Rapid detection of bacteria & Sterilization

1. In the name of God
Rapid detection of bacteria
And
Methods of sterilizations
Maryam kazemi –Pharm D
Ph.D student of pharmaceutics
Shiraz university of medical sciences

2. Source of contamination

3. ENUMERATION OF MICROORGANISMS
1. Direct Count of Cells
Direct Count Using a Counting Chamber
Direct Count Using Fluorescent Dyes
2. Indirect Count of Cells
Viable Count
The Most Probable Number (MPN)

6. Rapid detection of bacteria
Time and sensitivity of analysis are the most
important limitation related to the usefulness of
microbial testing .

7. Membrane filteration –direct epiflourescent technique
(DEFT)
Membrane can be made from :
-nitrocellulose
- cellulose acetate ester
-nylon
-Polyvinyl chloride
-Polyesters
The purpose of the membrane :
1)To concentrate target M.O
2)To remove growth inhibitors
3)To transfer M.O between growth media without physical injury
through resuspension

8.  DEFT is a direct method used for enumeration of
microbes based on binding properties of flurochrom
acridine orang .
 Samples are pretreated with detergents and
proteolytic enzymes ,filtered on to a polycarbonat
membrane stained with acridine orange and
examined with fluorescent microscope .
 The number of viable cells is determined based on
the count of orange cells on the filter
 Can be performed in 10 min

10. biosensors

13. Types of biosensor

15. Flow cytometry
 This may be considered as the form of automated fluorescence
microscopy in which instead of sample being fixed to slide ,it is
injected to a fluid (dye).
 The cells are carried by laminar flow of water through a focus of
light .
 Each cell emits a pulse of fluorescence and the scattered light
is collected by lenses and directed on selective detectors
 These detectors transform the light pulses into an equivalent
electrical signal
 The light scattering of the cells gives information on their size ,
shape and structure.

17. WHAT IS STERILIZATION:
Sterilization can be defined as any process that effectively
kills or eliminates transmissible agents (such as fungi,
bacteria, viruses and prions) from a surface, equipment,
foods, medications, or biological culture medium.

19. S.no METHOD
MECHANISM
MERITS DEMERITS APPLICATIONS
1 Heat
sterilization
Destroys
bacterial
endo toxins
Most widely
used and
reliable
method of
sterilization,
involving
destruction of
enzymes and
other essential
cell
constituents
Can be
applied only
to the
thermo
stable
products
Dry heat is applicable for
sterilizing glass wares and
metal surgical instruments
and moist heat is the most
dependable method for
decontamination of
laboratory waste and the
sterilization of laboratory
glassware, media, and
reagents.

20. S.no METHOD
MECHANISM
MERITS DEMERITS APPLICATIONS
1
2
Gaseous
sterilization
Radiation
sterilization
Alkylation
Ionization of
nucleic acids
Penetrating
ability of
gases.
It is a useful
method for
the industrial
sterilization of
heat sensitive
products
Gases being
alkylating
agents are
potentially
mutagenic and
carcinogenic.
Undesirable
changes occur in
irradiated
products,an
example is
aqueous
solution where
radiolysis of
water occurs.
Ethylene oxide gas has been used
widely to process heat-sensitive
devices.
Radiation sterilization is generally
applied to articles in the dry state;
including surgical instruments,
sutures, prostheses, unit dose
ointments, plastics

21. S.no METHOD
MECHANISM
MERITS DEMERITS APPLICATIONS
1 Filtration
sterilization
Does not
destroy but
removes the
microorganisms
It is used for both
the clarification
and sterilization of
liquids and gases
as it is capable of
preventing the
passage of both
viable and non
viable particles
Does not
differentiate
between viable
and non viable
particles
This method is Sterilizing
grade filters are used in the
treatment of heat sensitive
injections and ophthalmic
solutions, biological
products and air and other
gases for supply to aseptic
areas

22. Moist heat
 Both temperature and pressure should be used to monitor the process.
 Irrevesible denaturation of vital enzymes that results in cell death.
 If water vapor is peresent ,much lower tempretures are required in
heat sterilization.
 Autoclaving (steam under pressure) is the most effective method of
moist heat sterilization. The steam must directly contact the material
to be sterilized.
 Fifteen psi, 121° C for fifteen minutes kills all vegetative bacteria
and endospores.

24.  Sterilization can be effectively achieved at a temperature above 100oC
using an autoclave. Water boils at 100oC at atmospheric pressure, but
if pressure is raised, the temperature at which the water boils also
increases.
 To destroy the infective agents associated with spongiform
encephalopathies (prions), higher temperatures or longer times are
used; 135oC or 121oC for at least one hour are recommended.

25. Standard autoclave cycles

28. Dry heat
 Sterilization by dry heat may be suitable for non-aqueous liquids or
dry-powder products and materials (glassware ,metal parts)
 Use it only with materials that can withstand temperatures at least as
high as 170o C (338o F). Temperatures above 170o C are required to
kill endotoxins (pyrogens) as well as some bacterial spores. Some of
the new dry heat sterilizers can attain temperatures up to 210o C
(410o F).
 The process used should include air circulation within the chamber
and the maintenance of a positive pressure to prevent the entry of
non-sterile air.

29. DRY-HEAT STERILIZATION PROCESSES
The two types :
1) dry-heat batch sterilization/oven sterilization
2) dry-heat tunnel sterilization

30. dry-heat batch sterilization/oven sterilization
 the type of dry heat unit widely used in the pharmaceutical
industry
 There are four stages involved in oven sterilization.
1) Drying
2) heat-up
3) exposure
4) Cool down
 it uses the principle of convective heat transfer to heat the load.

31.  The two types of convection heating include :
• Natural: as a result of the buoyancy forces generated by differences
in density
• forced convection heating :(HEPA)-filtered air is heated by
passage over the electric heating elements of the oven and then this
heat is transferred from the air to the product by forced convection.

32. a modern forced-convection batch sterilizer

34. Dry-Heat Tunnel Sterilization
 Dry-heat tunnel sterilization is a continuous conduction
process in contrast to the batch processes in ovens.
 is only found in large-scale processes, and the main
application of this process is in the sterilization and
depyrogenation of glass.
 When a product is sterilized in a tunnel, it is dried, heat-
treated by radiant heat or hot air, and then cooled. A
typical tunnel sterilization exposure time is 3 or 4 min at
300C or more.

36.  Infrared tunnels transmit heat by exposing the surface of items to radiation
by direct rays. This is accomplished by infrared heaters, located in the roof
of the tunnel; when products pass through the tunnel, their surfaces are
heated along with the internal surface of the tunnel itself. This heat is then
diffused throughout the product by radiation.
 The development of the infrared radiation tunnel in recent years has
opened new possibilities for using dry heat in high-temperature short-time
sterilization processes.
 The main advantage of dry-heat sterilization is its penetrating power. It is
not as corrosive as steam for metals and sharp instruments.

37. Sterilization by radiation
 Sterilization by radiation is used mainly for heat-sensitive
materials and products.
 Many pharmaceutical products and some packaging materials
are radiation-sensitive.
 Ultraviolet irradiation is not an acceptable method for terminal
sterilization.
 During the sterilization procedure the radiation dose should be
measured.

38. Sterilization by gases and fumigants
 Sterilization by gases and fumigants should only be used for finished
products where there is no suitable alternative.
 Various gases and fumigants may be used for sterilization (e.g.
ethylene oxide and hydrogen peroxide vapour). Ethylene oxide
should be used only when no other method is practicable.
 Direct contact between gas and microorganisms is essential

39. Filteration

40. filteration
 Certified by the manufacturer to retain at least 107 microorganisms of
a strain of Brevundimonas diminuta per cm2 of filter surface area
 Certain solutions and liquids that cannot be sterilized in the final container
can be filtered through a sterile filter of nominal pore size 0.22 micron (or
less), or with at least equivalent microorganism-retaining properties, into a
previously sterilized container.
 Such filters can remove bacteria and molds, but not all viruses or
mycoplasmas.
 Filtration alone is not considered sufficient when sterilization in the final
container is possible. Of the methods currently available, steam sterilization
is preferred.

41.  The fibre-shedding characteristics of filters should be minimal
(virtually zero). Asbestos-containing filters should not be used
under any circumstances.
 Filtration is the passage of a liquid or gas through a filter with
pores small enough to remain microbes.
 Microbes can be removed from air by high -efficiency
particulate air filters. HEPA
 SAL for sterilizing a solution by filtration is normally accepted
as 1:1000 (10-3)

42. Membrane filters composed of nitrocellulose or cellulose acetate
are commonly used to filter out bacteria, viruses, and even large
proteins. Range from 0.22 um – 0.45 um for bacteria but
sometimes mycoplasma or spirochetes can pass through.
Sizes can go as low as 0.01 um to filter viruses and proteins.

44. radiation
• The effects of radiation depend on its wavelength, intensity and
duration.
 Ionizing radiation (gamma rays, X-rays and high-energy electron
beams) has a high degree of penetration and exerts its effect
primarily by ionizing water and forming highly reactive hydroxyl
radicals.
Destroys DNA.
 Non-ionizing radiation causes damage to DNA.
Ultraviolet causes formation of thymine dimers.

49. Z value , F value & d value
D value
decimal reduction time and is the time required at a given
condition (e.g. temperature), or set of conditions, to kill 90% (or 1
log) of the exposed microorganisms.
z Value
– The increase in temperature required to reduce D
to 1/10 its value or to reduce it by one log cycle
• F value
– Time in minutes at a specific temperature needed to kill a
population of cells or spores usually 121 o C

53. Referencess
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novel electrochemical biosensor based on dynamic polymerase-extending
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Pflug, I. J., Textbook for an Introductoty Course in the Microbiology and
Engineering of Sterilization Processes, University of Minnesota, 19825.
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