Control of microbial growth/ Sterilization

1. Control of Micro organisms‐
Sahaya Asirvatham

2. Prevent contamination
Prevent transmission of pathogen
To prevent decomposition & spoilage of product
To prevent contamination in aseptic areas, processes like
production of pharmaceuticals by fermentation.
To maintain aseptic condition in operation theaters, filling
area of non sterile pharmaceuticals.
Reasons for Controling Microorganisms

3. Sterilization
Sterilization is defined as the process by which an article, surface or the
medium is freed of all living microorganism either in vegetative or spore
state.
Essential stage in processing of any product used for parenteral
administration, broken skin, mucosal surface or internal organ.

4. Fundamentals of Microbial Control
Important terms:
Thermal death point (TDP)
It is the lowest temperature at which all of the microorganism present
in the liquid suspension will be killed in just 10 minutes.
Thermal death time (TDT)
It is the minimum length of time whereby all microorganism present in
the liquid culture medium will be killed at given temperature.

5. Fundamentals of Microbial Control
Disinfection: A treatment that reduces the total number of microbes on an
object or surface, but does not necessarily remove or kill all of the microbes

6. Sanitation: Reduction of the microbial population to levels
considered safe by public health standards
Antiseptic: A mild disinfectant agent suitable for use on skin
surfaces

7. –cide: to kill Bactericide: chemical that destroys bacteria
Fungicide: a chemical that can kill fungal spores, hyphae, and yeasts
Virucide: a chemical that inactivates viruses
Sporicide: can destroy bacterial endospores
Germicide and microbicide: chemical agents that kill microorganisms
Stasis and static: to stand still
Bacteristatic: prevent the growth of bacteria
Fungistatic: inhibit fungal growth
Bactericidal Vs Bacteristatic

8. Death (in terms of microorganisms)
Irreversible loss of ability to reproduce.
Determined by inoculating culture to the medium

9. Pattern of Death in a Microbial Population
Bacterial populations die at a constant
logarithmic rate
Microorganisms were previously considered to be
dead when they did not reproduce in conditions
that normally supported their reproduction
However organisms can be in a viable but
nonculturable (VBNC) condition
Once they recover they may regain the ability to
reproduce and cause infection Pattern of Microbial death-an exponential
plot of survivors against mins of exposure to heating at 121 0
C.

10. Conditions affecting Antimicrobial Activity
1. Type of agent:
Physical or chemical
2. Nature of heat
3. Concentration, Dose, Time & temperature of the agent
4. Bioburdon
5. Mechnism of Action of the agent
6.Environment:
Physical & chemical properties of the medium or substance carrying the organism
Effectiveness of heat in acid is greater than in alkali
Consistency of the material (viscous: less penetration)
Concentration of carbohydrates increases thermal resistance
Presence of extraneous organic matter (inactivation of agent or protective effect)
Action of chemical agent Increases with increase in temperature.

11. 7. Kind of Microorganism:
Microbial spp. Differe in susceptibility to physical & chemical agents
Vegetative cell of spore forming bacteria are more susceptible than spores
Spores are most resistant
8. Physiological State of Cells:
Young actively metabolizing cells are more susceptible
In dormant cells the agent which causes damage through the interference with
metabolism, non growing cell would not be affected

12.
How Antimicrobial Agents Work: Their
Modes of Action??
1. Damage to the cell wall or inhibition of cell­wall
synthesis
2. Alteration of permeability of cytoplasmic membrane
3. Alteration of physical /chemical state of proteins &
nucleic acid
4. Inhibition of proteins & nucleic acid synthesis

13. Survivor Curve
When exposed to a killing process, populations of microorganisms
generally lose their viability in an exponential fashion,
independent of the initial number of organisms.
Of the typical curves obtained, all have a linear portion which may
be continuous (plot A), or may be modified by an initial shoulder
(B) or by a reduced rate of kill at low survivor levels (C).
Short activation phase, representing an initial increase in viable
count, may be seen during the heat treatment of certain bacterial
spores.
Survivor curves have been employed principally in the examination
of heat sterilization methods, but can equally well be applied to any
biocidal process.
Fig. 20.1 Typical survivor curves for bacterial spores
exposed to moist heat or gamma­radiation.

14. Expressions of resistance
D­value:

The resistance of an organism to a sterilizing
agent can be described by means of the D­value.

For heat and radiation treatments, respectively,
this is defined as the time taken at a fixed
temperature or the radiation dose required to
achieve a 90% reduction in viable cells (i.e. a 1
log cycle reduction in survivors).

The calculation of the D­value assumes a linear
type A survivor curve, and must be corrected to
allow for any deviation from linearity with type
B or C curves.
Expressions of Resistance

15. In order to assess the influence of temperature changes on
thermal resistance a relationship between temperature and log D­
value can be developed leading to the
expression of a z­value.
which represents the increase in temperature needed to
reduce the D­value of an organism by 90% (i.e. 1 log cycle
reduction).
For bacterial spores used as biological indicators for moist heat
(B. Stearothermophilus) and dry heat (B. subtilis) sterilization
processes, mean z­values are given as 10°C and 22°C,
respectively.
The z­value is not truly independent of temperature but may be
considered essentially constant over the temperature ranges used
in heat sterilization processes.
Z­Value:

16. In food industry unit of lethality has been devised, called as F­value.
This is defined as the equivalent in minutes of 1210
C of all heat considered with respect
to its capacity to destroy spores or vegetative cells of perticular organism
F = D (log N0
– log N)
Where D = D­value at 1210
C of the organism
N0
= Initial population Number/unit volume
N = Final Population number/unit volume
It is used to calculate probable number of survivors remaining in a load
F­Value:

17. The inactivation factor is the degree to which the viable population of organisms is
reduced by the treatment applied and is obtained by dividing the initial viable count
by the final count
IF = 10t/D
Where
t = holding time at sterilizing temp.
D = D­value for the marker organism at that temp.
Inactivation Factor

19. Sterilization Methods
I. Physical Methods
1. Heat Sterilization methods
Moist Heat
Dry Heat
Low Temperatures

20. l
Moist Heat Sterilization
Mechanism of killing is a combinantion of protein/nucleic acid
denaturation and membrane disruption
Effectiveness Heavily dependent on type of cells present as well as
environmental conditions (type of medium or substrate)
Bacterial spores are much more difficult to kill than vegetative cells

21. Methods for Moist heat Sterilization
a) Temperature at 100° C – Boiling
b) Temperature below 100° C – Pasteurization
c) Steam Under Pressure – Autoclave
d) Steam at Atmospheric Pressure­ Tyndallization

22. Temperature at 100° C – Boiling
Most vegetative microorganism are
killed in 2­3 mins, but spores make take
2­3 hrs.
Endospores, protozoan cysts, and some
viruses can survive boiling

23. Pasteurization
Used to reduce microbial numbers in milk and other
beverages while retaining flavor and food quality of
the beverage
Retards spoilage but does not sterilize
Traditional treatment of milk, 63°C for 30 min
Flash pasteurization (high­temperature short term
pasteurization); quick heating to about 72°C for 15
sec, then rapid cooling

24. Tyndallisation
The media containing sugar and gelatin are exposed to 1000
C for 20 minutes on
three successive days is known as tyndallization or intermittent sterilization.
In first exposure it kills vegetative bacteria and spores, since they are in favorable
medium, will germinate and killed on subsequent occasion.

25. Steam under pressure : Autoclave
Principle:
The lethal effect of moist heat is due to the denaturation and
coagulation of protein.
When steam makes contact with the cooler articles in the
autoclave it condenses into water on their surface and in
their interstices.

26. This condensation has three effects of critical importance for sterilization
process
1. It wets the microorganisms on the articles and so provides this essential
condition for their killing by moist heat.
2. It liberates the very large latent heat of steam and so rapidly heats up the
articles to the sterilizing temperature.
3. It causes a great contraction in the volume of the stream and so promotes
the ingress of fresh steam.

27. Autocalave

28. Method :
Materials to be sterilized are placed in perforated diaphragm
The test­tubes are plugged with non absorbent cotton and covered with paper to avoid
drenching of plugs during the release of steam when condensation occurs
After adjusting the water level, lid is kept in position and is closed tightly by means of
screw clamps.
Autoclave is switched on, steam valve is kept open until all the air is expelled out and
steam comes out.

29. The steam valve is closed and the pressure begins to rise
As the pressure begins to rise to 15lbs/sq.in (psi), time is noted. One of the electrical
coils is switched off (when 2 coils are used) or steam valve is opened partially so as to
maintain the pressure constant at 15psi.
After 15 min the autoclave is switched off and is allowed to cool down
After all the steam has escaped from the open steam valves, autoclave is opened and
all the sterilized materials are removed

30. Pharmaceutical applications:
Sterilization of culture media, apron and rubber tubing etc
Sterilization of injections solution and suspensions.
Sterilization of surgical dressings and fabrics such as cotton wool, gauze swabs,
ribbon gauze, masks, and caps etc.
Sterilization of packaging materials such as containers like metal drum, card board
boxes and wrappings such as nylon film, paper etc.

31. Dry Heat Sterilization
Principle: The killing effect of dry heat is because of denaturation of protein,
oxidative damage and toxic effect of elevated levels of electrolyte.
Method: there are three methods for dry heat sterilization.
Flaming
Incineration
Hot air oven

32. Flaming: inoculating loop or
wire, tip of forceps and searing
spatulas are held in Bunsen
flame till they become red hot.
Incineration: it is excellent
method for safely destroying
materials such as contaminated
cloths, pathological materials etc.

33. Hot Air Oven
It consists of a double walled chamber insulated with
asbestos sheet or glass wool to prevent radiation of heat.
The chamber is heated electrically and maintained
thermostatically.
The temperature can be noted on the thermometer which
can be inserted from top.
The hot air oven can be operated at 150˚C for 150 min,
160˚C for 2 Hrs, 170˚C for 60 min and 180˚C for 30 min for
sterilization of the articles.

34. Hot air oven is generally used for sterilization of glassware, metal
devices and other articles which are spoiled by autoclaving.

35. Precautions:
All the glassware must be clean and dry to prevent internal
contamination after sterilization.
It is essential to wrap the apparatus before placing them into the oven.
The oven must be loaded when it is cool.
The temperature must not raise above 180˚C in any case otherwise the
cotton plugs and papers may be charred.
Before removing the apparatus, oven should be cooled to room
temperature.

36. Pharmaceutical Applications:
It is used to sterilize glassware’s forceps, scissors,
scalpels.
It is used to sterilize all glass syringes.
Pharmaceutical product such as liquid paraffin,
dusting powder, fats and grease can be sterilize.
Sterilization of anhydrous material.
Sterilization of powders.

37. Decrease microbial metabolism, growth, and reproduction
Chemical reactions occur slower at low temperatures
Liquid water not available
Psychrophilic microbes can multiply in refrigerated foods
Refrigeration halts growth of most pathogens
Slow freezing more effective than quick freezing
Organisms vary in susceptibility to freezing
Refrigeration and Freezing

38. Dessication is drying (98% of the water is removed) inhibits growth due
to removal of water
Lyophilization (freeze­drying)
Substance is rapidly frozen and sealed in a vacuum
Substance may also be turned into a powder
Used for long­term preservation of microbial cultures
Prevents formation of damaging ice crystals
Dessication and Lyophilization

39. The use of dessication as a means of preserving apricots

40. Lyophilization

41. Ionizing radiation: if the radiation ejects orbital electrons from an atom causing ions to
form
Nonionizing radiation: excites atoms by raising them to a higher energy state but does
not ionize them
l
Radiation as a Microbial Control Agent

42. Ionizing Radiation
Gamma radiation produced by Cobalt­60 source
Powerful sterilizing agent; penetrates and damages both DNA and
protein; effective against both vegetative cells and spores
Often used for sterilizing disposable plastic labware, e.g. petri dishes;
as well as antibiotics, hormones, sutures, and other heat­sensitive
materials
Also can be used for sterilization of food; has been approved but has not
been widely adopted by the food industry

43. Mode of action
Radiation can cause both ionization and excitation and their absorption is not
affected by structure of the molecule. It may act directly or indirectly.
Direct action:
Every microorganism and living cell having target region which is radiation
sensitive, a single ionization of radiation in this sensitive zone or region will kill the
microorganism.
Indirect effect:
Absorption of radiation by water, within or surrounding living cell produces free
radicals. these are powerful oxidizing and reducing agent capable of damaging
essential molecule and therefore causing death.

44. Sterilization with Ionization; Radiation machine which uses Cobalt 60 as a Gamma radiation to
sterilize fruits, veg, fish, meat, etc..

45. Applications
Sterilization of disposable surgical materials and equipments ex. Plastic syringes, catheters, hypodermic needle and scalpel
blade.
Sterilization of adhesive dressings.
Sterilization of single application capsule of eye ointment.
Sterilization of catgut
Sterilization of packaging materials such as plastic films and aluminium foil.
Sterilization of bacterial and viral vaccines.
Used in radiotherapy of cancer.

46. Increased shelf life of food achieved by Ionizing Radiation

47. Ultraviolet Radiation
DNA absorbs ultraviolet radiation at
260 nm wavelength
This causes damage to DNA in the
form of thymine dimer mutations
Useful for continuous disinfection of
work surfaces, e.g. in biological safety
cabinets
Non­Ionizing Radiation

48. Ultraviolet Radiations
Method:
The effectiveness of the method depends on the wavelength absorbed by the particular
region or component of microbial cell.
260nm is the wavelength giving 100% effectiveness because it is the adjacent
wavelength strongly absorb by nucleoprotein.

49. Applications
Irradiation of incoming and internal air of sterile filling area
of antibiotic plants.
Sterilization of thermolabile products.
Improvement of bacteriological water used to manufacture
non sterile pharmaceuticals.
As aid to asepsis in manufacturing houses and hospitals.
To prevent cross infection in hospitals and schools

50. Modes of Action of
Ionizing Vs
Nonionizing
Radiation

51. Filters are used to sterilize these heat­labile solutions.
Filters simply remove contaminating microorganisms from
solutions rather than directly destroying them.
The filters are of two types:
(a) Depth filters
(b) Membrane filters.
Filtration

52. Consist of fibrous or granular materials that have been bonded into a thick
layer filled with twisting channels of small diameter.
The solution containing microorganisms is sucked in through this layer under
vacuum and microbial cells are removed by physical screening or entrapment
and also by adsorption to the surface of
the filter material.
Depth filters

53. Depth filters are of the following types:
1. Candle filters:
These are made up of
(a) diatomaceous earth
(b) unglazed porcelain
They are available in different grades of porosity and
are used widely for purification of water for drinking and industrial uses.

54. 2. Asbestos filters
Made of asbestos such as magnesium silicate.
Eg: Seitz and Sterimat filters
These are disposable and single­use discs available in different
grades.
They have high adsorbing capacity and tend to alkalinize the
filtered fluid.
Their use is limited by the carcinogenic potential of asbestos.

55. 3.Sintered glass filters
These are made up of finely powdered glass
particles, which are fused together.
They have low absorbing property and are
available in different pore sizes.
These filters, although can be cleaned
easily, are brittle and expensive.

56. Membrane Filters
Porous membranes with defined pore sizes that
remove microorganisms primarily by physical
screening.
These filters are circular porous membranes and are
usually 0.1 mm thick.
Although a wide variety of pore sizes (0.015–12μm)
are available, membranes with pores about 0.2μm are
used, because the poresize is smaller than the size of
bacteria.
This has replaced Depth Filters.
Membrane Filter Sterilisation

57. Filtration equipment used for microbial control

58. The role of HEPA filters in Biological Safety Cabinets
High­Efficiency Particulate Arresting
(HEPA) air filters are used in medical
facilities, automobiles, aircraft, and homes.
The filter must remove 99.97% of all
particles greater than 0.3 micrometer from
the air that passes through.