TECHNIQUES IN HOMOGENATE

PREPARATION AND
LYOPHILIZATION

YAZMIN BUSTAMI, PhD (Modified By Prof. Dr. K. Sudesh Kumar)

SCHOOL OF BIOLOGICAL SCIENCES, USM
HOMOGENIZATION
In dictionary: a process of making things uniform or similar

Tissue homogenization
 to prepare a uniform dilution of a known quantity of tissue
suspended within a known quantity of a suitable diluents
and uniformly crush the tissue in such a manner as to
disperse the fragments of tissue evenly throughout the
mixture.
 a disorganization stage where a tissue is converted into a so-
called homogenate
Advantages/application of mixing
 To obtain uniform composition of the mixed components

 To enhance physical and chemical reaction of mixed components

 To improve dissolution and diffusion of mixture

 To get true solution after mixing two miscible liquids

 Mixing is essential to produce emulsion when two immiscible

liquids are mixed together with emulsifying agent.
Techniques in Preparing Homogenates and
Freeze Drying

➢Separation (centrifugation)
- Involves encouraging the cells to lyse / break apart to release their
contents.
- Preliminary stage for the separation of cellular components

➢Selection of homogenization methods

- Type of tissues and sample to disrupt
Selection of homogenization methods:

1) Osmotic lysis
• most gentle procedures for cell breakage
• suitable for the cells that have only a cell membrane and exist as single cells
such as bacteria and yeast
• not suitable for cells with strong carbohydrate-containing cell wall such as
prokaryotes bacteria, plants and fungi

a. lysozyme
• Works effectively for E. Coli
• Gram positive bacteria

b. chemical method
• Toluene (methylbenzene)
• Yeast
Detergent molecules interfere with the lipids and proteins on the cell membrane
and nuclear membrane, causing the DNA to be more exposed.
2) Grinding (some gentle and others more harsh)

a. Mortar and pestle

• gentle grinding
• limited to small sample size
• suitable for animal and plant tissues
• For bacteria and yeast: more efficient by adding
an abrasive to the mixture

Examples :
• Fine alumina is often employed to break bacteria
• Fine glass beads (45-50 µ diameter)
• adding liquid nitrogen (lower the temperature)-
to avoid denaturation/deactivation of compound
or protein
b. Tissue homogenizer
• glass or electric
• suitable for soft tissue → Animal tissue
(liver)

c. Blender
• relatively harsh method of breakage
• handles large and small samples
• for breaking plant and animal tissues
• Ineffective for yeast and bacteria
- add fine glass beads
Glass homogenizer
Electric homogenizer
-Samples are placed in the blender with
extraction buffer and then blended.

-The blades shear and cut tissues, reducing

tissues in size significantly.

Blender
d. Ultrasonic homogenizers
• also called disintegrators/sonificators
• are effective against bacteria and yeast at
sufficiently long periods of application
• the times may be shortened by including glass
beads in the suspension
• based on ultrasonic signal of high intensity
• The shock and vibration due to high
energy/ultrasonic waves → tissue destruction
• Various sized probes → samples from a few ml
up to 1 liter.
• Temperature must be carefully monitored and
the duration of sonification held to a
minimum, with intermittent cooling
e. Presses
• French press been used to disintegrate the biological samples
• by placing a cell suspension of small volume (5-50 ml) up to 40,000 psi
• forcing it through a small opening (1 mm or less) under a high pressure
• Gentle but restricted to small sample size

Concept:
As the sample passes through the valve, the cells experience shear stress,
resulting in cellular disruption. Also, as the cells move through the valve, they
experience decompression and subsequently expand and rupture.
French press cell lysis is a technique commonly used for lysing bacterial cells, and
other microorganisms for isolation of proteins and other cellular components.
French pressure cell
Advantages and disadvantages of different method of homogenization

Methods (mechanical) Advantages Disadvantages

Mortar & pestle - easy to use and -Throughput with mortar and pestle is low.
- inexpensive to purchase. -Contamination issues as
- With dry grinding, it is possible grinding will generate dust.
to generate very small particles. - many sets are made of glass or porcelain, which can
chip or crack if
dropped.

Glass homogenizer - inexpensive and easy to use. - will leave fibrous and membranous components
- They work relatively well and generate relatively intact.
very fine homogenate. - Certain tissues, even with prolonged grinding, are
- In single-step disruption experiments, difficult to disaggregate.
conical glass homogenizers liberate - Throughput with these homogenizers is also low unless
about half as much enzyme as multiple units are available.
compared to larger more expensive - Glass homogenizers are also prone to breakage.
high throughput homogenizers.
- They are very easy to clean and
decontaminate

French press - very effective and efficient tool. - Sample sizes are relatively small and throughput is very
- Homogenates generated by French low.
press rival - For any samples other than single cells
- ultrasonication in degree of or microbial cultures, a pre-homogenization step is first
thoroughness of disruption. necessary.
- sample homogenates are very uniform. - French pressure cells can be expensive relative
to the number of samples that can be processed. - Due
to the small orifice, the French press can clog.
Methods Advantages Disadvantages

Ultrasonication - Simple - Moderate energetic costs

- short extraction time - temperature rise
- high reproducibility - rigid cell wall hinders product release
- efficient - production of reactive hydroxyl radicals
- not applicable to large-scale

High speed - Simple effective - High energy consumption

homogenization - short contact time - temperature rise may lead to degradation of thermolabile
- can be applied on algal slurry compounds
- species-dependent effectiveness
- rigid cell wall may hinder product release
- contamination with abrasive materials

High-pressure - High efficiency - High energy consumption

homogenization - does not require biomass drying - temperature rise may lead to degradation of thermolabile
- easy scale-up compounds
- rigid cell wall may hinder product release
- very fine cell debris
Relative homogenizer efficiency
PRICE:
RM3,029.00

Example of
electric
homogenizer
T 18 BASIC ULTRA-
TURRAX
HOMOGENIZER
Components
Operating instruction of
homogenizer (T 18 basic ULTRA-TURRAX)

Loosen the corresponding clamps and lift up the

disperser.

Place vessel containing medium to be dispersed at

the middle of the plate stand.
Carefully immerse the dispersing tool into the liquid.
**the distance between the dispersing tool and the
vessel bottom should not be less than 10 mm
Make sure that the adjustment wheel is adjusted to
minimum speed (speed 1).

Push the on/off switch to start dispersion.

*Increase the dispersing speed gradually until a
suitable speed is attained (maximum speed is 6).

Medium will be completely homogenized in a few

minutes.

Switch off the instrument..

Loosen clamp, lift up disperser and remove medium

vessel from plate stand. Reattach disperser to clamp
and turn the adjustment wheel to minimum speed.
Precaution when using the homogenizer T 18
basic ULTRA-TURRAX
 Wash the dispersing tool before and after use of the instrument by repeating the
process with distilled water.
 Wipe the dispersion tool dry with lint free tissue.
 Do not start the instrument before immersing the dispersing tool in liquid.
 Never run the dispersion tool dry. Without cooling by the medium the bearing will be
destroyed. Always ensure that the dispersing element does not run dry in some cases
should spouts develop.
 Dispersing causes the medium to heat up.
 The fill level of the medium must be at least 10 mm above the lower overflow hole of
the shaft.
 While working with the disperser, the user must wear his personal protective equipment.
 Likewise the optimal dispersion duration and rotating frequency must be determined by
attempts.
 Longer application times bring hardly any improvements, increase however the sample
temperature substantially.
Example of analysis that required
homogenization method:
 Protein extraction for purification – using
ultrasonicator
 Enzyme activity measurement- using
French Press
Example: Purification of PhaCBP-M-CPF4
Bacterial Protein Expression
Ultracentrifugation
➢ E. coli/pET47 phaCBP-M-CPF4 ➢ Sonication ➢ 30,000 RPM
➢ Pellet sample in ➢ 40 minutes
lysis buffer ➢ 4 °C

Supernatant

Ion-exchange Affinity Chromatography

Size-exclusion
chromatography (His-Tag Protein Purification)
chromatography
Ni-NTA resin
Result : His-Tag Protein Purification (Ni-NTA Resin)
His-GP-PhaCBP-M-CPF4
No. of amino acids : 570 After optimization of
Molecular weight : 64147.94 Da sonication and lysis
Theoretical pI : 4.99 buffer

Protein Protein Eluates of affinity

Protein Protein Eluates of affinity marker Supernatant Pellet marker chromatography
marker Supernatant Pellet marker chromatography (In this case: Ni-NTA agarose purification)

~70 kDa (Suspected : Chaperone)

50 kDa 50 kDa
Target
protein

Target
protein

20 kDa Protein remain at the 20 kDa

pellet may due to
either overheat of
sample during
sonication or lysis
buffer was not
Guidelines for using sonicator for protein extraction
➢ Always keep the samples on ice. The energy from the sonicator which breaking the
samples also heats it up. Too much of heat can cause the protein extracts
denatured.
➢ Reduce the temperature of sample by pulsing.
➢ Avoid over-Sonicate.
➢ Sample volume and probe size. The probe you use can vary depending on your
sample size. Each probe has a recommended sample volume range.
➢ Limit foaming of the sample during sonication by submerged the probe properly in
the sample.
➢ Amplitude and intensity have a direct relationship. When users operate at a low
amplitude setting, they will deliver low intensity sonication and vice versa.
Sonication at lower amplitude for longer time will reduce heating of the sample
➢ Always clean the sonicator tip between samples. Cleaning the sonicator tip is
critical in limiting protein carryover. Wiping the sonicator probe with 70 %ethanol or
sonication of ethanol in a beaker is an effective way of cleaning the sonicator tip.
Adopted from : https://bitesizebio.com/30474/sonication-7-tips-mastering/
Example: Enzyme determination (using French press)
PHA biosynthesis
The culture was incubated for 24 h & 48 h

Bacterial cells were harvested after 24 h & 48 h

(Centrifugation: 8000 xg, 10 min, 4 °C

The cells were resuspended with 2 mL of suspension

buffer

The cells were lysed by using Thermo IEC French

Press Cell Press for three passages to obtain crude
cell extract

The crude cell extract was used for further

experiment
What is Lyophilization?

 A freeze-drying process that removes water from a

product after it is frozen and placed under vacuum.
 Sublimation: when a solid (ice) changes directly to a
vapour without first going through a liquid (water) phase
Introduction
• to concentrate the samples
• can be kept in an active or viable state for many years after lyophilization
• involves the removal of solvent from a frozen sample
• most effective methods for drying or concentrating heat-sensitive materials
• biological solution to be concentrated is “shell-frozen” on the wall of a round-bottom
or freeze-drying flask
• dry ice-acetone (-70oc) or liquid nitrogen (-170oc) → slowly rotating it as it held at a 45o
angle.
• Freezes in layers on the wall of the flask
• Provides a large surface area for evaporation of water
Process of lyophilization
• flask connected to freeze-dryer which consists of a refrigeration unit and a vacuum
pump
• Freeze: Maintains the sample at -40oc for stability of the biological materials and the
samples become completely frozen.
• Vacuum: applies a deep vacuum → 5 to 25 mmHg, well below the triple point of
water.
• Dry: Heat energy is then added to the product causing the ice to sublime.
• Ice formed from the aqueous solution sublimes and is pumped from the sample vial
(sublimation process)
• Most freeze-dried biological materials are stable for long periods of time and remain
viable for many years
 Product chamber: Freeze dryer equipment
manifold with
attached flask or a
larger chamber
with a system of Vacuum system:
shelves on which to separate vacuum
place product pump connected
to an airtight
condenser and
attached product
chamber

Control system:
complex control Condenser: Refrigerator system:
system. Basically To attract the Cools the ice condenser
has temperature vapors being
and sensing ability sublimed off
Precautions and limitations of lyophilization

✓ limited to aqueous solutions

✓ organic solvents lower the melting point of aqueous solutions and increase the
chances that the sample will melt and become denatured during freeze-
drying
✓ organic vapors will pass through the cold trap into the vacuum pump (cause
damage)
✓ use solvent trap or a special filter cartridges or liquid nitrogen traps
✓ safety consideration must be made when handling volatile and or potentially
harmful materials.
Example of Freeze
Drying machine
 LABCONCO FreeZone Freeze-
Dryer
Sample preparation for freeze
drying

Place the samples inside the Fast-Freeze Flask and stored at -

20°C overnight before freeze drying
Switching on freeze dryer

Take out the stopper of the drain hose

and release the water from the collector
chamber.

Wipe the collector chamber with

tissue/cloth if there is water inside the
chamber.

Close the lid of collector chamber

correctly.
Make sure all the vacuum valves of drying port are at OFF position.

On the main power switch at the left side of

the machine.

Do the setting accordingly on the machine in

term of the Vacuum and purge
Attached the Fast-Freeze Flask with the pre-
freezing samples to the vacuum valve by
using a Fast-Freeze Flask top.

Open the vacuum valve by turning it

clockwise to start the freeze-drying process.
References

 Pieracci, J.P., Armando, J.W., Westboy, M. and Thommes, J. (2018).

Chapter 9- Industry Review of Cell Separation and Product
Harvesting Methods. Biopharmaceutical Processing 165-2016
 Burden, D. B. (2012). Guide to the disruption of biological Sample.
Random Primers 12, 1-25
For more information:

 https://www.slideshare.net/BikashAdhikari26/pharmaceutical-
mixing-homogenization
 https://www.slideshare.net/ceutics1315/lyophilization-39635366