
of agar to 100 mL of distilled or deionized water. Add the above to a conical
flask.
2. Alongside the same, weigh out and add 1 g of tryptone, 0.5 g of yeast extract and
1 g of NaCl to 100 mL of distilled or deionized water. Add the above to a conical
flask, which can then be poured into test tubes for further use.

11
 3. Mix well and adjust the pH of both mixtures to 7.0 ± 0.2 using NaOH or HCl
solutions.
4. Cover the flask with a cotton plug wrapped tightly enough to cover the mouth, but
loose enough to allow steam to penetrate through it. Cover this plug with a piece
of paper and attach a rubber band to keep it in place before sterilization. Perform
the same plugging procedure for the test rubes and place them in a rack.
5. Prepare a stack of petri plates for the solid cultures that are to be sterilized in the
autoclave. Wrap them up with paper/inside a plastic pouch and utilize rubber
bands to clasp them inside the wrapping.
6. Place the LB agar conical flask, LB broth test tube rack and the petri plates in an
autoclave and initiate the autoclaving process for a period of ~17 minutes, at a
temperature of 121°C at 15 psi. This is done to kill any spore-resistant bacteria
that may be present in the media.
7. Retrieve the flask, rack and plates carefully and allow the media to cool to
approximately 50°C before using them for further purposes.

Figure 3.1.1: LB broth tubes, LB agar and autoclave

3.1.2 Agar plate/slant preparation

Given below is the nominal procedure for aseptically preparing agar plates or slants to be
used for further culturing.

Materials:

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Sterile petri plates, test rubes, sterile LB agar solution, Laminar flow hood, 70% ethanol
solution.

Procedure:

1. Disinfect the laminar flow hood with 70% ethanol and allow it to dry completely.
Leave the UV light on for 15 minutes under the air flow of the hood before any
procedure is carried out further.
2. Open the front window of the hood to a height sufficient enough to allow your
hands room to maneuver under it for carrying out any movements.
3. Place the LB agar flask, LB broth tubes and petri plates inside the LAF chamber.
Do not open unless further procedure is to be performed. Turn on the Bunsen
burner.
4. Unplug the LB agar solution and present the mouth of the flask to the Bunsen
burner for a couple seconds. This is done to kill any surface microbes.
5. Remove the lid from the agar plates and hold them slightly ajar using your non-
dominant hand. Pour the molten LB agar solution carefully along the sides of the
plates, making sure to sufficiently tilt and straighten to avoid spillage.
6. Place the lid on top again and let the plates cool till the agar media is sufficiently
solidified for further use. Repeat the same steps for test tubes to make slants.

Figure 3.1.2: LB agar slants with B.

subtilis colonies

3.1.3 Aseptic Culturing of LB broth and LB agar media

Given below is the procedure to be followed for aseptically inoculating the above
prepared media with microbial specimens as desired.

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Materials required:

Inoculation loop, LB agar plates/slants, broth tubes, microbe broth culture.

Procedure:

1. Flame-sterilize the inoculation loop until it glows red hot and allow it to cool for
a few seconds.
2. Hold the sterile loop with your dominant hand and the LB agar plate, slants or
LB broth tube with your non-dominant hand.
3. Open the lid of the LB agar plate or plug of the slants or LB broth tube with your
non-dominant hand and hold it slightly ajar. Flame-sterilize their mouths.
4. Use the sterile loop to pick a loopful of microbes from the culture or to gently
scrape some growth from the surface of the broth in the tube.
5. Immediately close the lid of the culture or tube plug and return it to the laminar
flow hood.
6. Flame-sterilize the loop again before replacing it in its sterile container.
7. For LB agar plates, streak the loop on the surface of the agar in a zigzag pattern
to create isolated colonies or streak for isolation. For LB broth tubes, gently swirl
the loop to mix the cells and distribute them evenly in the broth.
8. Close the lid of the agar plate or tube and return it to the laminar flow hood.
9. Disinfect the inoculation loop by flaming it until it glows red hot and allow it to
cool before returning it to its sterile container.
3.1.4 Microscopy of cultured media

Procedure for examining the specimens under a microscope followed the Standard
Operating Procedure depending on the type of staining used. For the purpose of this
training, students were made to revise their knowledge of the Gram-staining protocol
[4]. Having used an E. coli culture for the media preparation, colonies were reported to
be Gram-negative.

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 Figure 3.1.3: Gram-negative E. coli colonies under a microscope

3.2 Microbiological Quality Control Procedures

The tests described below were performed for qualification of samples of the materials
and chemicals used in the manufacturing process. Each of these procedures were done
in accordance with the 10 rules of Good Manufacturing Practices (GMP) [5], as well as
the established Standard Operating Procedures (SOP), instructions and warnings
enlisted by manufacturers on the packaging/instruction manuals of the materials.

3.2.1 Growth Promotion Testing (GPT)

Growth promotion testing is an essential part of Quality Control in microbiology. The

purpose of this test is to verify that the culture media being used is capable of
supporting the growth of the microorganisms it is intended to detect.

Background information - MacConkey’s Agar

MacConkey's agar is a selective and differential culture medium that is used in

laboratories to isolate and selectively promote Gram-negative bacteria, particularly
Enterobacteriaceae. The medium contains peptone, lactose, bile salts, and crystal violet,
which inhibit the growth of most Gram-positive bacteria while allowing the growth of
Gram-negative bacteria. The lactose in the medium serves as a fermentable substrate
that differentiates lactose-fermenting bacteria from non-lactose-fermenting bacteria.
Lactose-fermenting bacteria produce acid, which lowers the pH of the medium, leading

15
to the development of pink or red colonies. Non-lactose-fermenting bacteria do not
produce acid and form colorless colonies [6].

MacConkey's agar is used in the microbiological examination of water, food, and dairy
products to monitor for the presence of coliform bacteria. The properties of
MacConkey's agar make it a useful tool for identifying and characterizing gram-
negative bacteria in pharmaceutical microbiology and in public health settings as well.

The US Pharmacopeia (USP) has established performance standards for MacConkey's

agar using Escherichia coli ATCC 25922 as a test microorganism. According to the
USP, the MacConkey's agar medium should support the growth of E. coli ATCC 25922
when inoculated with a concentration of 100 colony-forming units (CFU) per plate [7].
The medium should also yield a minimum of 50-200 CFU per plate when tested with
the same strain of E. coli. Additionally, the USP requires that the pH of the medium be
between 7.1 and 7.5, and that the color of the colonies formed by E. coli be pink to red
due to the fermentation of lactose in the medium.

Materials:

MacConkey Agar plate, Test microorganism: Escherichia coli ATCC 25922 culture,
Sterile cotton swabs or inoculating loops, Incubator

Procedure:

1. The dehydrated MacConkey’s media stipulates that 49.53g of the medium must
be dissolved in 1000 mL of water. To prepare a 20 mL agar solution, we take
49.53
× 20=¿ 0.99 ≈ 1 g of the dehydrated media with 20 mL of distilled water
1000
and subject to sterilization and plating as described in Chapter 3.1.1 and 3.1.2.
2. Inoculate the plate with 1mL of standard strain of the test microorganism
Escherichia coli ATCC 25922 by swabbing the agar surface with a sterile cotton
swab or inoculating loop that has been dipped into a suspension of the organism.
3. Incubate the plate at 37°C for 48-72 hours and interpret the results.

16
 Result: Presence of yellow colonies indicate contamination by unknown species,
while pink regions indicate E. Coli growth. The total colony forming units
(CFU/mL) on the plate were reported to be 121 CFU. This passes the test.

Figure 3.2.1: MacConkey’s Agar plate for GPT

3.2.2 Pathogen Testing/Microbial Limit Test (MLT)

Pathogen testing involves the measurement of the bioburden of a pharmaceutical sample

with respect to established Pharmacopoeia standards. It is used when an evaluation must
be made for the presence of specific native, deleterious species such as yeast, molds,
and certain bacterial species such as coliforms (E. coli), Pseudomonas spp., Salmonella
spp., and Staphylococcus aureus [8]. The test is performed in duplicates to confirm the
absence of the above-mentioned pathogens and must be done under strictly aseptic
conditions to avoid contamination in a clean-room setting.

Background information:

The culture media used for MLT are selective media that allow microbiologists to
isolate and monitor the presence of the above species. The media used for this
evaluation were as follows:

Name of selective Purpose (selected Microbial Limit Amount of

agar media microbial species) (CFU/mL) media for
40mL (g)
Eosin Methylene Used for detecting the <100 CFU/mL 1.43
Blue (EMB) Agar presence of enteric and
17
 fecal coliforms
Cetrimide Agar Used for detecting the <10 CFU/mL 1.81
(CA) presence of
Pseudomonas
aeruginosa from the
used sample
Mannitol Salt Agar Used for detecting the <100 CFU/mL 4.41
(MSA) presence of
Staphylococcus aureus
from the used sample
Bismuth Sulphite Used for detecting the 0 CFU/mL 2.09
Agar (BSA) presence of
Salmonella spp.,
commonly Salmonella
typhi
Table 3.2.1: Media preparation criteria for pathogen testing

Procedure

Outlined below is a general procedure for pathogen testing using the above media:

1. Prepare the media as prescribed by the manufacturer. On adjusting the amount

of dehydrated media needed for the agar plates for a 40 mL solution, we obtain 4
flasks for each of the test media and autoclave, sterilize and prepare the agar
plates according to the protocol established in Chapter 3.1.1 and 3.1.2.
2. Inoculate the media with the sample from the unidentified GPT colony using a
sterile loop or swab.
3. Incubate the plates at 37°C for 48-72 hours to allow colony formation.
4. After incubation, examine the plates for growth and morphology of the
microorganisms.
4.1 Cetrimide Agar: Look for the presence of Pseudomonas aeruginosa, which
will appear as a bluish-green fluorescent colony. Other organisms may grow

18
 on the media, but they can be differentiated from P. aeruginosa based on
their morphology and color.
4.2 Bismuth Sulphite Agar: Look for the presence of Salmonella and some
Shigella species, which will appear as black colonies. Other organisms may
grow on the media, but they can be differentiated from Salmonella and
Shigella based on their morphology and color.
4.3 Mannitol Salt Agar: Look for the presence of Staphylococcus aureus, which
will appear as yellow colonies. Other organisms may grow on the media, but
they can be differentiated from S. aureus based on their morphology and
color.
4.4 EMB Agar: Look for the presence of Escherichia coli and some other Gram-
negative bacteria, which will appear as dark purple to black colonies with a
metallic sheen. Other organisms may grow on the media, but they can be
differentiated from E. coli based on their morphology and color.
5. Record the results of the tests and compare them to the acceptance criteria
provided by the US Pharmacopoeia.

Results:

Figure 3.2.2 (L to R): Mannitol salt Agar, EMB Agar, Cetrimide Agar, Bismuth Sulphite Agar

The mannitol salt agar showed minor growth; however, they did not display the
characteristic pale-yellow coloration that indicates S. aureus colonies. The EMB agar
indicated a true negative for E. coli, since the colonies do not report a metallic green
sheen that is characteristic of the medium. Cetrimide and bismuth sulphite agar
indicated absence of any of their characteristic colonies. The conclusion drawn is that
19
the species were suspected and verified to be Bacillus subtilis colonies, which are not to
be considered of pathogenic nature.

Figure 3.2.3: B. subtilis colonies as observed after Gram staining

3.2.3 Water Analysis

Assessing the microbial quality of water is an important procedure to estimate the

number of bacteria present in samples for which water is used with different purposes,
such as potable water, purified water and water for injection (WFI). Methods of
examination of the same involve the use of some form of plating technique, which is
done to quantify a heterotrophic mix of bacteria, regardless of its characteristics or
morphology unless required. Indication of species such as E. coli and P. aeruginosa
may indicate the presence of sewage in or around the manufacturing unit [9].

For the purposes of this training, we used drinking water as a marked equivalent for
potable water samples and tap water as the marked equivalent for non-potable water.
Potable water according to the US Pharmacopoeia, has a microbial count threshold of
<100 CFU/mL to be considered safe. Non-potable water has a threshold of <500
CFU/mL to be considered acceptable for use. We shall use the same for the test.

Materials:

Drinking water sample, tap water sample, 50 mL test tubes, Soyabean Casein Digest
Medium, Soyabean Casein Digest Agar (SCDA) plates, inoculation loop.

20
Procedure:

1. Prepare SCDM broth and SCDA plates as is prescribed by the manufacturer on the
dehydrated media container. Since the prescribed amount indicates 29.77 g of
29.77
SCDM and 40 g of SCDA for 1000 ml of solution, we take × 40 and
1000
40
× 40=1.12 g∧1.6 g g of dehydrated media each in two 50 mL test tubes and a
1000
conical flask for SCDA in this procedure.
2. On adding 40 ml of sterile water to the flask, we then subject the media along with
petri plates to autoclave sterilization as described in Chapter 3.1.1 and 3.1.2.
3. On cooling down the solution to ~50°C, 5 mL of the drinking and tap water samples
are taken in test tubes, from which 500 µl of each sample is taken through a
micropipette and added to the petri plates prior to adding the SCDA solutions. This
method is known as pour plating. Following this, the media are allowed to solidify.
4. Once the media are solidified, the plates are incubated at 37°C for 48-72 hours, and
the isolated colony formations are counted.
5. Compare the colony counts to the US Pharmacopoeia standards and present
validation results.

Results:

Colony counts for the drinking water sample were reported at 32 CFU/mL, and the tap
water sample reported 224 CFU/ml. Both the water samples were adjudged to be safe
for use by USP standards.

3.2.4 Bioburden Monitoring

Bioburden, or bioload, is the aggregate quantity of microbes present on the surface of a

solid or equipment/device, inside a liquid, or on material in contact with a
pharmaceutical product of concern. Keeping a check on the amount of bioburden is a
crucial cornerstone of Quality Control since it involves evaluating the manufacturing
pipeline along all stages: the procurement of raw material, the process equipment, the
chemicals used and the finished product itself [10]. For the purposes of this test, we

21
 shall perform a monitoring assessment of starch as a raw material, and paracetamol
tablets (Dolo 650) as a finished product.

Materials:

Soyabean Casein Digest Medium (SCDM), Soyabean Casein Digest Agar (SCDA),
starch powder, 10-piece tab of Paracetamol (Dolo 650), inoculation loop

Procedure:

1. Preparation of media: Prepare SCDM and SCDA according to the procedure

established for water analysis in 3.2.3. Sterilize the media by autoclaving at
121°C for 15 minutes.
2. Preparation of samples: For raw material (starch), weigh 5 g of the material and
add it to a 50 mL SCDM tube. For finished product (Paracetamol tablets), remove
~7 tablets and add them to the other 50 mL SCDM tube. Using a sterile swab or
L-shape rod, streak the suspensions onto the surface of the SCDA plates. Make
sure to spread the suspension evenly across the surface of the agar plate. Incubate
the tubes and plates at 30-35°C for 48-72 hours.
3. Colony counting: After incubation, count the number of colonies on each plate.
For SCDA, the bioburden should be less than 10 CFU per gram or milliliter of
sample.

Result: Starch: 322 CFU/mL (failed), Paracetamol 12 CFU/mL (pass). The reason for the
raw material not passing the QC test would be unsterile conditions when utilizing it by
other students at the laboratory; the batch should be sterilized before use or discarded.

22
 Figure 3.2.4: SCDA assays of starch and Paracetamol

3.2.5 Lactobacillus (probiotic) Assay

Oftentimes, dairy and clinical specimens may contain the presence of lactic acid
bacterial species. Food products such as probiotics (dehydrated tablets, for example)
contain pre-made cultures of healthy bacteria that are meant to enrich the gut
microbiome, and provide important metabolites as well as improve nutrient utilization.
For this purpose, the Lactobacillus assay is intended to quantify the number of colonies
present in a probiotic product and validate it according to the prescribed QC standard.

The media used for this purpose is known as deMan, Rogosa and Sharpe (MRS) agar; it
is a selective growth medium used for isolating Lactobacillus species due to its optimal
constituents conducive to their enumeration under aerobic as well as anaerobic
conditions [11]. The same is to be utilized for this experiment. We shall use Yakult (65
mL, Lacticaseibacillus Shirota (LcS), ~ 3.2 × 108 CFU/mL) for quality control in this
experiment.

Materials:

Dehydrated MRS agar plates, conical flask, probiotic product (Yakult), test tubes,
micropipette.

Procedure:

1. Prepare duplicate MRS Agar according to the manufacturer's instructions. Since

the manufacturer stipulates 65.13 g of dehydrated media per 1000 mL, we utilize
23
 65.13
× 40=2.6 g of the powder for a 40 mL solution in a conical flask. Sterilize
1000
the MRS Agar by autoclaving and preparing plates according to the procedure
outlined in Chapter 3.1.1 and 3.1.2.
2. For the purpose of sample preparation, take 1 mL of Yakult in a test tube and
dilute to 10 mL with sterile water. Following this, perform serial dilution till a
factor of 10-5 is obtained for the experiment, by taking 1 mL from each
preceding test tube and diluting it to 10 mL in the succeeding tubes.
3. Once the required dilution factor is obtained, take the 105 dilution factor (DF)
tube and draw 100 µL of sample to inoculate on the MRS agar plate. Drop the
sample in the middle of the agar plates and spread by swirling and using sterile
L-shape rod for a spread plate culture.
4. Incubate the plates at the appropriate temperature and time as specified by the
manufacturer. Lactobacillus grows best at around 37°C for 24-48 hours and in
an anaerobic environment. We achieve an anaerobic environment by placing the
plates in a jar or container that has been purged with nitrogen gas, or by using an
anaerobic chamber.
5. After incubation, examine the plates for growth of lactobacilli. Lactobacilli will
appear as small, white, opaque colonies with a convex shape.
6. Count the number of colonies and record the results.
7. Calculate the number of lactobacilli per gram or milliliter of sample. This can be
done by dividing the number of colonies by the volume of the sample or the
weight of the sample. The equation for the same is as follows:

No. of counted colonies

N umber of Lactobacill i (CFU /m L )= ×D F
volume of sample taken∈ml

Results:

After incubation, the number of colonies counted were reported at 654 and 588
respectively. On averaging the two, we get 621 colonies. To obtain the final colony count
in CFU/mL, we use the above equation. This gives us:

24
 621 colonies
× 105 = 3.105 × 108 CFU/mL.
0.2 ml

This is in accordance with described colony counts of the probiotic and passes QC check.

Figure 3.2.5: Lactobacillus assay duplicates for Yakult.

3.2.6 Antibiotic Sensitivity Testing

Antibiotic sensitivity testing, also known as antimicrobial susceptibility testing, is an

important component of quality control in the pharmaceutical industry, healthcare
settings, and research laboratories. The purpose of this testing is to determine the
effectiveness of antibiotics against a specific bacterial strain in order to guide
appropriate treatment and prevent the spread of antibiotic-resistant bacteria.

The procedure typically involves growing a bacterial lawn culture on a nutrient agar
plate, and then applying antibiotic discs to the surface of the plate. The antibiotics
diffuse through the agar, creating a zone of inhibition around the disc. The diameter of
the zone of inhibition is then measured and compared to standard diameters for different
antibiotics, which are set in guidelines like the US Pharmacopoeia (USP).

The USP sets standards for a wide range of antibiotics, including ampicillin,
ciprofloxacin, gentamicin, and tetracycline, among others. The standards take into
account factors such as the mechanism of action, the spectrum of activity, and the
dosage and administration of each antibiotic. For the purpose of this experiment, we
used 6 antibiotics to test their sensitivity against E. Coli, whose sensitivities are as
follows:

Name of antibiotic Dosage used Diameters of zones of inhibition for E. coli (mm);
25
 (abbrev.) in disc (µg) susceptible (S), intermediate (I), resistant (R)
Streptomycin (S) 25 ≥ 23 mm (S), 16-22 mm (I), ≤ 15 mm (R)
Amphotericin B (AP) 100 Resistant
Imipenem (IMP) 10 ≥ 23 mm (S), 16-22 mm (I), ≤ 15 mm (R)
Tetracycline (TE) 30 ≥ 19 mm (S), 15-18 mm (I), ≤ 14 mm (R)
Amoxicillin (AMC) 30 ≥ 22 mm (S), 19-21 mm (I), ≤ 18 mm (R)
Clindamycin (CC) 10 Resistant
Table 3.2.2: Antibiotic sensitivities against E. coli in terms of zones of inhibition

Materials:

Mueller Hinton Agar (MHA) plates, antibiotic disks: streptomycin, amphotericin B,

imipenem, tetracycline, amoxicillin, and clindamycin, sterile cotton swabs, 24 hours
incubated E. coli culture broth, incubator set at 35°C ± 2°C

Procedure:

1. Prepare and sterilize Mueller Hinton Agar (MHA) plates according to the
manufacturer’s instructions and protocol established in Chapter 3.1.1 and 3.1.2.
Since we use 38 g of dehydrated MHA for 1000 mL of solution, we use
38
× 40=¿ 1.52 g of MHA for 40 mL of the solid media preparation.
1000
2. Once the plates are prepared and cooled, inoculate the MHA plate with the culture
of E. coli, using a sterile cotton swab, thus inoculating a lawn culture over the
entire solid media surface. Allow the plate to dry for a few minutes. Make sure to
cover all parts of the plate to ensure complete colonization by the bacteria.
3. Place 3 antibiotic disks on each of the MHA plates using sterile forceps in a
triangular fashion, ensuring that they are well spaced apart from each other and the
plate walls to clearly observe antibiotic inhibition. Press them in slightly with the
forceps to properly attach them without any air gaps.

26
4. Incubate the plate at 35°C ± 2°C for 18 to 24 hours. Make sure to keep the plates
well covered and upright to prevent the discs from falling out or getting displaced
from their position.
5. After incubation, observe the MHA plate for the presence or absence of a zone of
inhibition around each disk. Measure the diameter of the zone of inhibition for
each disk using a caliper or ruler. In case of ingrowth within the zone, measure
from the outermost diameter to ensure accuracy.
6. Compare the diameter of the zone of inhibition to the appropriate standards or
breakpoints established for each antibiotic. Record the results and interpret the
antibiotic sensitivity of the organism to each antibiotic tested.

Results:

The results obtained are tabulated as follows:

Name of antibiotic Diameter of zone of Sensitivity level (susceptible/

(abbrev.) inhibition (mm) intermediate/resistant)
Streptomycin (S) 23 Susceptible
Amphotericin B (AP) 0 Resistant
Imipenem (IMP) 26 Susceptible
Tetracycline (TE) 18 Intermediate
Amoxicillin (AMC) 21 Susceptible
Clindamycin (CC) 0 Resistant
Table 3.2.3: Result of antibiotic sensitivity testing assay

27
 Figure 3.2.6 (L to R, clockwise): AP, S, IPM, TE, AMC and CC discs on E. coli lawn culture.

3.2.7 Minimum Inhibitory Concentration (MIC) of antibiotics

Minimum inhibitory concentration (MIC) is a measure of the sensitivity of

microorganisms to antimicrobial agents. It is defined as the lowest concentration of an
antimicrobial agent that completely inhibits the visible growth of a particular
microorganism in a liquid growth medium or on a solid agar medium. MIC testing is a
critical aspect of quality control in the manufacturing and testing of pharmaceuticals, as
well as in clinical microbiology and infectious disease management.

The determination of MIC values provides important information about the

susceptibility of microorganisms to antimicrobial agents, which can guide the choice of
appropriate treatment regimens for infections caused by these organisms. MIC testing is
typically performed using standardized methods, such as the broth microdilution
method, in which serial dilutions of an antimicrobial agent are prepared in liquid growth
medium, and a standardized inoculum of the test organism is added to each well of a
microtiter plate containing the dilutions of the antimicrobial agent.

Given below is a general procedure for determining the minimum inhibitory

concentration (MIC) of azithromycin according to US Pharmacopoeia (USP) guidelines:

Materials:

Sterile broth medium (Mueller-Hinton broth), antibiotic (azithromycin) stock solution,

sterile test tubes, inoculating loop, E. coli broth culture, incubator set at 35°C ± 2°C,

Procedure:

1. Prepare a fresh culture of E. coli using nutrient broth and incubate it at 35°C ±
2°C until it reaches the exponential phase of growth.
2. Prepare a series of doubling dilutions of the antibiotic stock solution(s) in test
tubes containing Mueller Hinton broth medium, typically ranging from 0.1 to 30
μg/mL, depending on the antibiotic being tested. For this, 1 mg of azithromycin
powder was dissolved in 10 mL of solution, giving us a 100 μg/mL stock solution.

28
 The remaining tubes were prepared using the balanced equation, a sample of
which is given below:
If 100 μg/mL is present in 10 mL of stock solution, in order to get 0.1 μg/mL, we
need ‘x’ mL of stock solution.

0.1 ×10
x= =0.01 ml=10 μl of stock solution.
100

Subsequently, prepare test tubes for 0.5, 1, 5, 10, 20 and 30 μg/mL solutions.

3. Inoculate the test tubes with a small amount of the E. coli culture using a sterile
inoculating loop.
4. Incubate the test tubes at 35°C ± 2°C for 18 to 24 hours.
5. After incubation, observe the test tubes for the presence or absence of bacterial
growth. The MIC is the lowest concentration of antibiotic that inhibits visible
growth of the bacteria. Determine the MIC by identifying the well with the lowest
concentration of antibiotic that shows no visible growth.

Results: The MIC of azithromycin against E. coli was found to be at 20 μg/mL. Thus,
the predicted range of antibiotic breakpoint is found to be between 16-32 μg/mL.

3.3 Equipment and Work Environment Validation Tests

Equipment and environment validation testing is a crucial aspect of ensuring the quality
and safety of pharmaceutical products. The US Pharmacopoeia sets standards and
guidelines for the validation of equipment and environment in pharmaceutical
manufacturing facilities. The purpose of equipment and environment validation testing
is to ensure that all tools and conditions under which the products are manufactured are
operating under controlled parameters, which are essential for the quality and safety of
the final product.

The validation of equipment involves the verification of the equipment’s performance

under specific operating conditions, as well as the consistency of the environment for
performing microbiological assays. This is done through a series of tests to assess and
consolidate information about the experimental parameters of concern. Some of the
tests done are as outlined below.
29
3.3.1 Disinfectant and preservative efficacy testing

Disinfectant and preservative efficacy testing is an important aspect of quality control in

the pharmaceutical industry. It involves the evaluation of the effectiveness of
disinfectants and preservatives in preventing the growth of microorganisms. The
purpose of this testing is to ensure that the disinfectants and preservatives used in
pharmaceutical products are effective in controlling microbial contamination.

The US Pharmacopoeia provides guidelines for the testing of disinfectants and

preservatives. The testing procedure involves the preparation of different dilutions of
the disinfectant or preservative in test tubes, along with a control. The test organism is
then inoculated into each of the test tubes and incubated under appropriate conditions.
The tubes are observed over a period of time to determine the effectiveness of the
disinfectant or preservative in inhibiting the growth of the test organism.

Materials required:

Sterile test tubes, disinfectant (Lysol), preservative (citric acid), E. coli broth culture,
nutrient broth preparation, inoculation loop, incubator.

Procedure:

1. Prepare 0.001%, 0.01%, 0.1%, 1% and 10% (v/v) solutions of Lysol to be tested in
separate test tubes. For this, we first sterilize and prepare a stock nutrient broth as
outlined in 3.1.1 and 3.1.2. Following this, we calculate the amount of disinfectant
needed per test tube to establish the above concentrations, a sample calculation for
which is as follows:
For a 0.01% (v/v) disinfectant solution of 10 mL in a test tube, we take
0.01
×10=0.001 mL=1 µL of Lysol and dilute up till 10 mL with sterile water.
100
Subsequently prepare solutions of 0.001% to 10% (v/v) solutions in separate test
tubes. Prepare one test tube with no disinfectant to act as a control.
2. Inoculate each of the test tubes with a loopful of E. coli. Incubate the tubes at 37°C
for 24-48 hours to allow colony formation to take place.
30
 3. Compare the turbidity of the test tubes with the control solution without any
disinfectant to determine the efficacy of the disinfectant at each concentration.

Figure 3.3.1: Disinfectant efficacy test tubes of varying concentrations with control (L)

Results:

The minimum disinfectant concentration required to observe a lack of microbial growth

was reported at 0.1% (v/v).

Procedure for Preservative Efficacy Testing:

1. Prepare 0.001%, 0.01%, 0.1%, 1% and 10% (w/v) solutions of citric acid in
separate test tubes. For this, we first sterilize and prepare a stock nutrient broth as
outlined in 3.1.1 and 3.1.2. Following this, we calculate the amount of preservative
needed per test tube to establish the above concentrations, a sample calculation for
which is as follows:
For a 0.01% (w/v) preservative solution of 10 mL in a test tube, we take
0.01
×10=0.001 mL=1 µ g of citric acid and dilute up till 10 mL with sterile
100
water.
Subsequently prepare solutions of 0.001% to 10% (w/v) solutions in separate
test tubes. Prepare one test tube with no preservative to act as a control.

31
 2. Inoculate each of the test tubes with a loopful of E. coli. Incubate the tubes at 37°C
for 24-48 hours to allow colony formation to take place.
3. Compare the turbidity of the test tubes with the control solution without any
preservative to determine the efficacy of citric acid at each concentration.

Figure 3.3.2: Preservative efficacy test tubes of varying concentrations with control (L)

Results:

The minimum disinfectant concentration required to observe a lack of microbial growth

was reported at 0.1% (v/v).

3.3.2 Personal hygiene monitoring

Personal hygiene monitoring is a critical aspect of quality control in pharmaceutical

manufacturing, as it helps ensure the safety and efficacy of the products produced. The
US Pharmacopoeia has set standards for personal hygiene monitoring to be followed in
pharmaceutical facilities to maintain a clean and sterile environment.

The purpose of personal hygiene monitoring is to ensure that personnel working in the
manufacturing environment maintain appropriate standards of hygiene to prevent the
contamination of the products. The areas of the body that require monitoring include
hands, elbows, and behind the ears. The process involves the collection of samples from
these areas, followed by microbiological testing to detect any bacterial or fungal
growth.

32
 The US Pharmacopoeia requires that personal hygiene monitoring be conducted at
regular intervals, and that the procedures for collecting and testing samples be
documented and followed consistently. The materials required for personal hygiene
monitoring include sterile swabs, test tubes containing transport medium, and
microbiological media such as Soybean-Casein Digest Agar or Plate Count Agar.

Materials required:

Soyabean Casein Digest Agar plates, sterile cotton swabs, transport medium tubes.

Procedure:

1. Sterilize the swabs and test tubes containing transport medium before use.
2. Collect the samples from the behind the ear and the elbow, using a sterile swab for
each area. The swab should be rubbed firmly over the skin surface for a couple
seconds to ensure that any microorganisms present are collected.

3. Transfer the swab to a sterile test tube containing transport medium. Cap the test
tube and label it with the sample collection details.
4. Under the LAF bench, inoculate the samples onto Soybean-Casein Digest Agar
plates. For hands, directly imprint your fingertips onto the surface of the agar
plate, spacing the fingers sufficiently apart.
5. Incubate the plates at 37°C for 24 to 48 hours. Examine the plates for the presence
of bacterial or fungal growth, and record the results.

Results:

33
 Figure 3.3.3 (L to R): Hand, ears and elbow samples on SCDA plates.

Presence of fungal colonies on hand sample indicates contact with contaminated surface
prior to inoculation. The growth of bacteria on the ear samples indicates lack of
addressal of sanitation behind the ears. The elbow sample has little to no growth,
indicating fair hygiene practice.

3.3.3 Lab environment monitoring

Lab environment monitoring is a crucial part of maintaining a high-quality laboratory. It

involves checking the laboratory environment for various factors, such as temperature,
humidity, and air quality, among others, to ensure they are within acceptable limits. The
lab environment can affect the accuracy and reliability of laboratory tests, which can
have an impact on the validity of the results obtained. Therefore, it is important to
maintain a clean and controlled environment that meets the specified standards.

These parameters can affect the growth of microorganisms and the stability of
chemicals used in laboratory tests. The US Pharmacopoeia recommends that the
temperature in the laboratory should be between 20°C and 25°C, while the humidity
should be between 45% and 65%. In addition, air quality is another important aspect of
laboratory environment monitoring. Air quality testing involves monitoring the levels of
airborne contaminants, such as dust, particles, and microorganisms. Proper ventilation
and air filtration systems are essential for maintaining good air quality in the laboratory.
For this purpose, we shall evaluate the laboratory conditions in the LAF benches at the
offices.
34
Materials required: Soyabean Casein Digest Agar plates.

Procedure: Without allowing the presence of any other personnel, place the SCDA
plates near the edge of the LAF benches without turning on any equipment or adjusting
the environment in any way. Allow them to remain exposed to the air in the room for
15-20 minutes without making any contact with the plate surface. Following this, cover
the plates and incubate for 24-48 hours at 37°C. Observe the plates and report the
findings.

Results:

Figure 3.3.4: Environment monitoring plates in LAF rooms.

The plates reported 6 and 1 colony each, averaging out to 4 CFU. The US
Pharmacopoeia standard for a safe environment is set at 5 CFU, which indicates that the
LAF rooms are safe for use.

3.3.4 Autoclave validation and thermal death time (TDT)

Before the commencement of any of the microbiological procedures, sterilization of

equipment is paramount to the reliability of the results obtained. If a laboratory engages
in the use of faulty sterilization equipment, this can pose a hazard not only to the
experiment being carried out, but also to personnel operating any equipment or
chemicals in a pharmaceutical setting. For this purpose, we shall evaluate the efficiency
of the autoclave as well as assess the minimum time required to administer all microbes
that may be present to be rendered ineffective under sterilization conditions, which is
also known as the thermal death time of the species under consideration.
35
Materials:

Unsterile test tubes, nutrient broth preparation, cotton plugs, B. subtilis broth culture,
inoculation loop.

Procedure:

1. Prepare test tubes marked for placement at the top, middle and bottom of the
autoclave. Along with the same, prepare test tubes to be placed for sterilization
periods of 5 minutes, 10 minutes and 15 minutes, with one control test tube that
is not to be sterilized in any way.
2. Fill each test tube with 10 mL of nutrient broth. Inoculate B. subtilis colonies
from the broth culture into each of the test tubes.
3. Place three of the validation test tubes in such a manner that one is present on
the top, one in the middle and one at the bottom of the autoclave. Use other
dummy apparatus for this purpose.
4. Place the remaining test tubes within the autoclave and start a timer for counting
how long they are subjected to heat at 121°C at 15 psi, once the conditions are
achieved in the autoclave. After every time period mentioned above, extract the
test tube after their specific durations, except for the 15-minute test tube, which
shall complete the standard sterilization time with the remaining tubes.
5. Once the autoclaving process is complete, place each test tube in a rack and put
them inside an incubator at 37°C for 48-72 hours.
6. Observe the results with reference to the control test tube and report the
findings.

Results - Thermal Death Time:

36
 Figure 3.3.5 (L to R): Control; periods of 5, 10 and 15 minutes respectively.

On comparison with the control test tube, the 5-minute tube was found to show a fair
level of turbidity, indicating that there was not enough heat and pressure subjection to
prevent colony formation. The 10-minute test tube indicated mild turbidity, implying
that even though sterilization action was observed, it was not critical enough to ensure
complete sterilization. The 15-minute test tube was clear, indicating full sterilization.
Thus, it is considered as a standard for the thermal death time (TDT) of E. coli

Autoclave validation:

Each of the test tubes that were placed in different parts of the autoclave reported no
colony formation after 15 minutes of sterilization, indicating uniformity of the heat
distribution mechanism and pressure conditions throughout the autoclave.

Figure 3.3.6: Autoclave test tubes from top and bottom of chamber.

3.3.5 Effect of growth parameters on microbial cultures

Without appropriate levels of temperature, pH and salt concentration, any microbial

colony is capable of exhibiting inhibited to negligible levels of growth, regardless of
other conditions being met. The presence of radiation such as ultraviolet light (UV) can
37
also pose a risk to the growth of colonies, which is why the method is considered to be
effective in sterilization of LAF chambers prior to use by any operating microbiologist.
These parameters are meant to be consistently met in a pharmaceutical facility
according to the purpose for which the experiment is being conducted; for instance,
growth of acidophilic or alkaliphilic bacteria is only conducive under low or high pH
levels respectively; thermophilic bacteria can only be observed when temperature
conditions are higher than the standard temperature condition in order to enable their
growth.

Judging optimal conditions for the growth of the bacterial species of concern is
therefore essential to understanding the nature of the bacteria; without fully assessing
these parameters, no experiment or process of incubation can be carried out efficiently.

Materials required:

Incubator, HCl 1M solution, NaOH 1M solution, pH probe, sodium chloride, LAF

bench with UV light, nutrient broth, test tubes, nutrient agar plates, E. coli broth.

Procedure – Optimal temperature for microbial growth

1. Prepare 10 mL nutrient broth test tubes, each labelled for different temperature
conditions to be met. For the purpose of this evaluation, we shall assess the
effect of 20°C, 25°C, 35°C, 40°C and 50°C, along with one tube to be placed
under room temperature condition.
2. Do not alter the pH or salt concentration of the preparation, ensuring uniformity
in each test tube used. Inoculate each of the test tubes with E. coli using an
inoculation loop.
3. After this, place each test tube in an incubator set at the specific temperature
conditions mentioned above, and allow to form colonies for a time period of 24-
48 hours.
4. Observe the turbidity of the broth cultures after incubation and report the results.

Results:

38
 Figure 3.3.7 (L to R): Broth cultures incubated at room temperature, and from 20°C to 50°C

The turbidity of the columns were measured differently for each test tube, with the 50°C
tube showing almost no signs of growth. The 20°C and 25°C tubes showed minimal
growth; however, the test tubes between the temperature range of 35°C to 40°C showed
maximum growth characteristics, implying that the optimum temperature lies between
this range at ~37°C.

Procedure – Optimal salt concentration for microbial growth

1. Prepare 10 mL nutrient broth test tubes, each labelled for different salt
concentrations to be met. For the purpose of this evaluation, we shall assess the
effect of 100 mg, 250 mg, 500 mg, 1 g and 2.5 g of salt.
2. Do not alter the pH or other constituents of the preparation, ensuring uniformity
in each test tube used. Inoculate each of the test tubes with E. coli using an
inoculation loop.
3. After this, place each test tube in an incubator set at 37°C, and allow to form
colonies for a time period of 24-48 hours.
4. Observe the turbidity of the broth cultures after incubation and report the results.

Results:

39
 Figure 3.3.8: Broth cultures incubated at salt concentrations between 100 mg to 2.5 g.

As expected, the broth culture with 100 mg of salt showed highest turbidity compared to
the subsequent concentrations, which were of decreasing turbidity. The basis for this is
that as salt concentrations increase, the osmolarity of the medium increases which can
lead to growth suppression of the incubated colonies.

Procedure – Optimal salt concentration for microbial growth

1. Prepare 10 mL nutrient broth test tubes, each labelled for different pH levels to
be met. For the purpose of this evaluation, we shall assess the effect of pH 4, 5,
6, 7 and 8. To meet these levels, we shall make use of a pH probe, and adjust the
pH of distilled water using 1M HCl and 1M NaOH solutions to calibrate the
solutions to the desired pH. The sensitivity of the probe is of importance in
ensuring the correct pH is attained, and requires time to stabilize on addition of a
specific solution.
2. Do not alter the other constituents of the preparation, ensuring uniformity in
each test tube used. Inoculate each of the test tubes with E. coli using an
inoculation loop.
3. After this, place each test tube in an incubator set at 37°C, and allow to form
colonies for a time period of 24-48 hours.
4. Observe the turbidity of the broth cultures after incubation and report the results.

Results:

40
 Figure 3.3.9 (L to R): Broth cultures of pH 4, 5, 6, 7 and 8.

The test tube with the highest characteristic turbidity was the pH 6 and 7 tubes. The
remaining test tubes indicated reduction in growth due to milder cloudy appearance;
however, this did not completely inhibit their growth in any manner.

CHAPTER 4
41
 CONCLUSION

Over the course of this training, the importance of every microbiology technique made
me more aware of the types of risks and hazards that can be posed to products and
personnel alike in the pharmaceutical setting. The role of every minute participant in the
manufacturing process is tantamount to its safety and reliability, especially those that
are often overlooked in a standard microbiology laboratory such as those in schools or
in college.

To that very end, the purpose of various Pharmacopoeias explains the urgency and need
for standardization of every protocol in a manner that is consistent not only with
regulation but also the science behind the manufacturing process; to accept even the
minutest excesses over any established threshold can pose a grave danger to the
consistency of the process. This is evidently seen in the types of risk assessments taken
for materials such as water, nutrient media, disinfectants and the environment that the
operators work in themselves.

My learnings over one month may not be absolute in the sense of fully knowing the ins
and outs of the Quality Control protocol, but the experience gained from the module I
undertook allowed me to get a better hold of my laboratory techniques as well as
understand the chronology of a risk assessment. The linearity and variety of the tests
taken indicate the comprehensiveness of the validation process, and is an indicator of
the need for manual comprehension of the assays made during the process. Future
developments may minimize the involvement of manual labor throughout the process,
but it shall in no way redefine the standards set throughout the pharmaceutical
development pipeline by regulatory authorities, especially when dealing with organisms
that cannot meet our naked eye.

CHAPTER 5
42
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44