LAB MANUAL

EXPERIMENT 1: THE USE OF MICROSCOPE

OBJECTIVE

1. To learn the correct method of using light microscope

2. To examine specimen samples under light microscope

THEORY

A microscope is an optical instrument for viewing objects that are too small to be seen by naked eye.
The science of investigating small objects using such an instrument is called microscopy. The term
'microscopic' means minute or very small, not visible with the eye unless aided by a microscope.
Microscopes can largely be separated into three classes namely, optical theory microscopes, electron
microscopes, and scanning probe microscopes (SPM).

The most common type of microscope is optical microscope (e.g. light microscope). This is an optical
instrument containing one or more lenses that produce an enlarged image of an object placed in the
focal plane of the lens (es). Regardless of numerous designs, optical microscopes function through the
optical theory of lenses in order to magnify the image generated by the passage of a wave through the
sample. Optical microscopes, through their use of visible wavelengths of light, are the simplest and
hence most widely used type of microscope. Optical microscopes typically use refractive lenses of
glass and occasionally of plastic or quartz, to focus light into the eye or another light detector. Mirror-
based optical microscopes operate in the same manner. Typical magnification of a light microscope,
assuming visible range light, is up to 1500x with a theoretical resolution limit of around 0.2 microns
or 200 nanometers.

Common lab available, light microscope has 3 magnifications (Table 1.1). Each objective lens will
have written the magnification. In addition to this, the ocular lens (eyepiece) has a magnification. The
total magnification is the ocular mag. x objective mag. The following Figure 1.1 shows the parts of
typical light microscope.

Table 1.1: Magnification for light microscope

Total
Magnification Ocular lens Magnification

Scanning 4x 10x 40x

Low Power 10x 10x 100x
High Power 40x 10x 400x

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 Figure 1.1: Light microscope and its part

EQUIPMENT AND MATERIAL

1. Microscope
2. Glass slides
3. Cover slips
4. Staining agent – Iodin, Methylene Blue
5. Scalpel/Razor blade
6. Tweezers
7. Specimen samples

EXPERIMENT PROCEDURES

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a) Preparing specimen/sample slide

1. Obtain a clean glass slide and cover slips. Ensure to hold the slide and cover slips by their edge.
Do not hold them by the surface as you will leave fingerprints which might compromise your
result.
2. Place the slide and cover slips on a clean working surface. It is advise for you to cover the
working surface with absorbing paper
3. Using tweezers, obtain the provided specimen samples and place it in the middle of the slide.
Ensure the specimen to lay flat on the slide.
4. If necessary, perform staining on your specimen
5. Cover your specimen with cover slips. Ensure there was no air bubble trapped between the slide
and the cover slips
6. Your slide is ready for viewing

b) Preparing microscope for examining samples

1. Plug your microscope cord to electrical socket. Make sure the cord is not entangled.
2. Place the prepared slide on the stage. Don’t use the stage clips first as you want to adjust the
position of the slide later in accordance to the view.
3. Always start and end with the Scanning Objective. Do not remove slides with the high power
objective into place - this will scratch the lens!
4. Odds are you will be able to see something on this setting. Use the Coarse Knob to focus, image
may be small at this magnification, but you won't be able to find it on the higher powers without
this first step. Do not use stage clips, try moving the slide around until you find something.
5. Once you are satisfied with the positioning of the slide, you may use the stage clips
6. Once you've focused on Scanning, switch to Low Power. Use the Coarse Knob to refocus.
Again, if you haven't focused on this level, you will not be able to move to the next level.
7. Now switch to High Power. (If you have a thick slide, or a slide without a cover, do NOT use
the high power objective). At this point, ONLY use the Fine Adjustment Knob to focus
specimens.
8. If the specimen is too light or too dark, try adjusting the diaphragm.
9. If you see a line in your viewing field, try twisting the eyepiece, the line should move. That's
because it’s a pointer, and is useful for pointing out things to other persons.
10. Sketch the image you saw under the microscope
11. Repeat step 2 – 10 for different specimen.

RESULT AND DISCUSSION

1. Report and discuss on all images from all sections in this experiment
2. In some section, a cover slip may not necessary to be used. WHY is it so?
3. Explain some precaution steps that are essential in a) using microscope b) preparing samples and c)
examining sample.

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 EXPERIMENT 2: STAINING AND CELL IDENTIFICATION

OBJECTIVE

1. To choose suitable staining agent for inspection of different types of specimens

2. To prepare slide using Gram staining methods

THEORY

Staining techniques are widely used to visualize those components that are otherwise too difficult to
see under an ordinary light microscope either because of the lack of color contrast between the object
under examination and the background or because of the limited resolving power of the light
microscope. In addition, staining techniques are useful in detecting the presence or absence of certain
cell components, thus allowing a relatively simple scheme of differentiation or identification of
microorganisms.

The Gram staining method is named after Hans Christian Gram, a Danish bacteriologist who originally
devised this technique in 1844. It is one of the most important staining techniques in microbiology,
primarily applied in first identification of bacteria.

The staining agent color in the Gram's method is crystal violet. Methylene blue may also be substituted
as it is equally effective. The microorganisms that retain the crystal violet-iodine complex appear
purple brown under microscopic examination. These microorganisms that are stained by the Gram's
method are commonly classified as gram positive or gram non-negative. Others that are not stained by
crystal violet are referred to as gram negative.

A mixture of S. cerevisiae and E. coli will be examined in this experiment, although Gram's stain is
usually applied to differentiate bacteria, which are usually too small to be seen clearly under a light
microscope. S. cerevisiae cells will be stained by the crystal violet-iodine complex and should appear
purple-brown in color. In contrast, the much smaller E. coli, cells should appear pink, the color of
safranin. Repeat the entire process until a satisfactory slide is prepared and properly focused in a
microscope for the approval of the instructor. Note the improvement in cell visualization as compared
to a plain unstained slide.

PART A

EQUIPMENT AND MATERIAL

1. Bunsen burner
2. Microscope
3. Glass slide
4. Cover slips
5. Tweezers
6. Water bottle
7. Crystal violet (95% dye content)

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 8. Methylene blue (90% dye content)
9. Ethanol, 95%
10. Acetone
11. Ammonium oxalate
12. Iodine
13. Potassium iodide
14. Sodium bicarbonate
15. Safranin O
16. S. cerevisae
17. E.coli

EXPERIMENT PROCEDURES

1. PREPARING REAGENTS

a. Gram Crystal Violet solution

1. Dissolve 20 g of crystal violet in 100 ml of ethanol to make a crystal violet stock solution.
2. Dissolve 1 g of ammonium oxalate in 100 ml of water to make an oxalate stock solution.
3. Working solution is obtained by mixing 1 ml of the crystal violet stock solution with 10 ml of
water and 40 ml of the oxalate stock solution.
4. Store the working solution in a drop bottle.

b. Methylene Blue solution

1. Dissolve 1 g of methylene blue in 100 ml of ethanol; this is Solution A.
2. Mix 0.03 g of KOH in 300 ml of water; this is Solution B.
3. Mixing Solutions A and B yields the working solution.

c. Gram Iodine Solution

1. Dissolve 1 g of iodine, 2 g of potassium iodide, and 3 g of sodium bicarbonate in 300 ml of
water.

d. Gram Decolorizer Solution

1. Mix equal volumes of 95 % ethanol and acetone.

e. Gram Safranin Solution

1. Dissolve 2.5 g of safranin O in 100 ml of 95 % ethanol to make a stock solution.
2. Working solution is obtained by diluting one part of the stock solution with five parts of water.

2. PREPARE A SLIDE SMEAR

1. Transfer a drop of the suspended culture on a slide with an inoculation loop. If the culture is to be
taken from a Petri dish or a slant culture tube, first add a drop or a few loopful of water on the
slide and aseptically transfer a minute amount of a colony from the Petri dish. Note that only a

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 very small amount of culture is needed; a visual detection of the culture on an inoculation loop
already indicates that too much is taken.
2. Spread the culture with an inoculation loop to an even thin film over a circle of 1.5 cm in diameter,
approximately the size of a 5 cent coin. Thus, a typical slide can simultaneously accommodate 3
to 4 small smears if more than one culture is to be examined.
3. Hold the slide with tweezers. Air-dry the culture and fix it or over a gentle flame, while moving
the slide in a circular fashion to avoid localized overheating. The applied heat helps the cell
adhesion on the glass slide to make possible the subsequent rinsing of the smear with water
without a significant loss of the culture. Heat can also be applied to facilitate drying the smear.
However, ring patterns can form if heating is not uniform, e.g. taking the slide in and out of the
flame.

3. GRAM STAINING

1. Add about 5 drops of crystal violet stain over the fixed culture. Let stand for 60 seconds.
2. Pour off the stain and gently rinse the excess stain with a stream of water from a faucet or a plastic
water bottle.
3. Add about 5 drops of the iodine solution on the smear, enough to cover the fixed culture. Let stand
for 30 seconds.
4. Pour off the iodine solution and rinse the slide with running water. Shake off the excess water
from the surface.
5. Add a few drops of decolorizer so the solution trickles down the slide. Rinse it off with water after
5 seconds. The exact time to stop is when the solvent is no longer colored as it flows over the
slide. Further delay will cause excess decolorization in the gram-positive cells, and the purpose of
staining will be defeated.
6. Counterstain with 5 drops of the safranin solution for 20 seconds.
7. Wash off the red safranin solution with water. Blot with bibulous paper to remove the excess
water. Alternatively, the slide may be shaken to remove most of the water and air-dried.
8. Liberally wash off any spilled stain immediately with water to avoid leaving permanent marks in
the sink, lab bench, or glassware.
9. Examine the finished slide under a microscope.

RESULT AND DISCUSSION

Determine the concentration (%w/v) of the following substances

a. Crystal violet in stock solution
b. Ammonium oxalate in working solution
c. KOH in working solution
2. Report and discuss on all images you observed in this experiment. Preparing sketches with different
ink color to indicate different strain of bacteria may help.
3. Explain the mechanism of Gram’s staining by stating the significant of each staining step with
respect to the properties of the examined cells’ wall.

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4. List all necessary precaution steps in staining technique and provide justification for each
precaution step.

Part B

In this experiment, you are required to stain another type of cell, Algae. This cell is fluorescent when
staining using a suitable agent e.g. nile red, oil red and etc.

EQUIPMENT AND MATERIAL

Solution A
Oil red O 1 g
Propylene glycol 100 mL
Other suitable dyes are:–
Sudan III, Sudan IV, Sudan black B

PROCEDURE

Add the dye to the propylene glycol, and heat to about 100°C. Mix well until saturated.
Filter while hot using a fast paper. Cool to room temperature, and re-filter using a vacuum pump.
This solution keeps for a long time (years).

Method
1. Place sections into propylene glycol for 1-2 minutes.
2. Transfer sections into solution A for 10 minutes.
3. Transfer sections to 85% propylene glycol for 2-3 minutes to clear the background.
4. Transfer sections to 50% propylene glycol for 1-2 minutes.
5. Gently agitate in several changes of tap water at room temperature
6. Coverslip using an aqueous medium.
Expected results
– red (orange or black if other dyes were used)
Notes
1. If using sudan black B, nuclear fast red may be a more appropriate counterstain.
2. Although this method can be used with paraffin sections, it will obviously not
demonstrate those lipids that are removed by the dehydrating and clearing agents. Only
solvent resistant lipids can be seen, such as some lipofuscins or residual myelin.

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 EXPERIMENT 3: BIOMASS CONCENTRATION MEASUREMENT

OBJECTIVE

1. To prepare standard curve of biomass concentration based on optical density measurement

2. To perform cell quantification for a given biomass suspension

THEORY

The cell density can be quantified in two basic ways: as grams of dry or wet weight per liter of sample,
or as number of viable/dead cells per ml. The cells in a sample can be separated from the broth and
weighed while they are wet, or the cells may be thoroughly dried before weighing. The dry weight
measurement usually gives a much more consistent result than the wet weight. Alternatively, the
number of cells can be counted either by successively diluting the original sample and plating on a
Petri dish, with the help of a microscope and a counting chamber, or with an automated cell counter
such as a Coulter counter or a cytoflowmeter. The plating method detects only the viable cells;
whereas, the automated cell counters can only detect the total number of cells.

All of the above methods either require the availability of expensive equipment or the substantial
investment of time. In reality, the most often used method simply monitors the optical density of the
sample. The absorbance of the sample measured in a spectrophotometer is correlated to either the dry
weight or the number of cells per volume.

Biomass concentration is one of the most critically needed measurements in fermentation studies. It is
also one of the most difficult and unreliable ones. For example, all the above dry/wet weight methods
and all the automated counting equipment fail completely if the broth contains other insoluble
particulate matter, which is often the case in a practical fermentor. Similarly, the optical density
measurement only has limited usefulness if the fermentation broth is not clear. In addition, these
methods cannot distinguish the viable cells from the dead ones. On the other hand, the standard plate
count can detect viable cells among other particulate matters. However, the method requires elaborate
preparations, and it takes 24-48 hours for the cells to be incubated and counted; the cost of Petri dishes
and media can also be prohibitive. Consequently, the direct plate count is useless in feedback control
of a fermentation process; it is mainly used industrially to countercheck other measurements, especially
the optical density.

In this experiment, the cell density of a given sample will be measured using optical density or
absorbance value of biomass suspensions. A spectrophotometer measures the amount of light that
passes through a solution as compared to a "blank" solution. A blank solution is usually water, buffer,
or sterile broth. In solutions that have a lot of particulate matter (bacteria for example), little light will
pass through and the absorbance will be high (i.e. that the solution has absorbed most of the light that
hit it). Furthermore, the transmission value will be low because little light has been transmitted through
the solution. Alternatively, relatively clear solutions will absorb very little light (=low absorbance
value) and will transmit most of the incident light (=high transmission value). By measuring
absorbance values of a bacterial culture, we can monitor the increase in the cell number over time.

EQUIPMENT AND MATERIAL

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 1. Beaker
2. Graduated cylinder
3. Spectrophotometer
4. Spectrophotometer cuvette
5. Medicine dropper
6. Buffer solution
7. Dry yeast

EXPERIMENT PROCEDURES

a) Preparing standard curve

1. Measure x gram of dry yeast and mixed with y ml of buffer solution and mixed properly in a
beaker. Make sure the yeast is properly distributed all over the solution. Take the biomass
concentration of this stock suspension as x g/y ml.
2. Perform series of successive dilutions from this stock suspension. Do perform enough dilutions
so that you have sufficient data for generating calibration curve later.
3. Label each dilution properly.
4. Measure the absorbance value of each dilution.
5. Generate a calibration curve from the data you have collected.
Absorbance
value

Wavelength = ___ nm

Biomass
conc. (g/ml)

b) Using spectrophotometer

1. Set the wavelength to a proper value by adjusting the wavelength control knob.
Always consistent on choosing wavelength value if you are preparing a
calibration curve.
2. The zero adjust knob should be used to set the absorbance to infinity
(transmission = 0%). There should be no tube present in the sample holder
3. The 100% adjust knob should be used to set the absorbance to 0 (transmission =
100%). There should be a tube containing just solvent (water, broth, buffer, etc)
in the sample holder.

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 4. Wipe the outside of the tube/cuvette with a cloth/tissue before placing it inside
the sample holder
5. Place a sample in the sample holder and measure the absorbance of your sample.
Always face the tubes in the same direction in the cuvette holder. 6. Reset the
zeros on the spectrophotometer every time you use it

c. Determining the unknown biomass concentration of a given sample

1. Check the absorbance reading of the given culture suspension. Determine the
biomass concentration of the given sample by cross referencing the absorbance
value in the calibration curve that you have constructed.

RESULT AND DISCUSSION

1. Report the cell density in appropriate units for the given sample.
2. Discuss the calibration curve that you have built. Report all errors and anomalies detected
upon analyzing your data.

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 EXPERIMENT 4: STERILIZATION

PLEASE WATCH THE VIDEO AND ANSWER A QUESTIONNAIRE BEFORE

CONDUCTING THIS EXPERIMENT!!!

OBJECTIVES

1. To learn correct method of using steam autoclave as one of aseptic techniques

2. To investigate the impact of improper autoclaving method on microbe culture preparation

INTRODUCTION

Autoclave is an equipment used for sterilizing articles with saturated steam under a pressure
which is higher than atmospheric pressure. Autoclaving clinical and biohazard waste is highly
recommended before further disposal. This is to kill hazardous pathogens and avoiding contamination
to the surroundings.
After commencement of operation, sterilizing water in the chamber is heated by the element
heater at the bottom of the chamber to generate steam. Steam generated through heating drives air from
the chamber and warms the inside of the chamber. When the temperature sensor in the chamber detects
that the temperature has risen to he specified level, this temperature is maintained for a specified time.
Residual air in the chamber is driven out through this operation. When the specified time has elapsed,
the valve closes to carry out heating.
When the temperature sensor detects that the temperature has risen to the specified level, the
timer is activated to maintain a constant temperature. When the set sterilizing time has elapsed, the
element heater stops. The sterilizing action is determined by the three elements: temperature, humidity,
and time applied to articles to be sterilized. During this time, when the temperature in the chamber
falls, the valve opens to return the pressure in the chamber to the atmospheric level. When an
abnormality is generated in the autoclave, it changes to a safer status and the abnormality is indicated
by an error code display and by a buzzer sounding.

Extra Notes on Precautions!

Practice the procedures, especially the closing and opening of the autoclave door, without
introducing any steam into the autoclave chamber until the entire sequence can be performed
without any difficulty. The student is authorized to use the autoclave only after he/she has
demonstrated to the instructor his ability without referring back to this write‐up. Do not use the
autoclave if the student is not absolutely sure of the proper operating procedures. The pressurized
steam is more likely to cause burns than boiling water. First, it is hotter than boiling water. More
importantly, the heat of vaporization released upon the condensation of steam causes much severer
damage than does the same quantity of boiling water. Always watch out for the hot metal parts on
the autoclave. Wear heat insulating (asbestos) gloves when handling hot autoclaved items.

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EQUIPMENT AND MATERIAL

1. Autoclave/High-Pressure Steam Sterilizer

a. VARIOKLAV Type 300/400/500 EH Steam Sterilizer – Located at
Biochemical Engg. Lab, KKA
b. Autoclave TOMY-SEIKO SX-300/500/700 – Located at Analytical Lab,
KKA
2. Erlenmeyer Flask
3. Nutrient broth (NB)
4. Microbes culture
5. Incubator

*Note that you will run Experiment 4 & 5 together. Please ensure you collect enough
glasswares and materials to cater both experiment.

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Figure 4.1: Main unit of steam autoclave TOMY-SEIKO SX-300/500/700

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Figure 4.1: Main unit of steam autoclave TOMY-SEIKO SX-300/500/700 (continued)

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Figure 4.2: Inner chamber part of steam autoclave TOMY-SEIKO SX-300/500/700

Figure 4.3: Piping diagram of steam autoclave TOMY-SEIKO SX-300/500/700

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Figure 4.4: Control panel display of steam autoclave TOMY-SEIKO SX-300/500/700

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Figure 4.5: Work monitor for steam autoclave TOMY-SEIKO SX-300/500/700

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Figure 4.6: Control panel key for steam autoclave TOMY-SEIKO SX-300/500/700

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EXPERIMENTAL PROCEDURE

a) Demonstration on checking autoclave condition

1. Your lab demonstrator will demonstrate on checking necessary items before starting
autoclaving
2. You are required to make notes on the procedure

b) Investigation on heat treatment impact on growth of microbes in media.

1. Prepare 3 sets of 50ml nutrient broth (NB) in 250ml shake flask and label accordingly.
2. Execute the following :

Set 1 – Autoclave at 121°C for 15 minutes. Cool to room temperature and

measure the OD at 600nm.
* Your lab demonstrator will demonstrate on autoclaving process.
You are required to take notes on the procedure

Set 2 – Cook/Boil for 5 minutes on hot plate. Cool to room temperature and measure
the OD at 600nm.

Set 3 – No thermal treatment. Cool to room temperature and measure the OD

at 600nm.

3. Use 2 types of blank when measuring the OD. Label as Blank 1 and Blank 2.
4. Incubate all shake flasks in an oven incubator or shake flask incubator at 36°C
overnight.
5. Measure the OD for each set after 12 hours or 24 hours.

RESULT AND DISCUSSION

1. Discuss the difference in the turbidity reading between set 1, 2 and 3 and state reasons
for this observation.
2. Why the OD measurements are taken at 600nm?
3. Explain on the significant on the use of Blank 1 and 2. In your opinion, which one is the
best blank to use?
4. What is the importance of steam autoclave in media preparation?
5. Why temperature setting of 121°C for 15 minutes was selected for the steam autoclave
cycle? Is this acceptable for other application? For instance, to sterilize biohazard waste
or bioreactor?
6. Suggest other method(s) that can be used to sterilize the media.

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 EXPERIMENT 5: DEVELOPMENT OF MICROBIAL STRAIN

OBJECTIVES

1. To demonstrate pure culture techniques with Petri dishes/flask

INTRODUCTION

In microbiological and biochemical engineering studies, one almost always deals

with a pure culture or a mixture of known cultures, except perhaps in wastewater
treatment studies. Unless aseptic culture techniques are followed strictly, an originally
pure culture will definitely become contaminated with other unwanted species. The
results of such a study will certainly be unreliable. Similarly, the use of a contaminated
culture with unknown microorganisms will only lead to incredible results that are of little
value. Thus, the isolation and maintenance of a pure culture is of utmost importance in
many microbiological studies.

Figure 5.1: Microbe culture on petri dish

The need for a clean working environment in biochemical engineering studies

must be stressed again, for cleanliness is the prerequisite to any meaningful work. Many
parts of the aseptic procedures require occasional exposure to the surrounding
environment. Since our laboratory cannot be made totally sterile economically, it is
imperative that the room be kept clean. The use of a laminar hood, which creates an air
curtain to reduce the chance of contaminants drifting into the working space enclosed by
the hood, is highly recommended if it is available. However, a laminar hood itself must
still be maintained in a clean condition.

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EQUIPMENT AND MATERIAL

1. Petri Dish
2. Potato Dextrose Agar (PDA)
3. Nutrient Broth (NB)
4. Microbes culture
5. Incubator

*Note that you will run Experiment 4 & 5 together. Please ensure you collect enough
glasswares and materials to cater both experiment.

EXPERIMENTAL PROCEDURES

a) Preparation of pure culture (streak technique)

1. Turn on the laminar flow cabinet.

2. Wipe the preparation area inside the cabinet with ethanol.
3. Flame the inoculation loop until the metal reddened and put away from Bunsen burner for
a few seconds.
4. Transfer a little amount of culture from culture source onto agar in your petri dish.
5. Streak the agar in the advised direction
6. After streaking, close the petri dish with its lid and wrap with paraffin film.
7. Store your petri dish in incubator at 32°C.
8. Report your observation after 24 hours. Illustrate the culture on your petri dish by taking
photo.

b) Preparation of pure culture (spread technique)

1. Turn on the laminar flow cabinet.

2. Wipe the preparation area inside the cabinet with ethanol.
3. Flame the L-rod and put away from Bunsen burner for a few seconds.
4. Pour three drops of culture suspension onto agar on your petri dish 5. Using L-rod, spread
the culture in 360 degree direction.
6. After spreading, close the petri dish with its lid and wrap with paraffin film.
7. Store your petri dish in incubator at 32°C.
8. Report your observation after 24 hours. Illustrate the culture on your petri dish by
taking photo.

RESULT AND DISCUSSION

1. Report your findings, any contaminations and the importance of aseptic technique
applied during the experiment.

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 EXPERIMENT 6: OPEN ENDED EXPERIMENT

 OPEN ENDED EXPERIMENT

 THE PROCEDURE WILL BE ANNOUNCED LATER

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 EXPERIMENT 7: PROTEIN CONCENTRATION MEASUREMENT

OBJECTIVE

1. To prepare standard curve of protein concentration using Bradford Protein Assay protocol
2. To determine the protein concentration of an unknown sample

THEORY

The determination of protein concentration is essential in biochemical works. For example, during
purification of protein, we need to know how pure the sample is. Several methods are available; each
having features that suit it to a specific use.

The Bradford assay protocol is fairly accurate and samples that are out of range can be retested within
minutes. The Bradford is recommended for general use, especially for determining protein content of
cell fractions and assessing protein concentrations for gel electrophoresis.
The assay is based on the observation that the absorbance maximum for an acidic solution of
Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. Both
hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color
change. Without protein, the solution is red-brown in its acidic solution. When protein binds, the pKa
of the dye will shift therefore turning it into blue. This change can be measured via its optical density.

In preparing standard curve, a known protein with different concentrations achieved via a series of
dilution is plotted against their absorbance value. Hence, by measuring the absorbance of unknown
sample, the protein concentration can be predicted

EQUIPMENT AND MATERIAL

1. Bradford Reagent
2. Beaker
3. Graduated cylinder
4. Spectrophotometer
5. Disposable spectrophotometer cuvette
6. Medicine dropper
7. Buffer solution – NaCl solution
8. Protein standards – Bovine Serum Albumin (BSA), Immunoglobulin Gamma (IgG)
9. Protein solution
10. Syringe

EXPERIMENT PROCEDURES

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a) Preparing standard curve

1. Measure x ml of BSA/IgG dry and mixed with y ml of NaCl solution and mixed properly in a
beaker. Stir until you obtain homogenous solution. Take the protein concentration of this stock
suspension as x ml/y ml.
2. Perform series of successive dilutions from this stock suspension. Do perform enough dilutions
so that you have sufficient data for generating calibration curve later.
3. Label each dilution properly.
4. Add 1ml Bradford reagent to 1ml of each dilution sample and incubate for 10 minutes
5. Measure the absorbance value of each dilution.
6. Generate a calibration curve from the data you have collected.
Absorbance
value

Wavelength = ___ nm

Protein conc.
(ml/ml)

b) Using spectrophotometer

2. Set the wavelength to 595nm value by adjusting the wavelength control knob. Always
consistent on choosing wavelength value if you are preparing a calibration curve.
3. The zero adjust knob should be used to set the absorbance to infinity (transmission =
0%). There should be no tube present in the sample holder
4. The 100% adjust knob should be used to set the absorbance to 0 (transmission = 100%).
There should be a tube containing just solvent (water, broth, buffer, etc) in the sample
holder.
5. Wipe the outside of the tube/cuvette with a cloth/tissue before placing it inside the
sample holder
6. Place a sample in the sample holder and measure the absorbance of your sample.
Always face the tubes in the same direction in the cuvette holder.
7. Reset the zeros on the spectrophotometer every time you use it

c) Determining the unknown protein concentration of given samples

1. Collect samples of protein solution.

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 2. Perform dilution on these samples. Report the dilution of your choice in the report and its
justification
3. Add Bradford reagent to the samples and let it stand for 10 minutes
4. Check the absorbance reading of your samples. Determine the protein concentration of the
provided sample by cross referencing the absorbance value with the calibration curve that you
have constructed in (b)

RESULT AND DISCUSSION

1. Report the result in appropriate units for the given sample.

2. What is the purpose of incubating the sample after reagent has been added to it?
3. Discuss the calibration curve that you have built. Report all errors and anomalies detected upon
analyzing your data.

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 EXPERIMENT 8: CELL SEPARATION METHOD

OBJECTIVES

1. To perform cell separation using centrifuge 2. To study factors that

affect centrifugation process.

INTRODUCTION

Sedimentation in centrifugal is given as

d2p 2

vc = (ρs −ρL )w r
18µ
and

w2r
Z= g

EQUIPMENT AND MATERIAL

1. Suspension sample A, B and C

2. Centrifuge
3. Centrifuge tube and cap
4. Vortex mixer

EXPERIMENTAL PROCEDURES

a) Centrifugation at different speed

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 1. Measure 5-10ml of sample and aliquot into centrifuge tubes 2. Centrifuge at
500rpm and 5000rpm for 10 minutes
3. Repeat step 1 & 2 for other samples.
4. Record the results

RESULT AND DISCUSSION

1. Discuss why separation occurred or otherwise for each sample observation

2. Explain the centrifugation equations presented in the theory
3. Nomogram for converting maximum relative centrifugal force (RCF, g-force) to RPM
centrifugation is presented as a unit of (rpm) or (x g). Give reason when each of this unit is
appropriate to use.

4.

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 EXPERIMENT 9: CELL DISRUPTION METHOD

OBJECTIVE

1. To investigate the effect of a) processing time b) ratio of glass beads over suspension volume
c) shaking speed in cell disruption using glass beads and effect of processing time in cell
disruption using sonication
2. To check protein release from cell disruption process

THEORY

Cell disruption revolves around the interest of obtaining the intercellular components.

MATERIAL & EQUIPMENT

1. E.coli culture suspension

2. Glass beads/ballotini
3. Shaker incubator
4. Sonicator
5. Spectrophotometer
6. Spectrophotometer cuvette (disposable)
7. Centrifuge tube
8. Centrifuge
9. Beaker
10. Pasteur pipette

EXPERIMENTAL PROCEDURE

*In this experiment, students are required to plan the execution of their experiment in terms of
choosing number of run, sample size, required volume of culture suspension. All of these settings
need to be clearly stated in the report, including justification for the choice made.

a) Cell disruption using glass beads in rotary shaker – Effect of processing time

1. Prepare enough amount of culture suspension to cater number of observation of your

choice
(Example: 30ml of culture suspension for 3 flasks)
2. Pour equal amount of culture suspension into Erlenmeyer flasks.
3. Put equal amount of glass beads into the same Erlenmeyer flasks. (Example: 1.5 g of
glass beads per 1 ml solution)
4. Run the flasks on rotary shaker at your chosen interval.
5. Obtain the solution after shaking process complete and centrifuge.
6. Estimate the protein concentration of the centrifuge supernatant. Use the solvent from
culture suspension as blank

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b) Cell disruption using glass beads in rotary shaker – Effect of ratio of glass beads over
suspension volume

1. Prepare enough amount of culture suspension to cater number of observation of your

choice
(Example: 30ml of culture suspension for 3 flasks)
2. Pour equal amount of culture suspension into Erlenmeyer flasks.
3. Put different amount of glass beads into each Erlenmeyer flasks.
4. Run the flasks on rotary shaker at a constant processing time 5. Obtain the solution after
shaking process complete and centrifuge.
6. Estimate the protein concentration of the centrifuge supernatant. Use the solvent from
culture suspension as blank

c) Cell disruption using glass beads in rotary shaker – Effect of shaking speed

1. Prepare enough amount of culture suspension to cater number of observation of your

choice
(Example: 30ml of culture suspension for 3 flasks)
2. Pour equal amount of culture suspension into Erlenmeyer flasks.
3. Put equal amount of glass beads into the same Erlenmeyer flasks.
4. Run each flask at different shaking speed
5. Obtain the solution after shaking process complete and centrifuge.
6. Estimate the protein concentration of the centrifuge supernatant. Use the solvent from
culture suspension as blank

d) Cell disruption using sonication – Effect of processing time

1. Prepare enough amount of culture suspension to cater number of observation of your

choice
(Example: 30ml of culture suspension for 3 flasks)
2. Pour equal amount of culture suspension into beakers
3. Run the sonication process at different processing time of your choice 4. Obtain the
solution after shaking process complete and centrifuge.
5. Estimate the protein concentration of the centrifuge supernatant. Use the solvent from
culture suspension as blank

RESULT AND DISCUSSION

1. Provide justification on your chosen experiment parameter. 2. Report your result accordingly.

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 EXPERIMENT 10: ENZYMATIC KINETICS

OBJECTIVES

1. To study the various parameters those affect the kinetics of amylase catalyzed hydrolysis of starch.

INTRODUCTION

Starchy substances constitute the major part of the human diet for most of the people in the world, as
well as many other animals. They are synthesized naturally in a variety of plants. Some plant examples
with high starch content are corn, potato, rice, wheat, and cassava. It is no surprise that all of these are
part of what we consume to derive carbohydrates.

Starch is generally insoluble in water at room temperature. Because of this, starch in nature is stored
in cells as small granules which can be seen under a microscope. Starch granules are quite resistant to
penetration by both water and hydrolytic enzymes due to the formation of hydrogen bonds within the
same molecule and with other neighboring molecules. However, these inter- and intra-hydrogen bonds
can become weak as the temperature of the suspension is raised. When an aqueous suspension of
starch is heated, the hydrogen bonds weaken, water is absorbed, and the starch granules swell. This
process is commonly called gelatinization because the solution formed has a gelatinous, highly viscous
consistency. The same process has long been employed to thicken broth in food preparation.

Depending on the relative location of the bond under attack as counted from the end of the chain, the
products of this digestive process are dextrin, maltotriose, maltose, and glucose, etc. Dextrins are
shorter, broken starch segments that form as the result of the random hydrolysis of internal glucosidic
bonds. A molecule of maltotriose is formed if the third bond from the end of a starch molecule is
cleaved; a molecule of maltose is formed if the point of attack is the second bond; a molecule of
glucose results if the bond being cleaved is the terminal one; and so on. The breakdown of large
particles drastically reduces the viscosity of gelatinized starch solution, resulting in a process called
liquefaction because of the thinning of the solution. The final stages of depolymerization are mainly
the formation of mono-, di-, and trisaccharides. This process is called saccharification, due to the
formation of saccharides.

Since a wide variety of organisms, including humans, can digest starch, alphaamylase is obviously
widely synthesized in nature, as opposed to cellulase. For example, human saliva and pancreatic
secretion contain a large amount of alphaamylase for starch digestion. The specificity of the bond
attacked by alpha-amylases depends on the sources of the enzymes. Currently, two major classes of
alphaamylases are commercially produced through microbial fermentation. Based on the points of
attack in the glucose polymer chain, they can be classified into two categories, liquefying and
saccharifying.

Because the bacterial alpha-amylase to be used in this experiment randomly attacks only the alpha-1,4
bonds, it belongs to the liquefying category. The hydrolysis reaction catalyzed by this class of enzymes
is usually carried out only to the extent that, for example, the starch is rendered soluble enough to
allow easy removal from starch-sized fabrics in the textile industry. The paper industry also uses
liquefying amylases on the starch used in paper coating where breakage into the smallest glucose
subunits is actually undesirable.

30
On the other hand, the fungal alpha-amylase belongs to the saccharifying category and attacks the
second linkage from the non-reducing terminals (i.e. C4 end) of the straight segment, resulting in the
splitting off of two glucose units at a time. Of course, the product is a disaccharide called maltose. The
bond breakage is thus more extensive in saccharifying enzymes than in liquefying enzymes. The starch
chains are literally chopped into small bits and pieces. Finally, the amyloglucosidase (also called
glucoamylase) component of an amylase preparation selectively attacks the last bond on the non-
reducing terminals. The type to be used in this experiment can act on both the alpha-1, 4 and the alpha-
1,6 glucosidic linkages at a relative rate of 1:20, resulting in the splitting off of simple glucose units
into the solution. Fungal amylase and amyloglucosidase may be used together to convert starch to
simple sugars. The practical applications of this type of enzyme mixture include the production of
corn syrup and the conversion of cereal mashes to sugars in brewing.

MATERIALS AND METHOD

1. Erlenmeyer flasks
2. Beakers
3. Graduated cylinder
4. Pipets, 1ml, 10ml 5. Test tubes
6. Temperature bath
7. Thermometer
8. Balance
9. Syringe
10. Spectrophotometer
11. Spectrophotometer disposable cuvette
12. Enzymes
a. Bacterial/Fungal amylase solution
13. Corn/Tapioca starch
14. HCl Stopping Solution, 0.1N HCl
15. Iodine Reagent Stock Solution (in aqueous solution) See Note 1.
a. Iodine: 5 g/l
b. KI: 50 g/l
16. Potassium Phosphate Buffers
a. KH2PO4 (monobasic phosphate) (FW=136.1)
b. K2HPO4·3H2O (dibasic phosphate) (FW=228.23)

EXPERIMENTAL PROCEDURES

Because there is a variety of kinetic studies in this experiment, work will be divided among the entire
class. Each student will be assigned responsibilities for different sections.
1. Prepare a 20 g/l starch solution.

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 1. Mix 20 g of soluble potato/corn starch in 50 ml of cold water.
2. While stirring, add the slurry to approx. 900 ml of gently boiling water in a large beaker.
3. Mix well and cool the gelatinized starch solution to room temperature.
4. Add more water to bring the total volume to 1 liter.
5. Put a few drops of the starch solution on a glass plate. Add 1 drop of the iodine reagent and see
that a deep blue color is developed. The blue color indicates the presence of starch in the
solution.

2. Effect of the pH

1. Prepare 0.1M pH buffer solutions ranging from pH=5 to pH=9 in increments of one pH unit.
(Note that phosphate buffer is only good for ph=4.5--9 due to the dissociation constant.)
2. Add an equal volume of one of the above buffer solutions to 5.0ml of the 20g/l starch solution
prepared in Step 1. The resulting solution should contain 10g/l of starch in a buffered
environment.
3. Start the enzymatic digestion process by adding 1.0 ml of amylase; shake and mix.
4. Let the hydrolysis reaction proceed for exactly 10 minutes.
5. Add 1.0 ml of the reacted starch solution to 5ml of the HCl stopping solution (0.1N)
6. Add 1.0 ml of the above mixture to 5ml iodine solution to develop color. Shake and mix. The
solution should turn deep blue if there is any residual, unconverted starch present in the solution.
The solution is brown-red colored for partially degraded starch, while it is clear for totally
degraded starch.
7. Measure the absorbance with a spectrophotometer at 620nm. See Note
2.
8. Carry out the same procedure for the other starch solutions buffered at different pH's. (Use your
time wisely; all the solutions can be handled simultaneously if you are familiar with the
procedure. Slightly stagger the sequential sample withdrawal so that there is enough time for
sample preparation and handling.)

3. Effect of Temperature

1. Adjust the temperatures of the temporary water baths for temperature of 25⁰CPrepare the starch
substrate by diluting the 20g/l starch solution prepared in Step 1 with an equal volume of pH=7.0
phosphate buffer solution. This results in a working starch concentration of 10 g/l. Add 5 ml of
the starch solution to each of the test tubes.
2. Allow the temperature of each of the starch solutions to come to equilibrium with that of the
water bath.
3. Add 1.0 ml of amylase to each of the thermostated test tubes to start the reaction.
4. Let the hydrolysis reaction proceed for exactly 10 minutes.
5. Add 1.0 ml of the reacted starch solution to 5ml of the HCl stopping solution (0.1N)
6. Add 1.0 ml of the above mixture to 5ml iodine solution to develop color. Shake and mix. The
solution should turn deep blue if there is any residual, unconverted starch present in the solution.
The solution is brown-red colored for partially degraded starch, while it is clear for totally
degraded starch.

32
 7. Measure the absorbance with a spectrophotometer at 620nm. See Note 2.
8. Carry out the same procedure for the other starch solutions buffered at different temperature
(37⁰C, 40⁰C, 60⁰C and 80⁰C). Use your time wisely; all the solutions can be handled
simultaneously if you are familiar with the procedure. Slightly stagger the sequential sample
withdrawal so that there is enough time for sample preparation and handling.

Notes on the Experimental Procedure

1. Dilute the stock solution 1:100 to obtain a working solution. Other dilutions may be used,
depending on the enzyme activity. Iodine does not dissolve much in water. Iodine (I2) alone or
iodide (I-) alone does not color starch. It is the triiodide complex ( I3-, formed by I2+I-) that
gives off the blue color when it is incoporated into the coil structure of starch.

2. Remember to take care of the background absorbance caused by the colored iodine solution.
The true absorbance should be roughly proportional to the starch concentration. The enzyme
solution may have to be diluted first if all the starch present in the sample is digested and all
the color disappears in 10 minutes. The most reliable results are obtained when the decrease in
the absorbance is approximately 20-70% of the absorbance of the original, undigested starch
solution. To measure the amount of starch digested, you need to know the absorbance
corresponding to the initial undigested starch solution by following the same procedure with a
sample in which plain water in lieu of the enzyme solution is added to the starch solution.

RESULT AND DISCUSSION

1. Plot the enzyme activity versus pH. From this curve, what is the optimal pH? Explain why
enzyme activities depend on the pH. Similarly plot the enzyme activity versus temperature.
Report the optimal temperature.
2. To what extent did the heat treatment affect the enzyme activities? What happens to an enzyme
when it is subjected to heat?
3. Comment on ways to improve the experiment.

33
 EXPERIMENT 11: MICROBE GROWTH IN BIOREACTOR

PLEASE WATCH THE VIDEO AND ANSWER A QUESTIONNAIRE BEFORE

CONDUCTING THIS EXPERIMENT!!!

Learning outcomes:

At the end of the experiment, you will be able to:

a) Design a suitable experiment to investigate the operation of bioreactor

b) Evaluate important parameters related to growth of cultures in bioreactors

Introduction

Bioreactors are important unit operation in any bioprocess or biochemical related processes. It is a
system or a device that is used to grow cells, perform fermentation or digestion processes and is
typically conditioned-controlled. The two main classes of bioreactors: aerobic and anaerobic. The
mode of operations of bioreactors vary from batch, fed-batch, or continuous. It is important to first
cultivate the microorganisms in prior to seeding them and growing them in the bioreactor.

It is very important to control the conditions and nutrients in the bioreactors to ensure growth. Different
strains of microorganisms might have different metabolic pathways, different production rate and
excrete out different products or by products. Nutrients feed and aeration strategies affect not only
rate but also product yield and product distribution in the bioreactor.

In this experiment you will develop a bioreactor system for E. Coli for a period of 48 hours. Your
team is required to formulate suitable medium to cultivate and grow the microorganism. Your report
should be comprised of the followings:

a) Detail descriptions of materials and methods

b) Detail experimental set up with proper references to conditions – constant and manipulated
parameters
c) Detail descriptions of medium compositions
d) The growth curves
e) Carbon source consumptions

Relationship between all the described parameters to the biomass concentration should be described
and elucidated in detail.

At the end of the project, you will be required to prepare and oral presentations on your findings

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