Product Sterilization –
Membrane Filtration Technique
Copyright ©2009 by Gary Fromert, M.S.
PETRI Project, Northampton Community College
After completing this exercise, you should be able to:
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Explain what is meant by filter sterilization.
Recognize the limits of filter sterilization.
Explain what is meant by filtrate.
Explain what is meant by retentate.
Express when membrane filtration is necessary as a means of sterilization.
Demonstrate an understanding of the filter sterilization technique.
While there is no substitute for aseptic technique, sterilization of liquids assures you that
media and reagents are not a source of bacterial cell contamination. Heat sterilization
(autoclaving) is not an option for many liquids. Liquids that would be damaged by heat,
irradiation or chemical sterilization can be sterilized by mechanical filtration. This
method is commonly used for sensitive pharmaceuticals and protein solutions in
biological research. Filtration, therefore, is the method of choice for sterilizing materials
that are heat labile (readily undergoing change or breakdown).
Filtration can physically remove organisms from a liquid as it passes through a
membrane filter. Filtration does not necessarily sterilize unless very small filter pores are
employed. A filter with pore size 0.2 µm will effectively remove bacteria. If viruses must
also be removed, a much smaller pore size around 20 nm is needed. Prions are typically
not removed by filtration. Solutions filter slowly through membranes with smaller pore
diameters and the speed and ease with which one may filter something is directly
proportional to pore size, with large pores filtration is much quicker and easier than with
small pores.
The sterilizing filtration process is designed to remove viable and nonviable particles
from the fluid passing through the filter. The particles removed from the fluid either
remain on the surface of the filter membrane or are trapped within the membrane matrix.
The separation of solid particles from a fluid/solids suspension by passage of the fluid
through a membrane that retains most of the solids on or within itself is called filtration.
The membrane is called a filter medium, and the equipment assembly that holds the
medium and provides space for the accumulated solids is called a filter. That which has
been filtered or the liquid which has passed through the filter in the process of filtration is
called the filtrate. The material that is trapped by or remains on the filter is called the
retentate.
The filtration equipment and the filters themselves may be purchased as presterilized
disposable units in sealed packaging. To ensure sterility, the filtration system must be
tested to ensure that the membranes have not been punctured prior to or during use.
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To ensure the best results, pharmaceutical sterile filtration is typically performed in a
room with highly filtered air (HEPA filtration) or in a laminar flow cabinet or "flowbox",
a device which produces a laminar stream of HEPA filtered air. Because each
manufacturer and the products of each manufacturer vary widely, the United States
Pharmacopoeia (USP) standard set for Filtration Sterilization is described in the USP
1229F monograph as follows:
USP <1229F> Filtration Sterilization
Responsibilities for control are shared between filter user and filter manufacturer.
Filter users should control their processes influencing filtration and monitor the prefiltration bioburden. Filter manufacturers must provide a filter product with performance
and characteristics consistent with that initially evaluated with the fluid. Microbial
retention is a function of fluid, filter and process elements.
“A rigorous evaluation of all of the relevant parameters within the context of an
individual filtration process is extremely cumbersome, impractical and largely valueless
in a real world situation.”
“Success with sterilizing filtration has been predominantly accomplished over the last 30
years using 0.2µm filters. When coupled with appropriate process controls and integrity
test methods there have been comparatively few incidents of contamination associated
solely with the filtration process. In specialized settings, larger and smaller pore size
filters may be appropriate.”
In this laboratory you will be using the membrane filtration technique to filter sterilize a
liquid media product containing a suspension of bacterial culture. You will determine the
effectiveness of the filtration sterilization process by making a spread plate of an aliquot
of the suspension prefiltration and then by making a spread plate of an aliquot of the
filtrate post filtration. The plates are then incubated at 37°C for 24 to 48 hours after
which the plates will be observed for the presence of growth and the results recorded.
Materials (per student)
1 Sterile Trypticase Soy or Nutrient Broth Tube
1 Trypticase Soy or Nutrient Broth Suspension of Escherichia coli
4 Nutrient Agar or Trypticase Soy Agar (TSA) Plates
2 Sterile Vacuum Filter Units 0.22m (preferred) or 0.45m (Note: units must be of the
type that allow for access to the filtrate)
2 Empty Sterile Broth Tubes with Caps (for filtrate if using filter unit s with pour spout)
Sterile 0.1 mL Pipettes
Glass Spreader or Sterile Disposable Plastic Spreaders
95% Ethanol (for flame sterilization if using glass spreader)
200 mL Beaker (for ethanol if flame sterilizing glass spreader)
China Marker or Sharpie
Bunsen Burner
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Procedure
Analysis for product sterility after membrane filtration sterilization.
Note: You will be instructed as to the setup and use of the sterile filter unit.
Sterilization - Membrane Filtration Technique
Note: Maintain aseptic technique throughout the laboratory exercise!
Acquire both the tube of sterile broth and the tube of broth containing a suspension of
Escherichia coli.
1. Mark the bottom of one each of the nutrient agar plates as follows: "Control - Pre",
"Control - Post", "Product - Pre", and "Product - Post" include your initials and the date
on all plates.
2. Remove the wrapping from the all the membrane filtration units according to the
instructions on the wrapper.
3. Mark the lid or side of one of the membrane filtration units "Control".
4. Mark the lid or side of the other membrane filtration unit "Product".
5. Aseptically transfer 0.1mL of the sterile broth (control) to the nutrient agar plate
labeled "Control - Pre". Aseptically transfer 0.1mL of the suspension of Escherichia coli
(product) to the nutrient agar plate labeled "Product - Pre".
6. If using the glass spreaders, place approximately 15mL to 20mL of ethanol into the
200mL beaker. Place the lower end of the "L" of the glass spreader into the beaker
containing the ethanol. Flame the glass spreader, allowing the ethanol to catch flame and
immediately remove from the flame of the Bunsen burner. Allow the ethanol flame to
extinguish. The glass spreader is now considered "flame sterilized". Note: Be sure to hold
the glass spreader in a slightly downward position once removed from the beaker and
during flaming as to not let the ethanol run down the spreader on to your hand! If using
the sterile disposable plastic spreaders, aseptically remove one from the package.
7. Make a spread plate by placing the lower end of the "L" of the spreader gently to the
surface of the agar and rotate the plate as to spread the inoculum over the entire surface
of the agar. Note: Be sure to flame sterilize the glass spreader or acquire a new
sterile plastic spreader prior to spreading the inoculum of the second plate. Set these
two plates aside.
8. Pour each of the liquid broth samples into their respectively marked filter unit funnel.
9. Apply a vacuum to the vacuum port on the unit until all of the liquid has passed
through the membrane.
10. Depending on your specific type of filter unit, either unscrew the media bottle base of
the filter unit from the upper funnel/filter assembly or access the pour spout on the side of
the filter unit and aseptically pour some of the filtrate into an empty sterile broth tube.
11. Aseptically transfer 0.1mL of the sterile broth filtrate (control) to the nutrient agar
plate labeled "Control - Post". Aseptically transfer 0.1mL of the suspension of
Escherichia coli filtrate (product) to the nutrient agar plate labeled "Product - Post".
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12. Again, make a spread plate by placing the lower end of the "L" of the spreader gently
to the surface of the agar and rotate the plate as to spread the inoculum over the entire
surface of the agar. Note: Be sure to flame sterilize the glass spreader or acquire a
new sterile plastic spreader prior to spreading the inoculum of the second plate.
13. Invert all the plates (lid side down), and incubate the plates at 37°C for 24 to 48 hours.
14. Observe all the plates for growth of viable bacteria that have grown on the surface of
the agar. Record your results.
Results and Observations
After examining your plates record your results in the following table.
Sample
Sterilization - Membrane Filtration Technique
Prefiltration
Post Filtration
Growth (+) or (-)
Growth (+) or (-)
Control
Product
Laboratory Review
1. Explain what is meant by filter sterilization.
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2. What are the limiting factors of filter sterilization?
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3. Explain what is meant by filtrate and retentate.
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4. When is it necessary to use filtration sterilization?
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