INTERNATIONAL ISLAMIC UNIVERSITY

MALAYSIA

Biochemical-Biotechnology Engineering

Lab Manual for

Biotechnology Engineering Lab IV

BTE 3212
 TABLE OF CONTENTS

CONTENTS Page

Preface ii
Laboratory Safety Regulations iii
Workplace Hazardous Materials Information System v
Biosafety Manual vi
Guideline for Laboratory Reports vii
Marking Scheme for Laboratory Reports xix
Content of Experiment (Body of Text) xix
xix
Part A: Animal & plant Experiments
Experiment 1: Animal Cell Culture. Aseptic techniques and procedures to study
growth of animal cells 2
Experiment 2: Plant Tissue Culture: Preparation of explants for callus culture.
Plant cell suspension culture 7
Part B: Operation of Bioreactors and Others Bioprocess Equipment
Experiment 3: Batch Fermentation of R-E.Coli in Bioreactor Using Different
Control Condition 14
Experiment 4: Cell Disruption by High Pressure Homogenizer 18
Part C: Water Quality Experiments 20
Experiment 5: Measurement of the Chemical Oxygen Demand (COD)
Experiment 6: Measurements of pH 23
Experiment 7: Determination of Total Suspended Solids (TSS), Volatile 26
Suspended Solids (VSS) 31
Experiment 8: Determination of Total Dissolved Solids (TDS) 38
Experiment 9: Determination of Biological Oxygen Demand (BOD5) 44
Experiment 10: Total Nitrogen (TN)
Experiment 11: Total Phosphorus (TP) 50
Experiment 112: Zinc
Appendix A 64
Appendix B 69
70

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Preface
-----------------------------------------------------------------------

This manual lab has been written for students taking the undergraduate of

Biotechnology Engineering (Bioprocess Laboratory) lab IV. It contains specific instructions

for completing the planned experiments involving mechanisms, procedures and materials, as

well as more general information covering laboratory conduct and safety rules.

This lab involves collection of many experiments from different resources. It

encourages a mixture of plant, animal and environmental experiments. After reading the

literature, listen to detailed discussion of laboratory techniques and reaction types, the student

should be able to form directions to run an experiment and not just blindly follow the

directions. It encourages students to achieve the primary objectives and the ability of

understanding the significant works of the experiments. Keep in mind as you work your way

through this manual there are specific purposes in each exercise. They will prepare you for

your first job in a biotechnology laboratory, so keep a careful record of your experience. If

you carefully document and archive your work, this information will be easy for you to

access later and your experiences will be more valuable in your later work.

Finally, this lab course is intended to provide an enjoyable, interesting experience for

students. Our wishes is not only meant to help students with the best learning possible, but

also to give support and confidence to students to consider professional careers involving the

practice or teaching of experimental science.

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 LABORATORY SAFETY REGULATIONS

GENERAL
1. Do NOT work with hazardous substances without a second person being
present
2. Do NOT eat, drink or smoke in the laboratory under any circumstances
3. ALWAYS Keep your working area clean and tidy and free of clutter
4. ALWAYS Keep benches tidy and gangways clear
5. ALWAYS support gas cylinders, and ALWAYS close cylinder valves after
use
6. ALWAYS label containers in plain English with the common known name
of the substance and the appropriate hazard warning sign
7. ALWAYS secure the tops of reagent bottles immediately after use
8. ALWAYS work with fume cupboard sashes as low as possible and
ALWAYS work towards the back of the cupboard
9. ALWAYS use double containment techniques where possible and drip
trays to limit the consequences of a spillage
10. ALWAYS clear up spillages immediately
11. Do NOT leave equipment using water, gas or electricity on overnight
without completing a Silent Running form, and ALWAYS ensure all water
hoses are secured with jubilee clips

PERSONAL PROTECTION
1. ALWAYS wear a lab coat and appropriate eye protection, e.g. safety
spectacles, goggles or face shield.
2. Lab coats should ALWAYS be buttoned up and NOT worn in amenity
areas of the University.
3. ALWAYS use the appropriate gloves whenever handling chemicals or
hazardous substances, and ALWAYS check their integrity before use,
ensuring they will give you protection against the substance being used
4. ALWAYS wear proper footwear, do NOT wear open toed footwear

EMERGENCIES
1. ALWAYS know where your nearest fire extinguisher and first aid kit are
2. ALWAYS know your emergency escape route and assembly point

STORAGE AND DISPOSAL

1. ALWAYS keep broken glassware and sharps separate from other waste
and ALWAYS dispose of in the appropriate containers
2. ALWAYS return stock bottles/jars/dewars etc of highly flammable liquids or
acids to their correct store cupboard after work has finished
3. Do NOT have more than 500 ml of a flammable solvent in use at any one
time on the bench.

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WORKPLACE HAZARDOUS MATERIALS INFORMATION SYSTEM

Using chemicals or other hazardous substances at work can affect peoples health. The CHIP
regulations (Chemicals Hazard Information and Packaging for Supply) place very specific
duties on suppliers of Chemicals in the workplace. They identify criteria by which hazardous
substances are classified, how to package the chemical in a suitable way and what
information needs to be displayed on the label and in the Safety Data Sheet. Hazardous
substances will display one or more of the following symbols on the label which identifies
the category of danger identified during the classification process as shown below:

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Biosafety Manual for Biotechnology Engineering Lab IV

This manual describes the combinations of code of practice, laboratory design &
facilities, and laboratory equipment constituting Biosafety Levels 1-2, which are
recommended for work with a variety of infectious agents such as bacteria, fungi, and yeast,
in various laboratory settings. These recommendations are intended to provide a voluntary
guide or code of practice as well as goals for upgrading operations. They also are offered as a
guide and reference in the construction of new laboratory facilities and in the renovation of
existing facilities. However, the application of these recommendations to a particular
laboratory operation should be based on a risk assessment of the special agents and activities,
rather than used as a universal and generic code applicable to all situations. The detailed
description here is confined on biosafety levels 1-2 only. However, biosafety levels 3-4 share
the same basic standards, but employ additional features that are not provided here due to the
lack of laboratories with such biosafety level.

Risk Groups

Classification of organisms according to risk group has traditionally been used to categorize
the relative hazards of infective organisms. The factors used to determine which risk group an
organism falls into is based upon the particular characteristics of the organism, such as

ility of effective preventive measures

These classifications presume ordinary circumstances in the research laboratory or growth

in small volumes for diagnostic and experimental purposes. Four levels of risk have been
defined as follows.
Risk Group 1 (low individual and community risk) Any biological agent that is unlikely to
cause disease in healthy workers or animals.
Risk Group 2 (moderate individual risk, low community risk) Any pathogen that can cause
human disease but, under normal circumstances, is unlikely to be a serious hazard to
laboratory workers, the community, livestock or the environment. Laboratory exposures

v
rarely cause infection leading to serious disease; effective treatment and preventive measures
are available, and the risk of spread is limited.

Risk Group 3 (high individual risk, low community risk) Any pathogen that usually causes
serious human disease or can result in serious economic consequences but does not ordinarily
spread by casual contact from one individual to another, or that causes diseases treatable by
antimicrobial or antiparasitic agents.

Risk Group 4 (high individual risk, high community risk)

Any pathogen that usually produces very serious human disease, often untreatable, and may
be readily transmitted from one individual to another, or from animal to human or vice-versa,
directly or indirectly, or by casual contact.

Containment Levels (Biosafety Levels)

Classification of organisms according to risk group is not meant to establish the actual
handling of biological hazards in the laboratory setting. For example, the risk group system
does not take into account the procedures that are to be employed during the manipulation of
a particular organism. Containment levels (also known as biosafety levels) are selected to
provide the end-user with a description of the minimum containment required for handling
the organism safely in a laboratory setting. In addition to the inherent characteristics of each
organism, the containment system includes the engineering, operational, technical and
physical requirements for manipulating a particular pathogen. These containment levels are
applicable to facilities such as diagnostic, research, clinical, teaching and production facilities
that are working at a laboratory scale. Four containment levels are described as follows:

Containment Level 1 (CL1)

This applies to the basic laboratory that handles agents requiring containment level 1. CL1
requires no special design features beyond those suitable for a well-designed and functional
laboratory. Biological safety cabinets (BSCs) are not required. Work may be done on an open
bench top, and containment is achieved through the use of practices normally employed in a
basic microbiology laboratory.

Containment Level 2 (CL2

This applies to the laboratory that handles agents requiring containment level 2. The primary
exposure hazards associated with organisms requiring CL2 are through the ingestion,

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inoculation and mucous membrane route. Agents requiring CL2 facilities are not generally
transmitted by airborne routes, but care must be taken to avoid the generation of aerosols
(aerosols can settle on bench tops and become an ingestion hazard through contamination of
the hands) or splashes. Primary containment devices such as BSCs and centrifuges with
sealed rotors or safety cups are to be used as well as appropriate personal protective
equipment (i.e., gloves, laboratory coats, protective eyewear). As well, environmental
contamination must be minimized by the use of handwashing sinks and decontamination
facilities (autoclaves).

Containment Level 3 (CL3)

This applies to the laboratory that handles agents requiring containment level 3. These agents
may be transmitted by the airborne route, often have low infectious doses to produce effects
and can cause serious or life-threatening disease. CL3 emphasizes additional primary and
secondary barriers to minimize the release of infectious organisms into the immediate
laboratory and the environment. Additional features to prevent transmission of CL3
organisms are appropriate respiratory protection, HEPA (High Efficiency Particulate Air)
filtration of exhausted laboratory air and strictly controlled laboratory access.

Containment Level 4 (CL4)

This is the maximum containment available and is suitable for facilities manipulating agents
requiring containment level 4. These agents have the potential for aerosol transmission, often
have a low infectious dose and produce very serious and often fatal disease; there is generally
no treatment or vaccine available. This level of containment represents an isolated unit,
functionally and, when necessary, structurally independent of other areas. CL4 emphasizes
maximum containment of the infectious agent by complete sealing of the facility perimeter
with confirmation by pressure decay testing; isolation of the researcher from the pathogen by
his or her containment in a positive pressure suit or containment of the pathogen in a Class III
BSC line; and decontamination of air and other effluents produced in the facility. A summary
of biosafety level requirements is shown in table 1.

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Risk Assessment
The backbone of the practice of biosafety is risk assessment. While there are many tools
available to assist in the assessment of risk for a given procedure or experiment, the most
important component is professional judgment. Risk assessments should be performed by the
individuals most familiar with the specific characteristics of the organisms being considered
for use, the equipment and procedures to be employed, animal models that may be used, and

viii
the containment equipment and facilities available. The laboratory director or principal
investigator is responsible for ensuring that adequate and timely risk assessments are
performed, and for working closely with the institutions safety committee and biosafety
personnel to ensure that appropriate equipment and facilities are available to support the work
being considered. Once performed, risk assessments should be reviewed routinely and
revised when necessary, taking into consideration the acquisition of new data having a
bearing on the degree of risk and other relevant new information from the scientific literature.
One of the most helpful tools available for performing a microbiological risk assessment is
the listing of risk groups for microbiological agents. However, simple reference to the risk
grouping for a particular agent is insufficient in the conduct of a risk assessment. Other
factors that should be considered, as appropriate, include:
1. Pathogenicity of the agent and infectious dose
2. Potential outcome of exposure
3. Natural route of infection
4. Other routes of infection, resulting from laboratory manipulations (parenteral, airborne,
ingestion)
5. Stability of the agent in the environment
6. Concentration of the agent and volume of concentrated material to be manipulated
7. Presence of a suitable host (human or animal)
8. Information available from animal studies and reports of laboratory-acquired infections or
clinical reports
9. Laboratory activity planned (sonication, aerosolization, centrifugation, etc.)
10. Any genetic manipulation of the organism that may extend the host range of the agent or
alter the agents sensitivity to known, effective treatment regimens .
11. Local availability of effective prophylaxis or therapeutic interventions.
On the basis of the information ascertained during the risk assessment, a biosafety level can
be assigned to the planned work, appropriate personal protective equipment selected, and
standard operating procedures (SOPs) incorporating other safety interventions developed to
ensure the safest possible conduct of the work.

Basic laboratories Biosafety Levels 1 and 2

For the purposes of this manual, the guidance and recommendations given as minimum
requirements pertaining to laboratories of all biosafety levels are directed at microorganisms

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in Risk Groups 14. Although some of the precautions may appear to be unnecessary for
some organisms in Risk Group 1, they are desirable for training purposes to promote good
(i.e. safe) microbiological techniques (GMT).

Diagnostic and health-care laboratories (public health, clinical or hospital-based) must all be
designed for Biosafety Level 2 or above. As no laboratory has complete control over the
specimens it receives, laboratory workers may be exposed to organisms in higher risk groups
than anticipated. This possibility must be recognized in the development of safety plans and
policies. In some countries, accreditation of clinical laboratories is required. Globally,
standard precautions should always be adopted and practiced. The guidelines for basic
laboratories Biosafety Levels 1 and 2 presented here are comprehensive and detailed, as
they are fundamental to laboratories of all biosafety levels. The guidelines for containment
laboratories Biosafety Level 3 and maximum containment laboratories Biosafety Level 4
(not shown here) are modifications of and additions to these guidelines, designed for work
with the more dangerous (hazardous) pathogens.

Code of practice
This code is a listing of the most essential laboratory practices and procedures that are basic
to GMT. In many laboratories, this code may be used to develop written practices and
procedures for safe laboratory operations. Each laboratory should adopt a safety or operations
manual that identifies known and potential hazards, and specifies practices and procedures to
eliminate or minimize such hazards. GMT is fundamental to laboratory safety. Specialized
laboratory equipment is a supplement to but can never replace appropriate procedures. The
most important concepts are listed below.

Access
1. The international biohazard warning symbol and sign (Figure 1) must be displayed on the
doors of the rooms where microorganisms of Risk Group 2 or higher risk groups are
handled.
2. Only authorized persons should be allowed to enter the laboratory working areas.
3. Laboratory doors should be kept closed.
4. Children should not be authorized or allowed to enter laboratory working areas.
5. Access to animal houses should be specially authorized.
6. No animals should be admitted other than those involved in the work of the laboratory.

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Personal protection

1. Laboratory coveralls, gowns or uniforms must be worn at all times for work in the
laboratory.
2. Appropriate gloves must be worn for all procedures that may involve direct or accidental
contact with blood, body fluids and other potentially infectious materials or infected
animals. After use, gloves should be removed aseptically and hands must then be washed.
3. Personnel must wash their hands after handling infectious materials and animals, and
before they leave the laboratory working areas.
4. Safety glasses, face shields (visors) or other protective devices must be worn when it is
necessary to protect the eyes and face from splashes, impacting objects and sources of
artificial ultraviolet radiation.
5. It is prohibited to wear protective laboratory clothing outside the laboratory, e.g. in
canteens, coffee rooms, offices, libraries, staff rooms and toilets.

xi
6. Open-toed footwear must not be worn in laboratories.
7. Eating, drinking, smoking, applying cosmetics and handling contact lenses is prohibited in
the laboratory working areas.
8. Storing human foods or drinks anywhere in the laboratory working areas is prohibited.
9. Protective laboratory clothing that has been used in the laboratory must not be stored in the
same lockers or cupboards as street clothing.

Procedures

1. Pipetting by mouth must be strictly forbidden.

2. Materials must not be placed in the mouth. Labels must not be licked.
3. All technical procedures should be performed in a way that minimizes the formation of
aerosols and droplets.
4. The use of hypodermic needles and syringes should be limited. They must not be used as
substitutes for pipetting devices or for any purpose other than parenteral injection or
aspiration of fluids from laboratory animals.
5. All spills, accidents and overt or potential exposures to infectious materials must be
reported to the laboratory supervisor. A written record of such accidents and incidents
should be maintained.
6. A written procedure for the clean-up of all spills must be developed and followed.
7. Contaminated liquids must be decontaminated (chemically or physically) before discharge
to the sanitary sewer. An effluent treatment system may be required, depending on the risk
assessment for the agent(s) being handled.
8. Written documents that are expected to be removed from the laboratory need to be
protected from contamination while in the laboratory.

Laboratory working areas

1. The laboratory should be kept neat, clean and free of materials that are not pertinent to the
work.
2. Work surfaces must be decontaminated after any spill of potentially dangerous material

and at the end of the working day.

3. All contaminated materials, specimens and cultures must be decontaminated before

disposal or cleaning for reuse.

xii
4. Packing and transportation must follow applicable national and/or international
regulations.
5. When windows can be opened, they should be fitted with arthropod-proof screens.

Biosafety management
1. It is the responsibility of the laboratory director (the person who has immediate
responsibility for the laboratory) to ensure the development and adoption of a biosafety
management plan and a safety or operations manual.
2. The laboratory supervisor (reporting to the laboratory director) should ensure that regular
training in laboratory safety is provided.
3. Personnel should be advised of special hazards, and required to read the safety or
operations manual and follow standard practices and procedures. The laboratory
supervisor should make sure that all personnel understand these. A copy of the safety or
operations manual should be available in the laboratory.
4. There should be an arthropod and rodent control programme.
5. Appropriate medical evaluation, surveillance and treatment should be provided for all
personnel in case of need, and adequate medical records should be maintained.

Laboratory design and facilities

In designing a laboratory and assigning certain types of work to it, special attention should
be paid to conditions that are known to pose safety problems. These include:
1. Formation of aerosols
2. Work with large volumes and/or high concentrations of microorganisms
3. Overcrowding and too much equipment
4. Infestation with rodents and arthropods
5. Unauthorized entrance
6. Workflow: use of specific samples and reagents.
Examples of laboratory designs for Biosafety Levels 1 and 2 are shown in Figures 2 and 3,
respectively.

Design features
1. Ample space must be provided for the safe conduct of laboratory work and for cleaning
and maintenance.

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2. Walls, ceilings and floors should be smooth, easy to clean, impermeable to liquids and
resistant to the chemicals and disinfectants normally used in the laboratory. Floors should
be slip-resistant.
3. Bench tops should be impervious to water and resistant to disinfectants, acids, alkalis,
organic solvents and moderate heat.
4. Illumination should be adequate for all activities. Undesirable reflections and glare should
be avoided.
5. Laboratory furniture should be sturdy. Open spaces between and under benches, cabinets
and equipment should be accessible for cleaning.
6. Storage space must be adequate to hold supplies for immediate use and thus prevent
clutter on bench tops and in aisles. Additional long-term storage space, conveniently
located outside the laboratory working areas, should also be provided.

7. Space and facilities should be provided for the safe handling and storage of solvents,
radioactive materials, and compressed and liquefied gases.
8. Facilities for storing outer garments and personal items should be provided outside the
laboratory working areas.

xiv
9. Facilities for eating and drinking and for rest should be provided outside the laboratory
working areas.
10. Hand-washing basins, with running water if possible, should be provided in each
laboratory room, preferably near the exit door.
11. Doors should have vision panels, appropriate fire ratings, and preferably be self-closing.
12. At Biosafety Level 2, an autoclave or other means of decontamination should be available
in appropriate proximity to the laboratory.
13. Safety systems should cover fire, electrical emergencies, and emergency shower and
eyewash facilities.
14. First-aid areas or rooms suitably equipped and readily accessible should be available.
15. In the planning of new facilities, consideration should be given to the provision of
mechanical ventilation systems that provide an inward flow of air without recirculation. If
there is no mechanical ventilation, windows should be able to be opened and should be
fitted with arthropod-proof screens.
16. A dependable supply of good quality water is essential. There should be no
crossconnections between sources of laboratory and drinking-water supplies. An
antibackflow device should be fitted to protect the public water system.
17. There should be a reliable and adequate electricity supply and emergency lighting to
permit safe exit. A stand-by generator is desirable for the support of essential equipment,
such as incubators, biological safety cabinets, freezers, etc., and for the ventilation of
animal cages.
18. There should be a reliable and adequate supply of gas. Good maintenance of the
installation is mandatory.
19. Laboratories and animal houses are occasionally the targets of vandals. Physical and fire
security must be considered. Strong doors, screened windows and restricted issue of keys
are compulsory. Other measures should be considered and applied, as appropriate, to
augment security.
Laboratory equipment Together with good procedures and practices, the use of safety
equipment will help to reduce risks when dealing with biosafety hazards. This section deals
with basic principles related to equipment suitable for laboratories of all biosafety levels.
Requirements for laboratory equipment pertinent to higher biosafety levels are dealt with in
the relevant chapters. The laboratory director should, after consultation with the biosafety
officer and safety committee (if designated), ensure that adequate equipment is provided and

xv
that it is used properly. Equipment should be selected to take account of certain general
principles, i.e. it should be:
1. Designed to prevent or limit contact between the operator and the infectious material
2. Constructed of materials that are impermeable to liquids, resistant to corrosion and meet
structural requirements
3. Fabricated to be free of burrs, sharp edges and unguarded moving parts
4. Designed, constructed and installed to facilitate simple operation and provide for ease of
maintenance, cleaning, decontamination and certification testing; glassware and other
breakable materials should be avoided, whenever possible.
Detailed performance and construction specifications may need to be consulted to ensure that
the equipment possesses the necessary safety features.

Essential biosafety equipment

1. Pipetting aids to avoid mouth pipetting. Many different designs are available.
2. Biological safety cabinets, to be used whenever:

xvi
 infectious materials are handled; such materials may be centrifuged in the open laboratory
if sealed centrifuge safety cups are used and if they are loaded and unloaded in a
biological safety cabinet
there is an increased risk of airborne infection
procedures with a high potential for producing aerosols are used; these may include
centrifugation, grinding, blending, vigorous shaking or mixing, sonic disruption, opening
of containers of infectious materials whose internal pressure may be different from the
ambient pressure, intranasal inoculation of animals, and harvesting of infectious tissues
from animals and eggs.
3. Plastic disposable transfer loops. Alternatively, electric transfer loop incinerators may be
used inside the biological safety cabinet to reduce aerosol production.
4. Screw-capped tubes and bottles.
5. Autoclaves or other appropriate means to decontaminate infectious materials.
6. Plastic disposable Pasteur pipettes, whenever available, to avoid glass.
7. Equipment such as autoclaves and biological safety cabinets must be validated with
appropriate methods before being taken into use. Recertification should take place at
regular intervals, according to the manufacturers instructions.

xvii
Guideline for Laboratory Reports
This section describes the format of laboratory report. Students may use any word processor
(such as MSword) they are familiar with to write their lab report and print the final version of
their lab manual. The lab report may vary from experiment to experiment, but in general they
are expected to contain the following:
(a) Title- Title of the experiment should be the same with stated in lab manual
(b) Introduction- Introduction about the experiment
(c) Objectives- Give the objectives of the experiment
(d) Procedure- Give a condensed version of what is in the manual. Please include the
exact weights of reagents/products you measured
(e) Results and Discussion- Give all the results obtained and discussed the results based
on knowledge and theory.
(f) Conclusion- Summary of the results obtained
(g) References

Marking Scheme for Laboratory Reports

This section describes the marking scheme for each report i.e.
Content Mark
Introduction 3
Objectives 3
Procedures 3
Result and Discussion 10
Conclusion 3
References 3

Total 25 pts

Content of Experiment (Body of the text)

The body of the text should be in the following order; Number of experiment, Title,
Introduction, Aim, Procedure, References. Please refer Appendix F for details.

xviii
 EXPERIMENT 1

Mammalian Cell Culture: Aseptic techniques, Thawing cells,

Subculturing of cells in T-flask and Cell Counting

INTRODUCTION
Mammalian cells are commonly used as host cells to produce proteins and biomolecules
which require complex post-translational modification. The routine maintenance of
mammalian cell culture involves several steps which are cell thawing, subculturing and
counting. Mammalian cell culture is very prone to contamination thus aseptic techniques
must be adhered at all times.

OBJECTIVES
1. To thaw frozen mammalian cells
2. To subculture monolayer cells in T-flask
3. To count cells using trypan blue dye exclusion method

METHODS
WORKPLACE PREPARATION
1. Make sure that the surface of biosafety hood is completely clear.
2. Switch on the hood. Swab the surface and inner glass window and with 70%
ethanol.
3. Leave the hood for at least 15 minutes for the ethanol to evaporate and laminar
flow to stabilize.
4. Wear glove and spray with 70% ethanol onto glove. Bring the items which are
required to use such as media, glassware, plasticware and 70% ethanols into the
hood.
5. Arrange the items. Make sure that you can reach them easily during experiment
while also minimizing obstruction of air flow.

1
 6. During the culturing work, minimize movement of hands in and out of the hood.
Swab hands (glove) each time before working in the hood.
7. Loosen all bottle caps in the hood before starting culturing work. Also ensure
pipette aids are working well. Make sure that pipette tip does not touch any
surface to prevent contamination.
8. After completion of experiment, tighten all bottle caps. Remove from hood. Swab
hood surface and inner glass window with 70 % ethanol before switching off the
hood. If necessary, switch on UV lamp overnight to decontaminate the hood.

MAIN EQUIPMENT
1. Biosafety hood
2. Carbon dioxide (CO2) incubator
3. Inverted phase contrast microscope

A) THAWING FROZEN CELLS

Materials:
4. Cell line of interest (e.g. CHO-KI, MCF-7)
5. Water bath
6. Culture medium (e.g. RPMI 1640 medium with 10% (v/v) Fetal bovine serum)
7. Centrifuge tubes
8. Centrifuge
9. Pippetes and pippeter
10. 25 cm3 T-flask

Procedures:
1. Take out a cryotube/ampoule from liquid nitrogen dewar or minus 150C freezer.
Use cryoglove and wear protective clothing.
2. The cryotube must be pre-warmed by rolling in both hands and immersing in a
water bath at 37C for maximum 2 minutes. It must be noted that the seal of tube
must be above the water level in case it burst open upon thawing.
3. Working in a biosafety hood, put 10 ml of medium into a centrifuge tube.

2
 4. Wipe ampoule with a tissue soaked in 70 % alcohol prior to opening. Transfer
thawed cell into the prepared centrifuge tube as above.
5. Centrifuge the tube at 1200 rpm for 3 minutes.
6. Pour off supernatant and resuspend in 10 ml fresh medium. Transfer to T-25
flask.
7. Place the flask in the CO2 incubator at 37C with 5% CO2 and 95% air.

B) SUBCULTURING MONOLAYER CELLS IN T-FLASK

Materials:
1. 70 % confluent monolayer cells in T-flask
2. 25 cm3 T-flask
3. Pipettes and pipette aid
4. Fresh medium (RPMI 1640 medium with 10% FBS)
5. Trypsin or accutase
6. Phosphate buffer saline (PBS)

Procedures:
1. Prepare all the items needed and prepare workplace as stated above.
2. Aspirate or pour off spent media from the 70 % confluent T-flask into waste
beaker.
3. Wash with 5-10 ml of PBS and discard into waste beaker.
4. Pipette a volume trypsin onto the monolayer cells and put flask in CO2 incubator
for 3-5 minutes to detach cells.
5. Put in same volume of fresh media to into the media. Resuspend the media and
inoculum to homogenize.
6. Use 10 ml disposable pippete to withdraw 9 ml of fresh medium and transfer into
a new T-flask.
7. Add 1 ml of inoculum (or any desired cell number) into the new T-flask.
8. Resuspend the media and inoculum to allow the mixture to homogenize.
9. Observe under inverted phase contrast microscope.
10. Place the T-flask in the incubator at 37C with 5% CO2 and 95% air.

3
 C) CELL COUNTING
Materials:
1. Pipettes and pipette aid
2. Micropipette and tips
3. Microcentrifuge tube
4. Hemacytometer and cover slip
5. Trypan blue dye

Procedures:
1. Put 10 l trypan blue into a microcentrifuge.
2. Take a drop of samples (from step B5) and transfer it the microcentrifuge tube.
Mix well by pipetting up and down 10-15 times.
3. Put cover slip onto a clean hemacytometer. Pipette the sample-dye mixture into
the chamber of the hemacytometer. The liquid shall fill up the chamber by
capillarity. Repeat with the other chamber.
4. Put the hemacytometer under inverted microscope for counting purpose.
5. Observe that dead cells are in blue colour while viable cells are translucent.
6. Select a 10X objective on the inverted microscope and focus on the grid lines in
the chamber. Count viable cells lying in the 1 mm2 central grid area (which
contains 25 smaller squares). Count cells systematically from left to right,
avoiding any cells on the three parallel lines on the four borders of the 1 mm2
area. For the subdivisions lines (smaller squares), count cells that lie on the top
and left-hand lines of each square, but not those on the bottom or right-hand lines,
in order to avoid counting the same cell twice.
7. Move to the second chamber and do the second count.
8. Calculate the average of two counts and derive the concentration of cells using

Formula c = n/v
where c = cell concentration (cells/ml)
n = number of cells counted

4
 v = volume counted (ml)
assuming only the central 1 mm2 area is used,
v = 0.1 mm x 1 mm x 1 mm
= 0.1 mm3 = 1 x 10 4 ml
thus; c = n/1 x 10 4 or c = n x 104 cells/ml

9. For total cell population, count all cells (viable and dead cells). For % of cell
viability, use the following formula

% percentage cell viability = (number of viable cells/number of total cell

population)*100

REFERENCES
Freshney, R.I. (2000). Culture of animal cells: A manual of basic technique, 4th edition.
Wiley Liss, New York.

5
 EXPERIMENT 2

Plant Cell and Tissue Culture: Preparation of explants for callus

culture. Plant cell suspension culture.

INTRODUCTION
Plant cell and tissue culture is the cultivation of plant cells, tissues and organs under
aseptic conditions in controlled environments. Plant cell and tissue culture is used widely
in agriculture, horticulture, forestry and biotechnology. There are several types of plant
cell and tissue culture such as callus culture, organ culture and plant cell suspension
culture.

Organogenesis refers to the development of plant organs such as leaves, roots or shoots from
undifferentiated callus. A callus is a mass of "unorganized" plant cells. Callus cultures are
usually initiated by placing a fragment of plant tissue (an explant) on solid culture media
under aseptic conditions. Callus is induced and formed from proliferating cells at the cut
surface of the explant tissue. Depending on the species, callus can be initiated from a
variety of tissues by employing the appropriate growth medium.

Plant cell suspension cultures are rapidly dividing suspensions of cells grown in liquid
medium. In general, suspension cultures grow more rapidly than callus cultures on agar
and are more amenable to experimental manipulation. Most suspension cultures are
comprised of cell aggregates as well as dispersed single cells. The time required to
initiate and establish a cell suspension culture depends on the species of plant and the
growth medium. However, in general, dicot species are easier to establish in cell
suspension culture than monocot species. Cell suspension cultures are usually initiated by
agitation a fragment of in vitro grown callus in a volume of liquid medium on an orbital
shaker.

6
OBJECTIVES
1. To cultivate adventitious shoot from explant (organogenesis)
2. To initiate callus from leaf
3. To establish cell suspension cultures from callus

METHODS
Organogenesis adventitious shoot formation
Plant material
Nicotiana tabacum leaf

Chemicals/Consumables
70% ethanol
10% sodium hypochlorite
Detergent
Murashige and Skoog basal medium (MS) with 1-2 mg/L BAP, 0.1-0.2 mg/L NAA and
2% (w/v) sucrose
8 g/L agar
Distilled water

Apparatus
Petri dish
Forceps
Scalpel
Parafilm
Growth cabinet (25C, 1000-lux intensity, 16 h light, 8 h dark)

Procedure
1. Take leaf explants from potted plants.
2. Gently wash with a mild detergent and then rinse under tap water.
3. Dip each leaf into 70% ethanol for 1 minute.

7
 4. Leaves are then surface sterilized with 10% commercial sodium hypochlorite for
5- 10 min followed by rinsing 3X in sterile distilled water.
5. Place the leaves in sterile Petri dish. Cut leaves into approximately 1 cm2 sections
using sterile forceps and scalpel, without vein in the explant.
6. Place the leaf sections on the following media: MS + 1-2 mg/L BAP + 0.1 0.2
mg/L NAA + 8 g/L agar, pH 5.7.
7. Seal the culture using parafilm. Incubate at 25C under 16 h photoperiod at 1000-
lux light intensity.
8. Observe the shoot formation after 3 weeks and record the data.

Callus formation from plant leaf

Plant material
Nicotiana tabacum leaf
Chemicals/Consumables
70% ethanol
10% sodium hypochlorite
Detergent
Murashige and Skoog basal medium (MS) with 1-2 mg/L 2, 4-D and 2% (w/v) sucrose
8 g/L agar
Distilled water

Apparatus
Petri dish
Forceps
Scalpel
Parafilm
Growth cabinet (25C, dark condition)

Procedure
1. Take leaf explants from potted plants.
2. Gently wash with a mild detergent and then rinse under tap water.

8
 3. Dip each leaf into 70% ethanol for 1 minute.
4. Leaves are then surface sterilized with 10% commercial sodium hypochlorite for
5- 10 min followed by rinsing 3X in sterile distilled water.
5. Place the leaves in sterile Petri dish. Cut leaves into approximately 1 cm2 sections
using sterile forceps and scalpel, without vein in the explant.
6. Place the leaf sections on the following media: MS + 1-2 mg/L 2, 4-D + 8 g/L
agar (MSN), pH 5.7.
7. Seal the cultures. Incubate the culture in dark at 25C. Callus will be produced in
3-4 weeks.

Initiation of suspension culture from callus

Plant material
Nicotiana tabacum callus culture
Chemicals/Consumables
1. 70% ethanol
2. Murashige and Skoog basal medium (MS) with 1-2 mg/L 2, 4-D (MSN) and 2%
(w/v) sucrose
3. Distilled water

Apparatus
1. Forceps
2. Scalpel
3. Parafilm
4. Temperature controlled room (25C)
5. Gyratory shaker, shaking at 125 rpm
6. Bunsen burner

Procedure
1. Cut small pieces of callus of ~0.5 g fresh weight and subculture on the same fresh
medium for proliferation.

9
 2. Prepare MSN + 2% sucrose (pH 5.7) liquid medium without agar. For the
experiment purpose, 15 mL medium is prepared in a 125 mL- Erlenmeyer flask.
Sterilize the opening of a flask with the flame of burner in the laminar flow
cabinet.
3. Transfer a piece of callus to a sterile Petri dish. Gently break the callus of ~2 cm
diameter with forceps into 20-30 small pieces.
4. Transfer the small pieces of callus to the liquid media using forceps.
5. Heat sterilize cap on the flask. Prepare replicate samples.
6. Incubate on a gyratory shaker set at 125 rpm which should be placed in a
temperature controlled room.
7. Subculture every week by transferring 1.5 mL of 7 day-old culture into 15 mL of
fresh liquid medium.

Abbreviations
BAP: benzylaminopurine
NAA: -naphthaleneacetic acid
2, 4-D: 2, 4-dichlorophenoxyacetic acid

Units for solution preparation

The concentration of a particular substance in the media can be expressed in various units
that are as follows:

Units in weight
It is represented as milligram per liter (mg/L)
10-6 = .0 (mg/L) or 1 part per million (ppm)
10-7 = 0.1 (mg/L)
10-8= 0.001 (mg/L) or 1 g/L

Molar Concentration
A molar solution (M) contains the same number of grams of substance as is given by its
molecular weight.

10
1 molar (M) = the molecular weight in g/L
1 mM = the molecular weight in g/L or 10-3 M
1 M = the molecular weight in g/L or 10-6 M or 10-3 M

References
Chawla, H.S. (2002). Introduction to plant biotechnology, 2nd edition. Science
Publishers.
Evans, D.E., Coleman, J.O.D., Kearns, A. (2003). Plant cell culture. Taylor and Francis.

11
 EXPERIMENT 3:

Batch Fermentation of E. coli Harboring Recombinant Enzyme in

Bioreactor

INTRODUCTION

Escherichia coli Bacteria

E. coli is classified as non-photosynthetic and mesophiles bacteria. There are hundreds of
different types of E. coli recognized by the combination of sugars and proteins displayed
on the bacterial surface. E. coli bacilli have long rods without separation when grown
under limiting conditions. Growth kinetic of E. coli (doubling time) has been recognized
at 40C and 0.35 hours. Meanwhile, the minimum, optimum and maximum growth
temperature of E. coli are 10C, 37C, and 45C respectively. As a neutrophile, pH
optimum of E. coli has been identified between 6 and 7 with the lower limit 4.4 and
upper limit 9.0.

There are several advantages of using E. coli. It is one of the most-studied organisms for
recombinant protein synthesis and the best-studied microorganism where its genetics and
physiology are far better understood than any other living organism, which greatly
facilitates genetic manipulations. E. coli also do not require any growth factors: they can
synthesize all essential purines, pyrimidines, amino acids and vitamins, starting with their
carbon source, as part of their own intermediary metabolism. In addition, a wide range of
mutations with specific characteristics and the well-defined vectors and promoters are
available, which has greatly speeded up the development of an appropriate biological
catalyst. Furthermore, it is easy to attain a very high volumetric productivity because E.
coli has a relatively high growth rate and it can reach a very high cell concentration (>50
g dry wt/L), as well as high expression levels by selecting a combination of specific
vector and promoter (about 25% to 50% more of total protein). Finally, the production
cost of protein is low since E. coli can grow on simple and inexpensive media, and the
operational cost of the fermentation process is also relatively low.

12
However there are also disadvantages of using E. coli. One major problem is it does not
normally secrete protein, therefore the active protein present intracellular is limited due to
the either proteolytic degradation or the formation of inclusion body and it makes the
product separation and purification difficult. This problem can be solved by protein
secretion. And also, production of recombinant proteins in E. coli typically, is severely
curtailed under slow-growth conditions, such as during the production stage of fed-batch
cultivation, due to the intense competition between normal cell function maintenance and
recombinant protein production for the limited available metabolic machinery. So, careful
medium selection and controlled growth rate is important.

Growth in Batch Culture

Batch culture is a closed culture system which contains limited amount of nutrient.
Growth phase of microorganism in batch culture consists of four distinct phases; 1) lag
phase, 2) exponential or log phase, 3) stationary phase and 4) death phase. During lag
phase, cell is adapting to the new environment. During this phase there will be no or very
little of growth. Once the cell adapted the new environment and synthesized new
component and ready to divide, exponential phase will started. In this exponential or log
phase the cell grows and divides at maximum rate possible until population growth
ceases as in the stationary phase, the growth curve becomes horizontal. Usually attained
at a population level of around 109 cells per ml. Reasons are nutrient limitation, limited
oxygen availability and accumulation of toxic waste products. And the final phase is,
death phase where decline in number of viable cells, caused by depletion of nutrients and
buildup of toxic wastes.

There is also mathematical consideration of growth. During the exponential phase each
microorganism is dividing at constant intervals. Thus, the population will double in
number during a specific length of time called the generation time or doubling time.
Because the population is doubling every generation the increase in population is always
2n, n is number of generations. The resulting population increases is exponential or
logarithmic.

13
It is important to develop high cell density culture along with high level of enzyme
expression. In a reference to any enzyme production such as phytase[1] or bromelain[2]
using recombinant E. coli, the fermentation process of the production is economically
attractive when high cell density culture is attained. Thus, when producing recombinant
enzyme, the aspect of high cell density culture must be taken into consideration. High cell
density is influenced by process conditions such as temperature, agitation speed, aeration
and acidity of the medium[3].

OBJECTIVE

To study the growth kinetics of E. coli in bench top bioreactor using different control
strategy.

METHODS
Chemical/Biochemical/Consumables
1. Ethanol 70%
2. Sodium hydroxide (NaOH)
3. Hydrochloric acid (HCl)
4. Antifoam
5. Phosphate Biorad protein assay reagent

ES & H Consideration and Hazards

Sodium hydroxide (NaOH) and hydrochloric acid (HCl) are corrosive and should be
handled with care.

Apparatus

Apparatus for bioreactor set-up

2-litre fermenter with DCU tower control equipped with;

1. pH sensor/probe

14
 2. dO2 / DO sensor/probe
3. Temperature sensor/probe
4. Agitator
5. Air sparger
6. Sampling bottle
7. Antifoam, acid and base bottles
8. Silicone tubes

Apparatus for analysis

1. Spectrophotometer
2. Cuvettes
3. Aluminum boat
4. Weighing scale
5. THOMA cell counting chamber
6. Microscope

Preparation of Apparatus
1. Connect pH, dissolve oxygen (DO), antifoam and temperature sensors/probes to DCU
controller.
2. Switch on DCU tower control unit.

Calibration & Standardization

Media Preparation

Glycerol medium
1. Dissolve 1.875 g yeast extract, 3.5 g KH2PO4, 2.5 g glycerol, 5 g peptone and 4.5 g
NaCl in 400 ml of distilled water.
2. Adjust the pH to 7.2 / 7.4 with sodium hydroxide (NaOH) and bring the volume up to
500 ml.

15
LB medium
1. Dissolve 10 g tryptone, 5 g yeast extract and 10 g NaCl 950 ml of dH20
2. Adjust pH to 7.2 / 7.4 with NaOH and bring the volume up to 1 L.

! The media must be autoclaved for sterilization and store at room temperature.

pH Calibration
1. At outside of the vessel, calibrate pH probe using standard solution of pH 4.0, 7.0 and
10.
2. Confirm the values at DCU controller.

DO Calibration
1. Equipped the bioreactor vessel with air sparger, sampling bottle, 1M sodium
hydroxide (NaOH) solution, 1M hydrochloric acid (HCl) solution and antifoam
solution before autoclave.
2. Screw the air sparger and probes (i.e. pH, temperature and DO probes) tightly to the
top cover of fermenter.
3. Fill in the media into the vessel and closed the top by screwing back the top plate.
4. Make sure all silicone tubes are tightly tied before autoclave.
5. Autoclave the bioreactor at 121oC for 15 min.
6. After autoclave, connect the DO probe (as well as other probes) to DCU controller
and allow DO probe to polarize for approximately 6 hours.
7. After 6 hours, to calibrate DO probe to 0%, briefly disconnect the cable from the
control station, or sparge nitrogen into medium at approximately 1 VVM (vessel
volume per minute) until the value stabilizes near zero.
8. Confirm the value at DCU controller.
9. To calibrate to 100% calibration, increase agitation to approximately 200-400 rpm
and increase the airflow to 0.5-1 VVM.
10. Confirm the 100% calibration at DCU controller.

16
Procedure

Preparation of E. coli cell culture

1. Take out the E. coli stock culture from -80C freezer and thaw the vial.
2. Prepare 10 ml of media (culture broth) in a bijou bottle.
3. Transfer E. coli by using a wire loop into culture broth.
4. Incubate the culture in an incubator shaker (rotary) for 12 hours at 37C, 250 rpm.

Preparation of inoculum

The first requirement for a batch scale process is sufficient inoculum culture to start full-
scale process. A one tenth volume (10%) inoculum will be used to achieve rapid growth
without a lag phase.
1. Using aseptic technique, transfer 1ml of E. coli liquid culture into a bijou bottle
containing 9 ml of media, and incubate for 4 hours at 37 C.

Cell Cultivation in Shake flask

1. Bring the prepared inoculum into a laminar flow hood.

2. Using aseptic technique, pipette 90 ml of media into each labeled shake flasks.
3. Transfer 10 ml of inoculum into the shake flask, resulting in final volume of 100 ml.
4. Cap the shake flask and swab with ethanol 70%
5. Incubate the flasks in a thermostatted rotary shaker set at 300 rpm and 37C for 4
hours before inoculate into bioreactor.

Bioreactor Experimental Design

1. Set up bench top bioreactor, 2-litre B-Braun fermenter, as shown in Figure 11.2 after
autoclave and calibrations.
2. By using aseptic technique, connect the connection tube of the inoculum flask to
inoculum pipeline of the bioreactor vessel.

17
3. Clamp-off the connection tube and flow the inoculum from flask through connection
tube by gravity action into the culture vessel.
4. Lift the inoculum flask high enough to ensure stable flow of the inoculum into
bioreactor vessel.
5. After all the inoculum has been transferred into the bioreactor vessel, aseptically
disconnect the connection tube from bioreactor vessel.
6. Set dissolved oxygen (DO), airflow and pH at DCU control unit to desired set point.
A proposed design of experiment is shown in Table 11.1.
7. Monitor dissolved oxygen level, temperature, pH, air pressure as well as the CO2 and
O2 (air outlet) during the fermentation process.

! Experiment must be aseptically handled to avoid contamination

Figure 3.2. A complete set-up of 2-litre fermenter with DCU controller.

Table 3.1. Reference for designed experiment for bioreactor condition optimization
F1 F2 F3
Parameters
(pO2, %) Temperature pH
Run 1 40 37 7.2
Run 2 40 35 7.4
Run 3 80 37 7.4
Run 4 80 2 7.2
F: Factor

18
Sampling

1. Close the airflow supply.

2. Connect the plunger/nozzle of syringe to outlet tubing of sampling bottle.
3. Remove hose/tube clamp from the connected tube to the culture vessel.
4. Clamp-off the outlet tubing of sampling bottle.
5. Pull slowly the plunger of syringe to suck the required amount of sample through the
sampling tube into the sampling reservoir/bottle.
6. Push back a little of the syringe plunger to empty the sampling tube from culture.
This is to avoid residues of previous samples in the sampling tube.
7. Remove hose/tube clamp from outlet tubing of sampling bottle and clamp-off the
hose/tube that connected sampling bottle to culture vessel.
8. Push slowly the plunger back to its original position to empty the sampling
bottle/reservoir and collect the sample into a collector (bijou bottle or yellow-capped
test tube).
9. Use flame and ethanol 70% during sample collecting for aseptic purpose.
10. Withdraw 5 ml sample for every 30 minutes into a bijou bottle during fermentation
for measuring the optical density/absorbance (OD, A600), substrate (glucose), and
total cell number.
11. Withdraw 5 ml samples for every 60 minutes into a yellow-capped test tube, and
centrifuged at 5000 rpm before undergo for enzyme activity and protein concentration
assay.

Methods of Analysis
Optical density (OD)
1. Take sample every 30 minutes.
2. Transfer 2 ml of culture into 3 ml cuvette.
3. Measure OD by using spectrophotometer at 600 nm; use media as blank.

Total Cell Number (TCN)

1. Take sample for every 1 hour.

19
2. Put 10 l of culture into THOMA counting chamber.
3. Calculate the cells under microscope at 40X
4. Do not count the cells at the border.
Count cell in the area labeled A,B,C, and D

Average cell number, N = A+B+C+D

A B 4

Total cell number,

TCN (cell/ml) = average cell number (N)
Volume chamber (V)

Volume, V (ml) = Area x depth

Area = 0.0025mm2
Depth = 0.1 mm

C D Thus, V = 0.0025 mm2 x 0.1 mm

1000 mm3/ml
= 2.5 x 104 ml

Dry Cell Weight (CDW)

1. Prepare the aluminum boat and dry in the oven at 80C for 6-8 hours.
2. After that, place the boat in a desiccator for 30 minutes and measure it weight (initial
weight, A).
3. Take 1.5 ml sample and put into centrifuge tube.
4. Centrifuge the sample at 5000 rpm for 30 min.
5. Discard the supernatant and collect the pellet.
6. Suspend the pellet in 1 ml distilled water and vortex for 30 sec to mix it well.
7. Transfer the solution into aluminum boat and dry in oven at 80C overnight.
8. Place the boat in a desiccator and immediately weight the aluminum boat (final
weight, B).

CDW = Final weight (B) Initial weight (A)

! Use forceps not hand when handling boat

Total protein (TP)

1. Take 1.5 ml sample and put into centrifuge tube.
2. Centrifuge sample at 5000 rpm for 30 minutes.
3. Discard the supernatant (will be used for glucose analysis).
4. Suspend the pellet in 1 ml distilled water and vortex for 30 sec to mix well.
5. Lyse the cell by using sonicator at 60% amplitude by 1 cycle for 15 sec, 3 times.
6. Centrifuge the sample at 10000 rpm for 30 minutes and collect the supernatant.

20
7. Take 200 l of supernatant; add with 1200 l of distilled water and 400 l of
Bradford into a cuvette.
8. Measure the OD by using spectrophotometer at 595 nm. For blank, add 1600 l of
distilled water and 400 l of Bradford reagent.
9. Calculate the protein concentration based on the protein standard curve (BSA
standard curve). Sample of standard curve is shown in Figure 11.3.

Figure 3.3. BSA standard curve

Glucose analysis
1. Transfer the supernatant into cuvette, put onto turntable of Innova YSI 2700 Select
Biochemical Analyzer for direct analysis of glucose (dextrose) concentration.
2. The glucose analysis is based on Glucose Oxidase that has been immobilized in the
YSI Dextrose Membrane (YSI 2365).

21
REPORT

Parameters for Run 1

Factors Responses
Time F1 F2 F3 OD CDW Protein Enzyme
(min) (pO2, %) Temp. pH
30
60
90
120
150
180
210
240
270
300
330
360

DISCUSSION
1. Plot the graph and compare for Run 1, 2, 3 and 4.
2. Determine observed yields YX/S, YP/S and YP/X.
3. Which condition to get the highest , td and yield coefficient?

REFERENCES

[1] N. Samsudin, A.-E.F. Gad, H.M. Salleh, Site Directed Mutagenesis to Improve E.
Coli Phytase Activity for Animal Feed, Australian Journal of Basic & Applied Sciences,
6 (2012).
[2] M.J.A. Jamaluddin, A. Amid, A.S. Azmi, M.E. Othman, Screening of Important
Autoinduction Medium Composition for High Biomass Production of E. coli Expressing
Recombinant Bromelain, Journal of Pure and Applied Microbiology, 8 (2014) 741-750.
[3] A.S. Azmi, C.G. Ngoh, M. Mel, Prediction of significant factors in the production of
ethanol by ragi tapai co-culture using Taguchi methodology, African Journal of
Biotechnology, 10 (2013) 18833-18841.

22
 EXPERIMENT 4:

Cell Disruption by High Pressure Homogenizer

INTRODUCTION

Homogenization technology is based on the use of pressure on liquids to subdivide

particles or droplets present in fluids into the very smallest sizes (submicron) and create a
stable dispersion ideal for further processing. Homogenization features a high
concentration of energy released on processed liquids by a combination of fluid
mechanical effects like local cavitation, turbulence, shear and impact to achieve a
homogeneous particle size distribution.

Figure 4.1 : Product before and after homogenization

The process is carried out in a special design valve, which represents the core of the
homogenizing process. The passage of fluid through the minute flow passages in the
valve under high pressure and controlled flow action subjects the fluid to conditions of
high turbulence and shear that creates the most efficient mechanism for particle and
droplet size reduction.

23
The high pressure homogenizer is a machine consisting of a high pressure plunger type
pumping section able to pump liquid products of low and high viscosity up to a defined
pressure level. This is combined with a specially designed adjustable valve able to create
the pressure and thereby the fluid dynamics effects used to micro size the processed
fluids.

Figure 4.2: Mixing chamber and homogenizing valve

OBJECTIVES
1. To investigates the effects of operating conditions in the release of intracellular
enzyme by high-pressure homogenization of E.coli suspension.
2. To understand the principles of homogenization and acquire relevant data for
enzyme release during operation of the homogenizer.

METHODS

Apparatus
Spectrophotometer, homogenizer, cuvettes, Eppendorf tubes, beaker, temperature probe

Reagent and Materials

100mM phosphate buffer pH 7 and pNP--D-glucuronide

24
ES & H Consideration and Hazard
1. The electrical power to the Bioprocess Engineering II can be shut off at the corner
of the laboratory, these will be identified
2. Isolation points for each of the equipment will be identified before experimental
work begins
3. Entry to the process area you are required to wear laboratory coat and a pair of
safety glasses
4. Note there is an emergency shower in the Bioprocess Engineering Laboratory at
the wash Station.
5. Good microbial Practice should be carried out in this area, e.g. use of latex gloves
for hand protection and washing of your hands at the wash station when leaving
the area
6. All work within the Bioprocess Engineering Laboratory will be supervised. Please
inform the supervisor or demonstrator if you need to leave the area and report
back on return
7. Do not touch any experimental equipment unless you have been asked to do so by
your supervisor or demonstrator
8. be aware of other operations within the Bioprocess Engineering Laboratory II

Preparation of Apparatus

The experimental system

The NS2006L Pony homogenizer is a pilot laboratory unit designed for continuous
operation up to 1500 bar, suitable for microsizing tests on many different fluids. It is
compact design for easy installation, use and maintenance and suitable for viscous
pumpable products. Homogenizing valve is a rupture type sharp-edge homogenizing
valve in special abrasion and wear resistant material and its pressure can be adjusted
manually by means of a hand-wheel. It has second homogenizing stage included with
flat profile homogenizing valve. This homogenizer is able to operate at an absolute
maximum pressure of 1500 bar. A minimum of 500ml sample is needed to operate this
machine and feeding into stainless steel feed funnel (5 L capacity) with connecting

25
pipework to the feeding pump (inclusive of 10 Mesh filter and Tri-clamp fittings). Lobe
type pump driven by a separate electric motor with built in variable speed drive unit can
be used to adjust the feed pressure according to the product viscosity. The standard
operation (SOP) of the machine is included in Appendix B.

Experimental Protocol

Run Pressure Pump Cycle

Speed
1 Low Low Low
2 Low High High
3 High Low High
4 High High Low

The released E. coli suspension will be collected and 100 mM phosphate buffer pH 7 will
be added into the suspension with the same volume.
Analyse release data to predict the breakage to be obtained with different cycle and flow
rate.

Enzyme activity assay

1. 350l of 100mM phosphate buffer pH 7 and 50l pNP--D-glucuronide (4mM

stock), which is the substrate, is pipetted into Eppendorf tubes and pre-incubated
at 75C for 3 minutes.
2. 100l supernatant is added into the tubes and then incubated for 5 minutes. The
reaction is stopped after 5 minutes by adding 500l of 200mM sodium carbonate
(Na2CO3) solution into the tubes.
3. The Eppendorf tubes then are centrifuged at 5000g for about 3 to 5 minutes. The
content of the Eppendorf tubes then transferred into cuvettes.
4. The spectrophotometer wavelength is set at 405nm and calibration of the
spectrophotometer is done using a blank, which consist of 350l phosphate
buffer, 50l pNP--glucuronide, and 100l distilled water.

26
REPORTS
Analysis
Cell Concentration: 9 cP

Run Pressure Pump Cycle Cell Protein Enzyme

Speed rupture, Conc. Conc.
(%) (g/L)
1 Low Low Low
2 Low High High
3 High Low High
4 High High Low

1. Estimate the Rm (the maximum amount of enzyme release)

2. Using data for enzyme release at the homogenizer pressure examined evaluate the
rate constant, k, for cell disruption using derived first order equation below:

3. Briefly discuss the pros and cons of using this method compare to other lysis
method and what parameters are crucial to the operation of the homogenizer.

REFERENCES
Harrison, R. G., Todd, P. W., Rudge, S. R., Petrides, D. (2003), Bioseparations Science and
Engineering, Oxford University Press.

Belter P. A., Cussler, E. L. & Hu, W. H. (2000), Bioseparations: Downstream Processing for
Biotechnology, John Wiley & Sons.

Dechow, F. J. (1989), Separation and Purification Techniques in Biotechnology, Noyes

Publications.

Ladisch, M. R. (2001), Bioseparations Engineering: Principles, Practice, and Economics, John

Wiley & Sons.

27
 EXPERIMENT 5

Cross flow filtration process

INTRODUCTION

Filtration is a pressure driven separation process that uses membranes to separate

components in a liquid solution or suspension based on their size and charge differences.
Filtration can be broken down into two different operational modes that are Normal Flow
Filtration and Tangential Flow Filtration. Normally, membrane based Tangential Flow
Filtration (TFF) unit operations are used for separation of cells from a product,
the concentration of cells, the removal of cell debris from cell, the concentration of
protein solutions, the exchange or removal of salts or salts in a protein solution, and the
removal of viruses from protein solutions [1, 2]. Tangential Flow Filtration is also called
as Cross Flow Filtration (CFF).

Cross flow filtration can be further subdivided into categories based on the size of
components being separated. One of the most widely used to separate protein from buffer
components for buffer exchange, desalting, or concentration is ultrafiltration.

In a CFF unit operation, a pump is used to generate flow of the feed stream through the
channel between two membranes surfaces. A schematic of a simple CFF is shown in
Figure 5.1. During each pass of fluid over the surface of the membrane, the
applied pressure forces a portion of the fluid through the membrane and into the filtrate
stream.

The result is a gradient in the feedstock concentration from the bulk condition at
the center of the channel to the more concentrated wall conditions at the membrane
surface. There is also a concentration gradient along the length of the feed channel from
the inlet to the outlet (retentate) as progressively more fluid passes to the filtrate side. In
Figure 4.1 illustrate the flows and forces describe with the parameters defined as; QF is

28
feed flowrate (L/h), QR is retentate flow rate (L/h), Qf is filtrate flow rate (L/h); Cb
is component concentration in the bulk solution (g/L), Cw is component concentration at
the membrane surface (g/L); Cfis component concentration in the filtrate stream
(g/L); and TMP is applied pressure across the membrane (psi).

Figure 5.1. Schematic of CFF system.

Before implementing of a CFF step for protein enzyme processing, the parameters
at which the step will operate must be defined. Key parameters to determine are cross
flow rate, transmembrane pressure, filtrate control, membrane area, and diafiltration
design. These parameters are typically arrived at through a combination of rule of
thumbs, experimentation, and consideration process requirements and limitations [2].

In filtration the flow of fluid through the filter medium is proportional to the
applied pressure difference (P) across the membrane. The rate of flow is dictated by the
resistance of the filter to the flow of fluid and any resistance associated with the trapped
particulate.

Permeability (Flux) = V / (t x A x P) (1)

29
where V, t, A and P denoted for permeate volume, time, surface area of
cartridge membranes and pressure gradient, respectively. In this event, the filtrate flux
is accurately predicted by the Hagen-Poiseulle Equation, which describes liquid flow
through cylindrical pores.

(2)

The Hagen-Poissuelle Equation, shown above, states that liquid flux is proportional to the
trans-membrane pressure (P) and inversely proportional to the liquid viscosity
(), which is controlled by the cell concentration and the temperature. Therefore,
increasing the pressure or the temperature results in an increase in the flux. When
the filter cake density is high, a gel layer does form, and filtrate flux decreases as
the density of the filter cake and retained-cell concentration increase.

OBJECTIVE

To study the cross flow filtration mechanism and factors affecting filtration process.

PROCEDURE

A. MICROFILTRATION
Although microfiltration (MF) membrane cartridges are shipped dry, without preservative
solutions, it is prudent to rinse cartridges before first process exposure or heat
sterilization. Follow New Cartridge Rinse Procedure for at least 5 minutes at 5 psig (0.3
barg) inlet pressure.
Sodium Hydroxide Sanitization and Depyrogenation
The initial "flush-up" of a cartridge, whether UF or MF, is an excellent time to circulate
an NaOH solution for sanitization and to depyrogenate the cartridge. A typical procedure
follows. This procedure should be optimized onsite to meet your particular needs.

30
1. Use clean water and make up a 0.1 to 0.5N NaOH solution. 100 ppm NaCI
will enhance results.
2. Recirculate solution 50oC, 5 to 10 psig inlet, 1 to 5 psig outlet for 30 to 60 minutes.
Make sure the NaOH solution contacts all areas within the cartridge.
3. Thoroughly drain the cartridge.
4. Flush to drain with purified water or buffer until pH on both permeate and retentate is
neutral.

B. ULTRAFILTRATION
Removal of Glycerol Preservative
Ultrafiltration (UF) membrane cartridges are pretreated with an alcohol/glycerol solution
within the pore structure to prevent drying of the membrane. This mixture
enhances wetting but may cause the fibers to appear wavy. Trace amounts of alcohol
(IPA) may remain when the cartridges are shipped. The glycerol must be thoroughly
rinsed from the cartridge prior to use. In addition to preventing drying, the glycerol
minimizes entrained air within the pore structure of the membrane wall which may
become "locked-in" reducing permeability until the air has been displaced by
liquid. Glycerol removal and "wetting out" will occur simultaneously when
performing the New Cartridge Rinsing Procedure.

New Cartridge Rinsing Procedure (Recommended for all UF membranes)

The New Cartridge Rinsing Procedure should be performed on all ultrafiltration
cartridges.
1. Use clean water (WFI or 10,000 NMWC UF permeate).
2. Adjust average transmembrane pressure of cartridge to 15 psig for 1,000 NMWC
and 3,000 NMWC pore sizes; 10 psig for 5,000 NMWC through 30,000 NMWC
pore sizes; and 5 psig for larger pore sizes.
3. Be certain retentate flow rate is at least 1/1Oth of the permeate flow.
4. Discharge both retentate and permeate to drain.

31
 5. Use room temperature or warm (up to 50oC) water for rinsing. Cold water will be
less effective. Addition of 100 ppm NaOCI to flush water will enhance glycerol
removal.
6. Continue rinsing for 90 minutes. Make sure NaOCI has been rinsed out
thoroughly before introducing process solution.

Procedure of experimental run for microfiltration/ultrafiltration

Experimental Run for Microfiltration/Ultrafiltration

Batch F1 F2 F3 P inlet Poutlet Viscosity TMP Optimum
(Random) (cP) (psi) Flux
(l/h.m2)
Run 1 L L L

Run 2 L H H

Run 3 H L H

Run 4 H H L

1. Insert the membrane at its position. Make sure the equipment can be run well and
smoothly.
2. The membrane is wetted by flushing 1 L deionized water into the system.
Run the filtration (Follow the recommendation of TMP using deionized water) and
measure the time for every 50 ml water from permeate tubing. Check the pH. It is
supposed to be pH neutral.
3. Run the filtration system using a sample at recommended condition.
4. The permeation rate is measured by putting a volumetric cylinder at the end
of permeate pipe and determine the time for every 50 ml sample liquid.
5. The filtration rate is measured according to the table and graph is drawn
6. Then, after finish, the cross flow as cleaned by 0.5 NaOH for 30 to 60 minutes.
7. Flush with deionized water (many times), check the permeation rate and water
pH.

32
Once, the permeation rate is 80% achieved its mean that the cleaning is confirmed.

Vol (mL) Time (s) Flow rate Flux

(mL/s) (L/m2.h)
50
100
150
200
250




1000
Note: Refer the membrane description for each membrane area.

ANALYSIS
1. Plot the graph of filtration rate (ml/s) versus filtration time (min)
2. From the graph, determine the three different zones on the graph. (Pore blocking
zone, Cake formation zone and Constant rate zone).
3. Compare the results of the four different operating conditions.
4. Discuss briefly the factors that usually affect the flux value during the filtration
process: a) concentration polarization, b) cake formation at membrane surface
and c) fouling.
5. From the theory, we know that as pressure increases, the shear increases and the
flux value increases (Situation A). Based on this two graph below, why the flux
value is reduced (Situation B) when further increase the TMP and shear rate (in
membrane structure in term of molecular orientation at active layer skin).

33
[1] Wang W.K. 2001. Membrane Separations in Biotechnology: Second Edition, Revised

and Expanded. Marcel Dekker, Inc. United States of America.

[2] Iverson, K. 2003. Troubleshooting Tangential Flow Filtration. CEP magazine,

Reactions and Separation; June, pp55-56

[3] Winston Ho, W.S and Sirkar K.K. (1992). Membrane Handbook. New York: Van

Nostrand Reinhold.

34
 EXPERIMENT 6

Measurement of the Chemical Oxygen Demand (COD)

INTRODUCTION

Chemical Oxygen Demand (COD) is widely used to estimate the amount of chemically
oxydiseable matter in wastewater. It is measurement of the oxygen equivalent of the
materials present in the wastewater that are subject to oxidation by strong chemical
oxidant (e.g. dichromate). COD differs from BOD in that it measures the oxygen demand
to digest all organic content, not just that portion which could be consumed by biological
processes.

COD is an important, rapidly measured variable for the approximate determination of the
organic matter content of water samples. Some water samples may contain substances
that are difficult to oxidise. In these cases, because of incomplete oxidation under the
given test methods, COD values may be a poor measure of the theoretical oxygen
demand. It should also be noted that the significance of the COD value depends on the
composition of the water studied.

The test is performed by adding the oxidizing solution of a dichromate salt (e.g.
potassium dichromate, K2Cr2O7) to a sample, boiling the mixture on a refluxing apparatus
for two hours, and then titrating the amount of dichromate remaining after the refluxing
period. The titration procedure involves adding ferrous ammonium sulphate (FAS), at a
known normality, to reduce the remaining dichromate. The amount of dichromate
reduced during the test--the initial amount minus the amount remaining at the end--is
then expressed in terms of oxygen. The test has nothing to do with oxygen initially
present. It is a measure of the demand of a solution or suspension for a strong oxidant.

35
The oxidant will react with most organic materials and certain inorganic materials under
the conditions of the test. For example, Fe(II) and Mn(II) will be oxidized to Fe(III) and
Mn(IV), respectively, during the test.

Generally, the COD is larger than the BOD exerted over a five-day period (BOD5), but
there are exceptions in which microbes of the BOD test can oxidize materials that the
COD reagents cannot. For a raw, domestic wastewater, the COD/BOD5 ratio is in the
area of 1.5-3.0/1.0. Higher ratios would indicate the presence of toxic, non-
biodegradable or less readily biodegradable materials.

The COD test is commonly used because it is a relatively short-term, precise test with
little interference. However, the spent solutions generated by the test are hazardous. The
liquids are acidic, and contain chromium, silver, mercury, and perhaps other toxic
materials in the sample tested. For this reason laboratories are doing fewer or smaller
COD tests in which smaller amounts of the same reagents are used.

OBJECTIVE

To estimate the oxygen demand of organic matter when it is subjected to oxidation by a

strong chemical oxidant in wastewater and tap water samples.

METHODS

Reagents/Apparatus:
Method 1: Titration Method
1. Ferroin Indicator Solution.
2. Ferrous Ammonium Sulfate solution.
3. Potassium Dichromate Standard Solution.
4. Sulfuric acid.

36
 5. Titration unit.

Method 2: Photometric Method

1. TestN Tube Reagent for COD,

2. Deionized water.
3. COD Reactor/Digester.
4. Spectrophotometer.

Procedure
Procedure for Method 2:
5. Homogenize 100 ml of sample for 30 seconds in a blender.
6. Turn on the COD Reactor. Preheat to 150 C. Place the plastic shield in front of the
reactor.
7. Remove the cap of COD Digestion Reagent Vial for the appropriate range:

Sample Conc. Range(mg/l) 0 to 40 0 to 150 0 to 1500 0 to 15000

COD Digestion Reagent Vial Ultra Low Low High High Range
Type Range Range Range Plus

4. Hold the vial at 45-degree angle. Pipet 2 ml (0.2 ml for the 0-1500 mg/l range) of
sample into the vial.

5. Replace the vial cap tightly. Rinse the outside of the COD vial with deionized water
and wipe the vial clean with towel paper.

6. Hold the vial by the cap and over a sink. Invert gently several times to mix the
contents. Place the vial in the preheated COD Reactor. Note that the vial will become
very hot during mixing.
7. Prepare a blank by repeating steps 3-6, substituting 2 ml deionized water for the
sample.
8. Heat the vial for 2 hours.

37
9. Turn the reactor off. Wait for about 20 minutes for the vials to cool to 120 C or less.
10. Invert each vial several times while still warm. Place the vials into a rack. Wait
until the vials have cooled to room temperature.
10. Note that if colour of the reacted sample is blue-green then repeat the test with a
diluted sample.
11. Switch On the spectrophotometer and select the program for COD test.
12. Clean outside of the vial that contain deionized water and put into the
spectrophotometer. Press zero.
13. Clean outside of the vial that contain sample. Put into the spectrophotometer and take
reading in mg/L.

38
 EXPERIMENT 7

Measurements of pH

INTRODUCTION

The term "pH" was originally derived from the French term "pouvoir hydrogne," in
English, this means "hydrogen power." The term pH is always written with a lower case
p and an upper case H. pH represents the effective concentration (activity) of hydrogen
ions (H+) in water. The activity of hydrogen ions can be expressed most conveniently in
logarithmic units. pH is defined as the negative logarithm of the activity of H+ ions:

pH = -log [H+]

where [H+] is the concentration of H+ ions in moles per litre. Because pH is defined as
log [H+], pH decreases as [H+] increases (which will happen if acid is added to the water).
Since pH is a log scale based on 10, the pH changes by 1 for every power of 10 change in
[H+]. The pH scale ranges from 0 to 14.

The pH of water can be measured with a pH meter, which is an electronic device with a
probe or sensor. The probe contains an acidic aqueous solution enclosed by a glass
membrane that allows migration of H+ ions. The electrical potential of the glass electrode
depends on the difference in [H+] between the reference solution and the solution into
which the electrode is dipped. pH can also be measured with pH paper or by adding a
reagent (indicator solution) to the water sample and recording the color change. The U.S.
Environmental Protection Agency (U.S. EPA) sets a secondary standard for pH levels in
drinking water: the water should be between pH 6.5 and 8.5.

39
OBJECTIVES
1. To conducted calibration of pH meter.
2. To measure the pH of wastewater samples using pH meter.

METHODS
Reagents/Apparatus:

1. Buffer solution of pH 4.1, 7 and 9.1

2. Deionized water

3. 25 ml beaker

4. pH meter

5. Conductivity meter.

Procedure

1. Prepare the HACH pH meter as directed in the instrument manual.

2. Calibrate (standardized) the meter for pH measurements using buffer solutions as in
the instrument manual.
3. Rinse the electrode thoroughly with deionized water.
4. Pipet about 25 ml of the sample into the beaker.
5. Immerse the tip of the electrode in the sample.
6. While stirring the sample, press the dispenser button once and read the pH value.
7. Rinse the probe thoroughly with deionized water after each measurement.

40
 Experiment 8

Determination of Total Suspended Solids (TSS), Volatile Suspended

Solids (VSS).

INTRODUCTION

Total Suspended Solids (TSS) is solids in water that can be trapped by a filter. TSS can
include a wide variety of material, such as silt, decaying plant and animal matter,
industrial wastes, and sewage. High concentrations of suspended solids can cause many
problems for stream health and aquatic life.

High TSS can block light from reaching submerged vegetation. As the amount of light
passing through the water is reduced, photosynthesis slows down. Reduced rates of
photosynthesis causes less dissolved oxygen to be released into the water by plants. If
light is completely blocked from bottom dwelling plants, the plants will stop producing
oxygen and will die. As the plants are decomposed, bacteria will use up even more
oxygen from the water. Low dissolved oxygen can lead to fish kills. High TSS can also
cause an increase in surface water temperature, because the suspended particles absorb
heat from sunlight. This can cause dissolved oxygen levels to fall even further (because
warmer waters can hold less dissolved oxygen, DO), and can harm aquatic life in many
other ways. High TSS can cause problems for industrial use, because the solids may clog
or scour pipes and machinery.

To measure TSS, the water sample is filtered through a washed, dried and pre-weighed
filter paper. The residue retained on the
the weight of the filter no longer changes. The increase in weight of the filter represents
the total suspended solids.

Volatile suspended solids are those solids lost on ignition (heating to 550C.) They are
useful to the treatment plant operator because they give a rough approximation of the

41
amount of organic matter (biomass) present in the solid fraction of wastewater, activated
sludge and industrial wastes.

OBJECTIVE
To measure the total suspended solids, volatile suspended solids and fixed suspended
solids from wastewater and river water samples.

METHODS

Reagents/Apparatus:

1. 10 ml cylinder, 100 ml cylinder, Pipet, Analytical balance.

2. Desiccators.
3. Aluminum dishes.
4. Magnetic filter holder.
5. 47 mm diameter filter paper.
6. 250 ml filter flask.
7. Deionized water.
8. Forceps for filter handling.
9. Vacuum pump.
10. Oven.
11. Muffle furnace.

(i) Experiment for measuring Total Suspended Solids (TSS, Nonfiltrate Residue):

Procedure:

1. Weigh a filter (Weight of the filter before filtration is A).

2. Place the pre-weighed filter into the filter holder with the wrinkled surface up.
3. Place the filter holder assembly in the 250 ml filter flask, and wet the filter with
deionized water to ensure adhesion to the holder.

42
4. Transfer 100 ml of well mixed water sample to the filtering apparatus, while applying
a vacuum, and follow that with 3 separate 10 ml washings of deionized water.
5. Dry the filter at the oven for 60 minutes at 103-105 C.
6. Take the filter out the oven and allow it to cool to room temperature in desiccators.
7. Weigh the filter, after drying to the nearest 0.1 mg using an analytical balance
(Weight of the filter after drying is B).

Calculations:

Total Suspended Solids (mg/l) = 1000 (B A)

Sample Volume (ml)

ii) Experiment for measuring Volatile Suspended Solids (VSS) and Fixed Suspended
Solids (FSS):

Procedure:
8. Weigh an aluminum dish to the nearest 0.1 mg using an analytical balance (C).
9. Transfer the filter from step 7 of the TSS procedure into the pre-weighed aluminum
dish.
10. Weigh the dish with filter to the nearest 0.1 mg using an analytical balance (Weight
of the dish and filter and solids before ignition is D).
11. Place the aluminum dish in muffle furnace at 550 C for 30 minutes.
12. Take the dish out of the furnace and let it cool in desiccators.
13. Weigh the dish to the nearest 0.1 mg using an analytical balance (weight of the dish
and filter and solids after ignition is E).

Calculations:
Volatile Suspended Solids (mg/l) = 1000 (D E)
Sample Volume (ml)

Fixed Suspended Solids (mg/l) = 1000 (E A C)

Sample Volume (ml)

43
 EXPERIMENT 9

Determination of Total Dissolved Solids (TDS).

INTRODUCTION

Total Dissolved Solids (TDS) are solids in water that can pass through a filter (usually
with a pore size of 0.45 micrometers). TDS is a measure of the amount of material
dissolved in water. This material can include carbonate, bicarbonate, chloride, sulphate,
phosphate, nitrate, calcium, magnesium, sodium, organic ions, and other ions. A certain
level of these ions in water is necessary for aquatic life. Changes in TDS concentrations
can be harmful because the density of the water determines the flow of water into and out
of an organism's cells. However, if TDS concentrations are too high or too low, the
growth of many aquatic lives can be limited, and death may occur.

Similar to TSS, high concentrations of TDS may also reduce water clarity, contribute to a
decrease in photosynthesis, combine with toxic compounds and heavy metals, and lead to
an increase in water temperature. TDS is used to estimate the quality of drinking water,
because it represents the amount of ions in the water. Water with high TDS often has a
bad taste and/or high water hardness, and could result in a laxative effect.

To measure TDS, the water sample is filtered, and then the filtrate (the water that passes
through the filter) is evaporated in a pre-weighed dish and dried in an oven at 180
until the weight of the dish no longer changes. The increase in weight of the dish
represents the total dissolved solids, and is reported in milligrams per liter (mg/l).

The U.S. Environmental Protection Agency (U.S. EPA) sets a secondary standard of 500
mg/l TDS in drinking water. High TDS concentrations can produce laxative effects and
can give an unpleasant mineral taste to water. High TDS concentrations in water are also
unsuitable for many industrial applications.

44
OBJECTIVE
To measure the total dissolved solids, volatile dissolved solids and fixed dissolved solids
from wastewater and river water samples.

METHODS

Reagents/Apparatus:
1. 50 ml cylinder, 100 ml cylinder, Pipet, Deionized water, Magnetic filter holder.
2. 47 mm diameter filter paper.
3. 250 ml filter flask.
4. Vacuum pump.
5. Analytical balance.
6. Desiccators.
7. Aluminum dish.
8. Forceps for filter handling.
9. Oven.
10. Muffle furnace.

(i) Experiment for measuring Total Dissolved Solids (TDS, filtrate residue):

Procedure:
1. Weigh an aluminum dish to the nearest 0.1 mg using an analytical balance (A).
2. Place a filter into the filter holder with the wrinkled surface up.
3. Place the filter holder assembly in the 250 ml filter flask, and wet the filter with
deionized water to ensure adhesion to the holder.
4. Transfer 100 ml of well mixed water sample to the filtering apparatus, while applying
a vacuum followed by 3 separate 10 ml washings of deionized water.
5. Slowly release the vacuum from the filtering flask and transfer 50 ml of filtrate (i.e.
the solution in the flask) to the pre-weighed aluminum dish (A).
6. Evaporate and dry the filtrate in an oven at 1802C for about 6 hours.

45
7. Take the dish out the oven and allow it to cool to room temperature in desiccators.
8. Weigh the dish to the nearest 0.1 mg using an analytical balance (Weight of the dish
after evaporation is B).

Calculations:
Total dissolved solids (mg/l) = 1000 (B A)
Sample Volume (ml)

46
 EXPERIMENT 10

Determination of Biological Oxygen Demand (BOD5)

INTRODUCTION

Microorganisms such as bacteria are responsible for decomposing organic waste. When
organic matter such as dead plants, leaves, grass clippings, manure, sewage, or even food
waste is present in a water supply, the bacteria will begin the process of breaking down
this waste. When this happens, much of the available dissolved oxygen is consumed by
aerobic bacteria, robbing other aquatic organisms of the oxygen they need to live.

Biological Oxygen Demand (BOD) is a measure of the oxygen used by microorganisms

to decompose this waste. If there is a large quantity of organic waste in the water supply,
there will also be a lot of bacteria present working to decompose this waste. In this case,
the demand for oxygen will be high (due to all the bacteria) so the BOD level will be
high. As the waste is consumed or dispersed through the water, BOD levels will begin to
decline.

Nitrates and phosphates in a body of water can contribute to high BOD levels. Nitrates
and phosphates are plant nutrients and can cause plant life and algae to grow quickly.
When the micro plants grow quickly, they also die quickly. This contributes to the
organic waste in the water, which is then decomposed by bacteria. This results in a high
BOD level. The temperature of the water can also contribute to high BOD levels. For
example, warmer water usually will have a higher BOD level than colder water. As water
temperature increases, the rate of photosynthesis by algae and other plant life in the water
also increases. When this happens, plants grow faster and also die faster. When the plants
die, they fall to the bottom where they are decomposed by bacteria. The bacteria require

47
oxygen for this process so the BOD is high at this location. Therefore, increased water
temperatures will speed up bacterial decomposition and result in higher BOD levels.

When BOD levels are high, dissolved oxygen (DO) levels decrease because the oxygen
that is available in the water is being consumed by the bacteria. Since less dissolved
oxygen is available in the water, fish and other aquatic organisms may not survive.

The standard BOD test takes 5 days to complete and is performed using a dissolved
oxygen test kit. The BOD level is determined by comparing the DO level of a water
sample taken immediately with the DO level of a water sample that has been incubated in
a dark location for 5 days. The difference between the two DO levels represents the
amount of oxygen required for the decomposition of any organic material in the sample
and is a good approximation of the BOD level.

OBJECTIVE
To determine the amount of oxygen necessary for biological oxidation of wastewater.

METHODS

Reagents/Apparatus:

1. Scaled pipet
2. BOD bottles
3. BOD Nutrient Buffer
4. Nitrification inhibitor
5. Dissolved Oxygen (DO) probe
6. Incubator

48
Procedure:

1. Prepare sample dilution water using a BOD Nutrient Buffer.

2. From the Table below, determine the sample size (ml) to be taken and diluted to

300 ml in standard BOD bottle.

Sample Type Estimated BOD mg/l ml of sample

Strong Trade Waste 600 1

300 2
200 3
150 4
Raw and Settled Sewage 120 5
100 6
75 8
60 10

50 12
40 15
Oxidized Effluents 30 20
20 30
10 60

6 100
Polluted River Waters 4 200
2 300

3. Use graduated pipet to measure series of 4-6 different volumes (use the Table above)

of well mixed sample and transfer to separate glass-stoppered, 300 ml BOD bottles.

Stir the sample with the pipet before pipeting each portion.

4. Prepare a separate BOD bottle with dilution water only. This will be the dilution

water blank.

49
5. If required, add 2 shots of nitrification inhibitor (approximately 0.16 g) to each bottle.

This will inhibit the oxidation of nitrogen compounds and the results will reflect only

the carbonaceous oxygen demand.

6. Fill each bottle with seeded or unseeded dilution water. When adding the water allow

it to flow slowly down the sides of the bottle to prevent bubbles from forming.

7. Stopper the bottle, being careful not to trap any air bubbles. Press on the stopper of

the bottle with your finger, and then invert several times to mix.

8. Determine the initial dissolved oxygen, DO (Dl).

9. Stopper the bottle again and add enough dilution water to the lip of the BOD bottle to

make a water seal.

10. Place a plastic overcap over the lip of each bottle and place bottles in an incubator at

201C. Incubate in the dark for 5 days.

11. After 5 days, determine the DO content (mg/l DO remaining) in each bottle, using the

DO probe (D2).

Interferences:

To eliminate small amount of residual chlorine, allow the sample to stand for 1-2
hours at room temperature. For large quantities, add sodium thiosulfate as
prescribed in the manual.
To eliminate the effect of phenol, heavy metals or cyanide, dilute the sample with
high quality distilled water.

50
 Optimum pH for BOD test is between 6.57.5. Adjust samples to pH 7.2 with
Phosphate Buffer Solution, 1 N sulfuric Acid, or Sodium Hydroxide Standard
Solution.
Cold samples may be supersaturated with oxygen and will have low BOD results.
Fill a one-quart bottle about halfway with cold sample and shake vigorously for 2
minutes. Allow sample temperature to reach 20C before testing.

Calculations:
When dilution water is not seeded:

BOD5 (mg/l) = (D1 D2)

P

When dilution water is seeded:

BOD5 (mg/l) = (D1 D2) (B1 B2) f

P
where:
D1 : DO of diluted sample immediately after preparation, mg/l.
D2 : DO of diluted sample after 5 days incubation at 20 C, mg/l.
P : decimal volumetric fraction of sample used.
B1: DO of seeded control before incubation, mg/l.
B2: DO of seeded control after incubation, mg/l.
f: ratio of seed in diluted sample to seed in seed control.

If nitrification is inhibited, report results as CBOD5.

Average results are in the acceptable range if more than one sample dilution meets all
these criteria:
a residual DO of at least 1 mg/l.
a DO depletion of at least 2 mg/l.
no evidence of toxicity at higher sample concentrations, and
no obvious anomalies.

51
 EXPERIMENT 11

Total Nitrogen (TN)

Persulfate Digestion Method

Range (0.5 to 25.0 mg/l)

INTRODUCTION

Nitrogen is one of the nutrients which is often present in water in various forms. The
most common forms of nitrogen related to water quality are ammoniacal nitrogen (NH3-
N), nitrate (NO3), nitrite (NO2), organic nitrogen, total kjeldahl nitrogen (TKN) and total
nitrogen (TN). Excess nitrogen in water may cause eutrophication by stimulating rapid
growth of algae and other aquatic plants. Provided other conditions (such as, sunlight,
turbidity of water, etc.) are fulfilled. Therefore, determination of nitrogen is essential to
protect the water bodies from eutrophication and other toxicity, such as blue baby
syndrome. The methodology to determine the total amount of nitrogen (TN) is discussed
in this Laboratory Manual. There are several methods available in the literature.
However, the HACH TestN Tube method is included in this manual for educational
purpose.

OBJECTIVE
To determine the total nitrogen in water and waste water

METHODS

Safety issue: Place a safety shield in front of the COD reactor to prevent injury.

Tips: Wipe the outside of the sample cells before each insertion in the cell holder.

52
 Procedure Steps Images Remark

1. Turn the COD reactor The optimal temperature is at

and place the safety 105C
shield, set the
temperature at 103C to
106C

2. Using a funnel add the Wipe off any reagent that may
content of 1Persulfate get on the lid or the tube
Reagent Powder Pillow threads.
to each of two Total
Nitrogen Hydroxide
reagent vials.

3. Add 2 ml of the sample Use only water that is free of

to a vial (prepared nitrogen containing species as
sample). a substitute for the deodorized
Add 2 ml of the
water provided.
deodorized water
included in the kit to a
second vial (Reagent
vial).

4. Cap both vials. Shake The persulfate reagent may not

vigorously for at least dissolve completely after
30 second to mix. shaking. This will not affect
accuracy.

5. Place the vials in the COD

Reactor. Heat for exactly 30
minutes.

53
 Procedure Steps Images Remark

6. Using a finger coat,

immediately remove the hot vials
from the reactor.

7. Touch HACH Program.

Select program:

350N, Total TNT.

Touch Start.

8. Remove the caps from the

digested vials and add the
content of 1 Total Nitrogen(TN)
Reagent A Powder Pillow to each
vial.

9. Cap the tubes and shake for 15

seconds.

10. Touch the timer icon than Three minutes reaction period
touch OK. will begin.

54
 Procedure Steps Images Remark

11. After the timer beeps, remove

the caps from the vials and add
one TN Reagent B Powder
Pillow to each vial.

12. Cap the tubes and shake for The reagent may not dissolve
15 seconds. completely after shaking. This
will not affect the accuracy.

The solution will begin to turn

yellow.

13. Touch the timer icon. Touch Two minutes reaction period
OK. will begin.

14. After the timer beeps, remove

the caps from two TN Reagent C
vials and add 2 ml of digested,
treated sample to one vial.
Add2ml of digested, treated
reagent blank to the second TN
Reagent C vial.

15. Cap the vials and invert ten Use slow, deliberate inversions
times to mix. for complete recovery. The
tubes will be warm.

55
 Procedure Steps Images Remark

16. Touch the timer icon. Touch Five minutes reaction period
OK. will begin.

The yellow color will intensify.

17. Wipe the reagent blank and

place it into the cell holder.

18. Touch Zero. The display will show 0.0 mg/l.

19. Wipe the reagent vial and Results will be shown in mg/L
place it into the cell holder. N.

The reagent blank can be used up to seven days within the same lots of the reagent. It has
to stored in a dark room and temperature (18 to 25C). If white floc appears, new blank
has to be prepared.

Interferences
- If the sample is known to contain large amount of organics, it should be diluted and
re-run to verify digestion efficiency.

56
- The samples have to be collected in a clean plastic or glass bottles, and have to be
preserved by reducing the pH to 2 and stored up to 28 days. Before running the
experiment the samples have to be warmed to room temperature and neutralized with
5N sodium Hydroxide.

57
 Experiment 12

Total Phosphorus (TP)

Molybdovanadate Method with Acid Persulfate Digestion

Range (1.0 to 100.0 mg/L PO4_3)
Method 10127
Test N Tube Vials

INTRODUCTION

Similar to nitrogen, phosphorus is also one of the nutrients which is frequently present in
water in various forms. The most common forms of phosphorus are dissolved
phosphorus, particulate phosphorus, inorganic phosphorus and organic phosphorus.
Depending on the aquatic environment, type and concentration of the phosphorus can
change from one form to another. Excess amount of phosphorus in water also may cause
eutrophication by stimulating rapid growth of algae and other aquatic plants. Therefore,
determination of phosphorus is essential to protect the water bodies from eutrophication.
Although different forms of phosphorus have different impact on the aquatic
environment, determination of total phosphorus (TP) gives an idea to the scientists or
engineers about the phosphorus content in the water. The methodology to determine the
total amount of phosphorus (TP) is given in this Laboratory Manual. There are several
methods available in the literature. However, the TestN Tube method of HACH is
included in this manual for educational purpose.

OBJECTIVE
To determine total phosphorus in water and waste water.

METHODS

Application: Water and waste water.

Safety issue: Place a safety shield in front of the COD reactor to prevent injury.

58
Tips: Reagent blanks can be used more than once but not more than one day.

Procedure:

Procedure Steps Images Remark

1. Turn on the COD Safety shield should be placed in
Reactor. Heat to front of the reactor.
150C

2. Touch: HACH .
Programs.
Select program

541P Total HR TNT.

Touch Start

3. Use TenSette Pipet

to add 5ml of
deionized water to a
total Phosphorus
Test N Tube Vial(
the blank)

4. Use TenSette pipett

to add 5ml of sample
to a Total
Phosphorus Test N
Tube Vial( Sample).

5. Use funnel to add to Cap tightly and shake to

add the content of dissolve.
one Potassium
Persulfate Powder
Pillow for
Phosphonate to each
vial.

59
 Procedure Steps Images Remark
6. Place the vials in the
COD Reactor.

7. Touch the timer icon 30 minutes heating period will

Touch OK. begin.

8. After the timer beeps Place them in test tubes racks

carefully remove the and allow them to cool to room
hot vials from the temperature(18-25C)
reactor.

9. Use TenSette Pipet to add 2ml Cap and invert them to mix
of 1.54N sodium hydroxide to
each vial.

10. Use Polyethylene dropper to Cap and invert them to mix.

add 0.5ml of Molybdovanadate
Reagent to each vial.

60
 Procedure Steps
11. Touch the timer icon. Touch
OK.
12. Wipe the vials with a damp
towel, followed by a dry one to
remove the fingerprints or other
marks.
13. When the timer beeps, place
the blank into the cell holder
14. Touch Zero
15. Place the prepared sample
into the cell holder.
Results will appear mg/l PO4-3
Images Remark
7 minutes reaction period will
begin.
Read the sample within 7 to9
minutes after adding the
molybdovanadate Reagent.
The reagent may not dissolve
completely after shaking. This
will not affect the accuracy.
The solution will begin to turn
yellow.
The display will show: 0.0 mg/l
PO4-3
.
61
Sample Preparation:
- The samples have to be collected in plastic or glass bottles that have been acid
washed with 1:1Hydrochloric Acid solution, and rinsed with deionized water.

- The samples can be preserved up to 28 days by adjusting the pH to 2 or less with

concentrated Sulfuric Acid and storing at 4C. Warm the sample at room temperature
and neutralize with 5N Sodium Hydroxide before analysis.

62
 EXPERIMENT 13
Zinc

INTRODUCTION

Zinc can appear in water from natural and man-made sources. Man-made sources are
industrial waste, metal plating, plumbing, domestic sludge, urban runoff, etc. Although
zinc is an essential micro nutrient, high level of zinc causes toxicity in various organisms
at different concentrations. As such, Zn is included in the EQA effluent discharge
standards and NWQS as a monitoring parameter. Zinc can be determined by various
methods. However, colour spectrophotometer and atomic absorption spectrophotometer
are commonly used for better accuracy of the results.

OBJECTIVE
To determine concentration of zinc in water and waste water.

63
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65
66
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68
 APPENDIX A

(Body of text-example)

EXPERIMENT 1 14 font size, centred, bold,

double spacing

Bradford Protein Assay

Introduction

In biochemistry, it is frequently necessary to know the total protein concentration in your

solution. A number of ......

Aim/ Objectives

To detect the total protein concentration in a solution

Methods

Chemical/Biochemical/Consumables
Note the chemicals, biochemicals and consumables used in the experiments. You may state
the total amount needed and the chemical company (brands) you can purchase from, eg
Sigma, Fisher etc.

Apparatus
Note all the apparatus needed to conduct the experiment

Procedure

Note the procedure used to conduct the experiments.

References

Russians. (1998). In T. L. Gall (Ed.), Worldmark encyclopedia of cultures and daily life
(Vol. 4, pp. 332-339). Detroit, MI: Gale Research.

69
 APPENDIX B

STANDAD OPERATION OF HOMOGENIZER

Operators position

1. To ensure the safe use of the machine during start-up and adjustment operations,
it is recommended that the operator should stand as indicated in Figure 1.
2. The control panel configured as shown in Figure 2 is on the left hand side of
the machine front; an EMERGENCY STOP PUSH BUTTON is always fitted on
each machine.
The controls on the panel are (see figure 2):
Emergency Stop push-button to halt the machine instantly in case of
emergency
On Push-button, identified by the symbol I, with a GREEN colored light
(C)
Off Push-button, identified by the symbol O, with a RED colored light
(D)
Setting knob (F) for adjusting rotation speed of feed pump, equipped with
integrated frequency converter.

Figure 1

70
 Figure 2

Start-up
3. After connecting the machine to the power mains, switch the Electric Board on
using the main switch which may be accessed from the rear part of the machine.
4. Verify that the red light indicating MACHINE STOPPED is on. Open all on/off
valves on utility lines (water, air, steam) and regulate the rate of flow if necessary
(adjustments are usually made at the factory during the final machine test)
5. Refer to Figure 3 and follow these steps:
Fill the infeed hopper (B) with product or water
Check once more that flywheels (H) and (K) of the two homogenizing stages
are completely loosened
Switch on the digital pressure gauge (J) by quickly pressing the ZERO/ON
button in the display
Set to the minimum the control knob which adjusts feed pump speed, located
on the control panel (A) item (F) in Figure 2
Press the START push-button located on the control panel (A) - item (F) in
Figure 2.

71
 Figure 3

Pump and Feeding System

6. This machine is equipped with an integrated feed pump (item F Figure 3)

housed inside the bodywork, complete with loading hopper (B) for feeding
product into the machine.
7. To adjust pump speed, check the value given by the suction pressure gauge (E).
8. Feed pressure must be adjusted in accordance with the values given in the table
below which shows the minimum pressure levels required:

72
 Table 1

For products with viscosity close to water (1 cP), and for washing conditions, an
infeed pressure of 1-2 bar is allowed with the pump on maximum speed.

Operation and Adjustment

9. Once the machine has been started, keep regular operation and in particular infeed
pressure value under control without increasing the homogenizingpressure,
according to Table 1
10. In particular it is possible to act on the 3-way valve (L) on the outfeed end of the
homogenizing assemblies to send the discharged product out through the outlet
pipe (C) or to be ricircled into the hopper (B) through pipe (M). We suggest you
keep the valve to the recircling position until a correcthomogenizing pressure has
been reached.
11. The manual regulator is provided with an handwheel (H, K) for the increase of the
homogenizing pressure (turning clockwise), and decrease (turning
counterclockwise) - (see Figure 3). To perform this operation rotate the
handwheel SLOWLY until the desired pressure reading is obtained on the
pressure gauge (J) installed on the compression block.
12. Once the operating pressure has been reached you can set the 3-way valve to
collect the treated sample through a discharge pipe (C).
Machine Stop

13. To stop the machine, follow these steps:

release the homogenizing pressure by turning counterclockwise the
handwheel(s)
press the stop push-button (if available on the machines control panel)
cut off power using the main switch.

73