Unit 2 : sterilization techniques

1. Faculty of Pharmacy
Babu Banarasi Das Northern India Institute of Technology, Lucknow
By :- Deepa Gupta
Assistant Professor
BBDNIIT, Lucknow
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2. Turbidimetric method
 Advantage : shorter incubation period for growth of test organisms. (usually 3-4
hour)
 Used : disinfectant and antibiotic assessment.
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3.  Media : Alternative Thioglycollate medium
a. L-cysteine – 0.5 g
b. Sodium chloride – 2.5g
c. Dextrose monohydrate/ anhydrous – 5.5g/ 5g
d. Yeast extract (water soluble) – 5g
e. Pancreatic digest of casein – 15g
f. Sodium Thioglycollate – 0.5g
g. Thioglycollic acid – 0.3 ml
h. Distilled water – 1000ml
i. pH of medium after sterilization – 7.1± 0.2
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4. Turbidimetric method /tube assay method
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5.  Place all tubes randomly in an incubator or water bath (37° C for 3-4 hour)
 After incubation, add 0.5ml of diluted formaldehyde solution on each tube.
 Measure the growth of test organism by determining the turbidity on tubes.
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6. In-use test
capacity use-dilution test (Kelsey-Sykes test)
 Firstly, it was capacity test
 Aim : to measure the capacity of the disinfectant to deal with successive bacterial
contamination.
 Here a bacterial inoculum was added to the disinfectant in three successive lots at
0, 1, and 5 minutes.
 This is the principle of a capacity test where the capacity or lack of capacity of the
disinfectant to destroy successive addition of a bacterial culture is tested.
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7.  It is a triple challenged test.
 Test organisms :
 Staphylococcus aureus
 Pseudomonas aeruginosa
 Proteus vulgaris
 Escherichia coli
 Disinfectant in dilution : water
 Note : any no. of dilution can be made, but it is important to include-
a) Dilution recommended by the manufacturer.
b) Dilution 20-25% weaker than a.
c) Dilution 20-25% stronger than a.
7 Kelsey-Sykes test

8.  The test is carried out under clean and dirty conditions.
 Clean conditions are simulated by using broth as suspending fluid.
 Dirty conditions are simulated by using yeast suspension (like CM test) or
inactivated horse serum as suspending fluid.
 In both the cases- final concentration of bacterial cells should be about 109/ml.
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10. Interpretation of results
 A disinfectant is satisfactory for use at initial concentration if –
a) No growth occurs in 2 or more of 5 tubes of 18 minute samples.( i.e. subcultures
taken after second incremental addition of bacteria)
b) There are not more than 2 colonies from the 5 drops on the agar plate.
 Tubes of disinfectant reaction should be kept for 5 days, without further inoculation
and further samples taken to confirm that undetected surviving cells have not
developed.
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11.  NOTE :
 Main source of error in this test is due to organism variation, but test will show the
stability of disinfectant dilutions and their long term effectiveness.
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12. ‘In-use’test by Kelsey & Maurer (1966)
 Samples are taken from situations in which the disinfectant is used, e.g., liquid from
mops, storage, rinse liquid from urine bottles, etc.
 1 ml of liquid is diluted with 9ml of ¼( one-quarter) strength Ringer’s solutions.
(plus inactivator if necessary)
 10 drops are placed on surface of agar plate with 50 dropper pipette.
 If not more than 5 out of 10 drops show growth after 48 hours at 30-32°C .
 The disinfectant is considered adequate.
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13. In-vivo test
 SKIN TEST
 This test must be applied if the antibacterial is intended to be used on living
tissue.
 If it is used on skin, disinfectant action can be evaluated by placing test organism
on skin e.g., on the back of the hand and test sample placed on the same area.
 After a given time interval , area swabbed with sterile cotton wool.
 And swab incubated in a suitable medium or washed in a suitable fluid.
 And viable counts are subsequently made.
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14.  HYGIENIC HAND DISINFECTION
 This term used to denote the killing and removal of transient microorganisms on
the skin. i.e. those germs that literally ‘come and go’
 It can be achieved on two ways:
1. Use of a hygienic hand rub
2. Use of a hygienic hand wash
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15. Use of a hygienic hand rub
 Suitable disinfectant or disinfectant-detergent is rubbed into dry hand for not more
than 30 seconds.
 A suitable test method is to compare a product with a standard (70% ethanol or
60% isopropanol).
 The product must not be less effective than the standard.
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16. Use of a hygienic hand wash
 Suitable disinfectant or disinfectant-detergent is rubbed into wet or dry hands for
not more than 30 seconds.
 And then washing the hands in water.
 A suitable test method is to compare a product with a standard (soap and water).
 The product must be significantly more effective than the control.
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17.  SURGICAL HAND DISINFECTION
 This term refers to pre-operative disinfection of surgeons’ hands, with the aim of
preventing surgical wound infection.
 Important criteria associated with surgical hand disinfection are-
i. Reduction of the resident skin flora to low levels.
ii. Prolonged effect (lasting several hours)
iii. Minimal irritation to the skin.
 NOTE : Principle of tests-
 Evaluating the efficacy of disinfectants on hand before and after surgical hand
disinfection.
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18. Tests on Aerial disinfectants
 Closed room – cubic dimensions & 1000 cu ft. capacity used.
 Fan – incorporated for uniform mixing of bacteria & bactericide.
 Test organism – Staph. albus (non-clumping strain).
 Collison inhaler – dispersion of organisms into air.
 Slit sampler – samples of air taken at suitable intervals.
 Bactericide achieving an 85% (or more) kill in 4 to 6 min, at 20° C is considered
satisfactory.
 Cyclopentanol-1-carboxylic acid has been suggested as are reference standard
for air disinfectant.
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19. Assessment of Bacteriostatic Activity
 Original method : portion of the preparation were placed on the surface of nutrient
agar inoculated with staphylococcus aureus and after incubation , the zone of
inhibition , if any measured.
 This test was modified later by – Incorporating 10% of horse serum in the agar (to
simulate condition in a wound)
 Cup-plate or ditch-plate techniques can equally well be applied to these types of
formulation.
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20. Assessment of Bactericidal Activity
 Modified phenol coefficient type tests can be used.
 Test organism must be well mixed with semi-solid preparation/formulations
samples taken at appropriate intervals and placed in a broth which is capable of
dispersing the base and nullifying antimicrobial activity of disinfectant.
 Sampling error using a loop are large.
 Skin tests should also be made.
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21. Virucidal activity
 Tissue culture or egg inoculation
 Plaque assays
 Acceptable animal model
 Duck hepatitis B virus
 Immune reaction
 Virus morphology
 Endogenous reverse transcriptase
 bacteriophage
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22. Sterility Indicator/ Validation
Parameters
 As the name indicate “Sterility indicator”, sterility indicators are those whose
indicate the efficiency of any sterilization process.
 Such can be achieved by use of physical, chemical, or biological indicators.
 Such indicators are frequently employed in combination.
 Types of sterility indicator :
1. Physical indicators
2. Chemical indicators
3. Biological indicators
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23. 1. Physical indicators : The display on the sterilizer or a recording device that show
the parameters like time, temperature and pressure associated with each
sterilization cycle.
2. Chemical indicators : It is a chemical which monitoring of a sterilization process
which is based on the ability of heat, steam, sterilants gases and ionizing radiation
to alter the chemical and or physical characteristics.
 Chemical indicators generally undergo melting or colour changes.
3. Biological indicators (BIs) : They are actually standardized bacterial spores used
in the form of suspension in water or culture media or of spores dried on paper or
plastic carries that are kept in the sterilizer.
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24. Physical indicators for monitoring
sterilization processes
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25. Bubble point pressure test
 It is used for determining the pore size of the filters
 Also to check the integrity of certain types of filter device (membrane and sintered
glass)
 Principle:
 Wetted filter paper disc, in its assembled unit, is subjected to an increasing air or
nitrogen gas pressure differential.
 And very gradually applying an increasing air pressure until the first bubble is seen
at the filter paper disc is related to the maximum pore size.
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26.  Bubble point pressure method is adopted by British Standard (BS) for measurement
of pore size.
 The pressure when the first bubble is seen indicates the maximum pore size.
 It is calculated from the equation :
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28. Bubble point pressure apparatus
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29. Bubble point pressure apparatus
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30. Diffusion rate test
 A modification of this test for membrane filters involves measuring the diffusion of
gas through a wetted filter at pressures below the bubble point pressure (diffusion
rate test)
 Note :
1. A faster diffusion rate than expected would again indicate a loss of filter integrity.
2. In addition, a filter is considered ineffective when an unusually rapid rate of
filtration occurs.
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31. DOP (Dioctylphthalate) test
 Efficiency testing of high-efficiency particulate air (HEPA) filters used for the supply
of sterile air to aseptic workplaces is generally achieved by-
 Generation upstream of dioctylphthalate (DOP) or sodium chloride particles of
known dimension, followed by detection in downstream filtered air.
 Retention efficiency is recorded as the percentage of particles removed under
defined test conditions.
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32. Chemical indicators
 It is a chemical which monitoring of a sterilization process which is based on the
ability of heat, steam, sterilants gases and ionizing radiation to alter the chemical
and or physical characteristics.
 This change should take place only when satisfactory conditions for sterilization
prevail, thus confirming that a sterilization cycle has been successfully completed.
 Chemical indicators generally undergo melting or colour changes.
 The changes recorded do not necessarily correspond to microbiological sterility
and consequently the devices should not never be employed as sole indicators in a
sterilization process.
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33. Chemical indicators for monitoring
sterilization process
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34. Browne’s tubes test
 Sealed tubed partly filled with a solution which changes colour at elevated
temperatures.
 Each tube consists of a sealed glass tube which contains a red fluid (an ester &
acid-base indicator) that changes to yellow, brown and finally green on heating.
 Note : rate of colour change is proportional to temperature.
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35. Browne’s tubes test
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37. Bowie-Dick Test
 The Bowie-Dick (Air-Removal) Test demonstrates proper air removal from the
chamber of a pre-vacuum autoclave or sterilizer chamber.
 Pockets of cool air act as a barrier that prevents steam from penetrating the load.
Therefore, the air must be removed by a vacuum.
 Bowie-Dick test packs consisted of 29-36 huckaback towels, each folded and
stacked to a height of 10 -11 inches, with autoclave tape in the middle of the pack.
 Today, laboratories use small disposable packs made
of thermochromatic (temperature sensitive) paper sandwiched between porous
substrates and reticulated foam.
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38.  To run the test, a pack is placed in an empty chamber on the lowest shelf above the
drain (the coldest point in the chamber) and a “Bowie-Dick Cycle” is initiated.
 The thermochromatic paper inside the pack will indicate if steam has penetrated
the porous load.
 A Bowie-Dick test pack that shows a uniform dark black color pattern indicates a
successful vacuum and full steam penetration, whereas no or partial color change
indicates an unsuccessful test cycle.
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39. Bowie-Dick Test
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40. Thermalog S indicating strips
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42. Chemical Indicator Strips
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44. Biological indicators
 The biological indicators are the standardized bacterial spore preparations which
are usually in the form of suspension in water or culture medium or of spores dried
on paper, aluminum or plastic carriers, they are placed in sterilizer.
 They are usually placed in dummy packs located at strategic sites in the sterilizer.
 Alternatively, for gaseous sterilization these may also placed within a tubular helix
(Line-Pickerill) device.
 After the sterilization process the aqueous suspension /spores are on carriers are
aseptically transferred to an appropriate nutrient medium, which is then incubated
and periodically seen for the growth.
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45.  D-value : (decimal reduction time)
 It is the rate of killing of microorganism.
 It is used for determining the thermal death time.
 Definition: it is time required for 90% reduction in the microbial population at
specified temperature or by a factor of 10.
Or
 Time required for to reduce the microbial population by one decimal point.
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46.  D-value is important in the validation of sterilization process for several reasons:
1. It is specific for each microorganisms in environment subjected to specific
sterilizing agent or condition.
2. The knowledge of D-value at different temperature in heat sterilization is necessary
for the calculation of z-value
 NOTE: Z-value – increase in temperature required to reduce D-value to 1/10 its
value.
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47. D-value formula
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48.  Spores of Bacillus stearothermophilus in sealed ampoules of culture medium are
used for steam sterilization monitoring,
 And these may be incubated directly at 55°C; this eliminates the need for an
aseptic transfer.
 Aseptic transfers are also avoided by the use of self-contained units where the
spore strip and nutrient medium are present in the same device ready for mixing
after use.
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49. Properties of biological indicators
 It must be non-pathogenic.
 It should possess above-average resistance to the particular sterilization process.
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50.  It should be realized that BIs may be less reliable monitors than physical methods
and are not recommended for routine use, except in case of gaseous sterilization.
 one of the long-standing criticisms of BIs is that – the incubation period required is
very long in order to confirm a satisfactory sterilization process.
 This problem has been overcome, with respect to steam sterilization at least, by use
of a detection system in which a spore enzyme, alpha-glycosidase(reflective of
spore viability), converts a non-fluorescent substrate into a fluorescent product in
as little as 1 hour.
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51.  Filtration sterilization require a different approach from biological monitoring, the
test effectively measure in the ability of a filter to produce a sterile filtrate from a
culture of suitable organism i.e., Serratia marcescens, a small gram negative rod
shape bacterium (minimum dimension 0.5 μm), has been recommended in the
Pharmaceutical Codex.
 Serratia marcescens and Brevundimonas diminuta formerly Pseudomonas
used as a biological indicator having a dimension 0.5 μm and 0.3 μm respectively
has been used for filters of 0.45 μm and 0.22 μm.
 The extent of the passage of this organism through membrane filter is enhanced by
increasing the filtration pressure.
 Thus successful sterile filtration depends markedly on the challenge condition.
 Such test are used as the part of filter manufacture characterization and quality
assurance process, and users initial validation procedure.
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52. Biological indicators for monitoring
sterilization processes
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53. Thank You. …….
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