Sterilization: Sterility Test

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Sterilization 

➢ The  ultimate  goal  in  the  manufacture  of  a  sterile  product  is  ​absolute  absence  of 
microbial contamination​. 
➢ Inactivation of MOs by physical or chemical means. 
➢ The  D  value  is  a  single  quantitative  expression  of  the  rate  of  killing  of 
microorganisms. 
➢ This  is  the  time  required  for  a  90%  reduction  in the microbial population. Hence, the 
time or dose it takes to reduce 1000 microbial cells to 100 cells is the D value. 
➢ Sterilization  Assurance  limit  (SAL)​:  The  product  will  be  sterilized  by  a  sterilization 
process  sufficient  to  produce  a  probability  of  non  sterility  of  one  out  of  1  million 
containers .  
➢ SAL= 10​-6​(1 viable microorganism in 10​6​ sterilized item of the final product). 
➢ Whenever  possible  terminal  sterilization  is  chosen,  sterilized  in  its  final  container  & 
possible with heat stable particles. 

❖ Sterility test 
➢ Sterility  test  is  applied  to  the  Pharmaceutical  preparations  that  are  required 
to sterile preparation like parenteral and ophthalmic preparation. 
➢ To detect the presence of viable form of microorganism. 
➢ Sterility  test  is  carried  out  at  aseptic  condition(grade  A  laminar  airflow 
cabinet) 
➢ The  microorganism  are  placed  in  a  culture  medium  which  provides  nutrition, 
water,  and  favourable  temperature/condition,  microorganism  will  grow  and 
their presence detected in culture medium. 
 
 

 
 

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Method of sterilization:- 
❖ Physical Method 
➢ (a) Thermal (Heat) methods 
➢ (b) Radiation method 
➢ (c) Filtration method 
❖ Chemical Method  
➢ (a) liquid  
➢ (b) Gases 
 

 
 
1. Steam sterilization/moist heat sterilization/autoclave 
● Used for aqueous & surgical materials. 
● most widely used and reliable method of sterilization 
● The  efficiency  to  inactivate  microorganisms  is  dependent  upon  the  degree  of 
heat, the exposure time and the presence of water. 
● Sterilization  is  carried  out  using  saturated  steam  under  pressure.  Saturated 
steam cause destruction of enzyme and other cellular component and protein. 
● Autoclaving also kills virus 
● not  suitable  for  rubber,  plastics,  and  equipment  that  would  be  damaged  by 
high temperatures 
 
Holding temp.   Holding time (minutes) 

115-118 ℃  30 minutes 

121- 124 ℃  15 minutes 

126- 129 ℃  10 minutes 

134-138 ℃  3 minutes 
 
2. Dry heat sterilization 

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● Used  for  sterilizing  glass-wares,  metal  surgical  instruments  &  sterilizing 
non-aqueous thermo-stable liquids and thermostable powders. 
●  
● Dry heat destroy bacterial endotoxin 
● Pyrogen can destroy at 600 degree centigrade for 60 second 
 
Holding temp.   Holding time  

160 ℃  2 hrs  

170 ℃  1 hrs 

180 ℃  30 minutes 
 
3. Sterilization by filtration: 
● Used for thermolabile/heat sensitive Drugs 
● The  size  of  filter  medium  pores  to  retain  micro-organisms  must  be  quite  small.  The 
0.20-  or  0.22  pore  size  filter  media  are  considered  to  be capable of producing sterile 
filtrates. 
● Both clarification and sterilization of liquid preparation 
● Filtration  process  does  not  destroy  but  removes  the  microorganisms  by  adsorption 
and trapping within the matrix of the filter material. 
● The  bubble  point  test  is  a  popular  single-point  physical  integrity  test  for  disc  filter 
membrane.A  filter  medium  is  wetted  with  a  liquid,  and  test  gas  pressure  is  slowly 
raised  until  a steady stream of bubbles appears from a tube or hose attached to the 
downstream  side  of  the  filter  and  immersed  in  water  (Fig.  9).  The  pressure  at  which 
the bubbles first appear is recorded as the bubble point and is related to the largest 
pores in the filter medium. 
 
 

 
 
4. Ethylene oxide ​/(CH​2​)​2​O): 
● Ethylene oxide is a colorless, odorless, and flammable gas. 
● Cause  alkylations  of  sulphydryl,  amino,  hydroxyl  and  carboxyl  groups  on  proteins 
and amino groups of nucleic acids/DNA. 
● produce acute toxicity including irritation of the skin, conjunctiva and nasal mucosa. 

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Efficacy  depends  upon  ​humidity,  time  of  exposure,  temperature  of  exposure, 

concentration of gas, nature of microorganism​. 
● Limitation:  Limited  ability  of  Ethylene  oxide  gas  to  diffuse  to  the innermost product 
(penetrability problem) 
5. Gamma radiation: 
● Cause ionization of DNA 
● Dose: 2.5 Mrads 
● Source of radiation: Cobalt 60 and cesium 137 
● Many  types  of  radiation  are  used  for  sterilization like electromagnetic radiation (e.g. 
gamma  rays  and  UV-260  nm  light),  particulate  radiation  (e.g.  accelerated 
electrons).The  major  target  for  these  radiation  is  microbial  DNA.  Gamma  rays  and 
electrons  cause  ionization  and  free  radical  production  while  UV  light  causes 
excitation. 
● Radiation  sterilization  is  generally  applied  to  articles  in  the  dry  state;  including 
surgical  instruments,  sutures,  prostheses,  unit  dose  ointments,  plastic  syringes  and 
dry pharmaceutical products. 
 
6. OTHERS 
● Hydrogen  Peroxide  Sterilization  -oxidizing  key  cellular  components,  which 
inactivates the microorganism​s. 
● HEPA (High efficiency particulate air):-  
○ filters can remove up to 99.97% of particles >0.3 micrometer in diameter. 
○ Application of filtration for sterilization of gases. 

Bio-logical indicators 
● Biological  indicators  (BIs)  are  live  spore  forms  of  micro-organisms  known  to  be  the 
most  resistant  living  organisms  to  the  lethal  effects  of  the  particular  sterilization 
process. 
● Provide  the  effectiveness  of  a  given  sterilization  process/validation  of  sterilization 
process. 
● Biological indicators are viable culture of known species of microorganism. 
 
S. Methods of Material to be Biological indicator
No. sterilization sterilized

1  Moist  Heat  Saturated steam under pressure  Aq.  &  Surgical  Bacillus 
(steam)  items  strearothermophilus 

2  Dry heat  Oven or tunnel  Non-aq.  &  B. subtilis 


powders 

3  Gas  Ethylene oxide    B. atrophaeus 


 

Vapour phase hydrogen peroxide    Bacillus 


stearothermophi 
lus 
 

4  Radiation  Ionization of DNA    B. pumilus 


(cobalt-60, 
cesium-137) 

5  Filtration   Adsorption   Thermolabile   Pseudomonas 

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aeruginosa 
 

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