Microbial death curve, Physical and chemical methods of microbial control, Sterility test and sterility assurance.

1. Control of
Microorganisms
(Dr.) Mirza Salman Baig
Assistant Professor (Pharmaceutics)
AIKTC, School of Pharmacy,New Panvel
Affiliated to University of Mumbai (INDIA)

2. Fundamentals of Microbial
Control

3. Concepts
• Death of microbe= Loss of Ability to
reproduce
• Microorganism die at constant rate
over a period of time
• Exponential death
• It take same time to kill last 9 cells as
that for killing initial 900,000 cells

4. Microbial Exponential death rate

5. Microbial Exponential death Curve

6. Microbial Exponential death Curve
for different population load

7. D - Value
• D-value refers to decimal
reduction time and is the time
required at a given condition (e.g.
temperature) to kill 90% (or 1 log) of
the exposed microorganisms.
• Thus after a colony is reduced by 1 D,
only 10% of the original organisms
remain

8. Z - Value
• The z-value is a measure of the
change of the D-value with varying
temperature
• Z - value is the number of degrees
the temperature has to be increased
to achieve a tenfold (i.e. 1 log10)
reduction in the D-value.
• Range is 10-15OC

9. Contd...
D-value gives the time needed at a
certain temperature to kill 90% of the
organisms,
Z-value relates the resistance of an
organism to differing temperatures

10. Conditions affecting
Antimicrobial activity
• Size of microbial population
• Concentration of antimicrobial agent
• Characterstic of microbe (Gm +ve
are more resistant)

11. Mechanism of microbial cell
damage
• Microbial cell = Cell oranels in a
colloidal state sorrounded by cell
membrane and cell wall
• Damage to DNA, Ribosome, Enzyme
may lead to death of microbe
• Disruption of cell membrane or cell
wall
• Precipitation of proteins in cell

12. Physical Methods of
Microbial Control

13. Definations
Sterilisation :
• It is a process by which an article, surface
or medium is made free of all
microorganisms either in vegetative or
spore form.
Disinfection :
• Destruction of all pathogens capable of
producing infections but not necessarily
spores.
• All organisms may not be killed but the
number is reduced to a level that is no
longer harmful to health.

14. Contd...
Antiseptics :
• Chemical disinfectants which can
safely applied to living tissues
and are used to prevent infection
by inhibiting the growth of
microorganisms.
Asepsis :
• Technique by which the occurrence
of infection (into an uninfected
tissue) is prevented.

15. Physical Methods
• High Temperature
– Moist heat (steam)
• Boiling water
• Autoclave
• Pasteurization (kill most cells but no sterlization)
– Dry Heat
• Incineration (Burning)
• Hot air oven
• Low Temperature (bacterio static, Inhibit
metabolism)
• Radiation
– Ionizing (Gamma ray, X-ray)
– Non ionizing (UV)
• Filteration
– Membrane filter
– HEPA filter
• Desiccation (bacterio static)

16. Use of Temperature to control Microorganism

17. Moist heat
• More effective than dry heat
• Denaturation and coagulation of
protein
• Application - To sterlize culture
media, utensil, surgical instrument
• Autoclave can sterilize in 15 min at
121.5OC

18. Autoclave

19. Dry Heat
• Less effective than moist heat
sterilization
• Kill microbe by oxidation of cell
component
• Application - Sterilize glasswares
• Hot air oven can sterilize glassware
in 2hrs at 160-180OC

20. Hot air oven

21. Ionizing Radiation
(cold sterilization)
• Ionizing radiation split water
molecule in hydroxil ion and proton
• Good penetration ability
• Economical
• Application - sterilize packaged food
and medical equipments
• X rays, Gamma rays

22. Non ionizing radiation
• Low penetration power compare to
ionizing radiation
• UV rays (295-400 nm) causes
damage to DNA
• Pyrimidine dimers are formed
because of UV exposure
• Application- to sterlize surface in
labs, aseptic rooms, kill microbe in
water.

23. Filteration Method
Steps
• Passage of solution through sterilize
bacteria proof filter
• Aseptic transfer of filterate to sterile
container
• Testing of sterility

24. Filteration Method
Types of Filters
• Asbestos Filter (Seitz filter) (0.01-5 micron)
• Sinterd glass filter (borosilicate glass
powder is partially fused for adhesion
and allowed to cool in a funnel)
• Filter Candels (ceramic/ made up of
porcelain or kieselguhr)
• Membrane Filter

25. Scintered glass filter

26. Membrane Filters
• Thin 150 micron
• Pore size 0.2micron
• pores with uniform diameter
• they are disposible
• Application- sterilization of eyedrops,
seperation of virus from bacteria

27. Membrane Filter Assembly

28. HEPA filters
• High efficiency particulate air filters
• Consist cellulose acetate pleated
• It capture 99% of particulate matter
from air
• For aseptic rooms
• Filling room of parentrals

29. HEPA Filter

30. Desiccation
• Stop metabolic activity
• Neisseria meningitidis is susceptible
to desiccation
• In lyophilization microbes are quickly
dried at freezing temperature and
sealed in container under vacuum
• Application - preservation of high
sugar concentration product
(Jam) due to removal of water from
bacterial cell by osmosis

31. (Chemical Agents)
Chemical Methods of
Microbial Control

32. Ideal properties of chemical agent
• Antimicrobila activity
• Solubility
• Stability
• Non toxic
• Homogeneous
• Active at room temp
• Detergency
• Economical

33. Chemical Agents
• Phenol (Alter permiability)
• Alcohol (denature protein/ solublise
lipid)
• Halogen
• Heavy metals (Zinc)
• Detergent
• Qutarnary ammonium compound
• Gaseous agents

47. Mechanism of microbial cell damage

48. Gaseous sterilization
• Ethylene oxide
• B- Propiolactone
• Formaldehyde

50. MOA
• The mechanism of antimicrobial
action of the two gases is assumed to
be through alkylations of sulphydryl,
amino, hydroxyl and carboxyl groups
on proteins and amino groups of
nucleic acids

53. Application
• Gaseous sterilization is used for
sterilizing thermolabile
substances like; hormones, proteins,
various heat sensitive drugs

54. Evaluation of antimicrobial
potency
• Tube dilution
• Agar plate
• Phenol coefficient

63. Sterility assurance
Inactivation factor
Sterilization Indicators
Test for sterility

64. Sterilization Control

67. Inactivation Factor
• It is the degree to which viable
population of organism is reduced by
treatment applied and it is obtained
by dividing initial viable count by
final viable count.
• IF=10t/D
• t= holding time for sterilization
• D= D value

68. Sterilization Indicators
• Physical
• Chemicals
• Biological (Bascillus stearothermophilus)

69. Physical Indicators
1.Moist heat- Master processes record
(MPR) & Batch processes record
(BPR) comparision (pressure guage)
2.Dry heat- Temperature record
3.Radio sterilization- Plastic dosimeter
4.Gaseous method- Temperature,
Pressure, concentration and humidity
5.Filteration- Bubble point pressure
technique use for pore size
determination and integrity.

70. Chemical Indicators
1. Browne's tube- for heat processes,
sealed tube with reaction mixture,
• Colour change from
red-> brown -> green
• Moist heat --> Black spot
• Dry heat --> Green spot

71. Chemical Indicator
2. Witness tube- Crystalline
substance with specific melting point
are used. Time required to move
molten compound from one section
to another through narrow
constriction is corellated with time of
exposure.
Ex- Sulphur 115O C
Benzoic acid 121O C

72. Chemical Indicator
3. Heat Sensitive tape- This is to
make sure all air has removed from
autoclave and steam penetration is
rapid and even. All the bars on tape
should change colour.

73. Chemical Indicator
4. Roye Sachet- Chemical indicator
for ethulene oxide indicator. It
consist of Magnesium chloride, HCl
and Bromophenol indicator
• Colour change, Yellow --> Purple
5. Chemical Dosimeter- Measure for
radiation dose, Made of radio
sensitive indicator.
• Colour Change, Yellow --> Red

74. Biological Indicators
• It consist of Suitable organism on
carrier distrubuted through sterlizer
load.
• At the end of sterilization process,
units are recoverd and inoculated to
determine culture growth.
• Organism used are resistant bacterial
spores.

75. Biological Indicators

76. Test for sterility
• Intended for detecting presence of
viable form of microorganism in
pharmaceutical preparation
• Final product testing for sterility
• Culture medium used
– Fluid thioglycollate medium
– Soyabean casein digest media

77. Test for sterility
• Method A- Membrane filteration test
• Method B- Direct inoculation test

78. Method A
• Oil
• Ointments
• Bacteriostatic substance
Method B
• Solutions
• Suspensions
• Sterile devices