Physical methods of microbial control

1. Methods of Microbial Control
in Healthcare
Principles to Practices
Dr. Rajarshi Gupta

2. Part 1 - Topics to be covered
1. Role / Need of/ for Microbial Control Methods in healthcare
2. Methods of microbial control-an overview
3. Choice of methods
4. Factors affecting methods of microbial control
5. Methods of Sterilization - an overview
6. Physical methods of sterilization

3. Need for microbial control in healthcare
settings
SOMETHING WAS WRONG
And then came
Semmelweiss:
Though ostracized by
medical fraternity at that
time, his work on the
role of handwashing in
reducing mortality due
to 'Childbed fever' was
monumental in infection
control.

4. Endoscopy associated infection outbreaks
1974-2004
Seoane-Vazquez E, et al. Endoscopy 2007;39:742-778

5. Pathogen Estimated surface survival
Acinetobacter spp. 3 days - 5 months
C.difficile spores 5months
E.coli 1.5 hours - 16 months
Enterococci(including VRE) 5 days - 4 months
Klebsiella spp. 2 hours - 30 months
M. tuberculosis 1 day - 4 months
P.aeruginosa 6 hours - 16 months
Staphylococci(including MRSA) 7 days - 7 months
HSV Minutes
Influenza Hours
Hepatitis A Days
HIV Days
Hepatitis B Months
With so many bugs
remaining viable for such a
long time,
lack of microbial control will
invariably lead to transfer of
these to susceptible hosts.

6. Microbial Control
Source
Control
Transmission
prevention
Sterilisation
Disinfection
Antisepsis
Anti-microbial therapy
Inanimate objects primarily
Mainly in animate

7. Definitions:
• Sterilisation: Elimination of all microbial forms ( vegetative cells + spores)
• Disinfection: Elimination of atleast pathogenic of microbial forms applied to inanimate
objects
• Antisepsis : Same principle as disinfection but applied to animate surfaces
• Cleaning: Elimination of visible dirt / other organic matter from animate or inanimate
surfaces mainly using surface tension lowering agents ( necessary for dislodging biofilms
with entangled bacteria where disinfectant or antiseptic can’t penetrate)
• Decontamination: Rendering an area / surface / instrument safe for handling
• Asepsis: It is a practice for prevention of contact with microorganisms

8. Classification of methods of microbial control
Physical Methods Chemical Methods
Heat
Filtration
Radiation
Age old methods
Osmotic pressure
(eg. high salt in pickles)
Refrigeration
( These only suppress
bacterial multiplication
but dont kill them) Better and quality assured methods for killing microbes

9. RATIONALE TO DISINFECTION AND
STERILIZATION
•Spaulding scheme: More than 30 years ago, Earle H. Spaulding
•Clear and logical classification scheme
•Retained, refined, and successfully used by infection control
professionals and others when planning methods for disinfection or
sterilization

10. RATIONALE TO DISINFECTION AND STERILIZATION
(Spaulding scheme from more than 30 years ago, by Earle H. Spaulding)
•Critical items - Enter sterile tissue or vascular system
(STERILISE ALWAYS)
•Semi-critical items - Contact mucous membranes or non-intact skin
( HIGH LEVEL DISINFECTION)
•Non-critical items – Contact with intact skin only
(LOW LEVEL DISINFECTION)

11. Changes in concepts since 1981 and
pitfalls of the Spaulding scheme
•Oversimplification of categories
(Unresolved in Spaulding scheme – What to do if semi-critical items are
used along with critical items?)
Eg. Bronchoscope + Punch biopsy forceps

12. •Increasing complexity in medical devices
1. Complex ventilator circuits
2. Advanced dialysis machines
3. Fibre-optic bronchoscopes
Complicating factors here are mainly
•DIFFERENTIAL SENSITIVITY OF MACHINE COMPONENTS TO HEAT
•NETWORK OF SMALL LUMENS REDUCE STERILISER PENETRATION

13. Sterilisation and disinfection for Prion diseases
Prions are resistant to common sterilisation/disinfection protocols
They are difficult to remove by cleaning as they stick to surfaces
Protocol : 1N sodium hydroxide (NaOH) and heat in an autoclave at 121°C for 30
minutes

14. • Understanding the importance of Biofilms in this context
•Bronchoscopes and endoscopy devices often get coated with biofilms
containing organisms embedded in matrix.
Sterilization or disinfection of such scopes is effective only if they are
‘Cleaned’ first because the sterilizer or disinfectant will not penetrate
the biofilm
In general prior ' CLEANING' is essential for any
sterilisation/disinfection process to be effective

15. “I thot I saw a tweety
bird”
“I thot I taw
a pussy cat”
“ But the biofilm
will protect us!”

16. •Intrinsic resistance, emergence of multi-drug resistant (MDR) bugs and their resistance to
disinfectants
Intrinsic resistance – Pseudomonas spp. and Burkholderia spp. To quarternary ammonium
compounds.
Conflicting results from studies and emergent hypotheses propose cross resistance between
disinfectants and antibiotics. But this isn't a problem if the disinfectant is used at recommended
concentrations
1.Gentamicin resistance might encode reduced susceptibility to propamidine, quaternary
ammonium compounds, and ethidium bromide.
2.MRSA strains might be less susceptible than methicillin-sensitive S. aureus
(MSSA) strains to chlorhexidine, propamidine,
and the quaternary ammonium compound cetrimide.

17. General factors affecting sterilization or
disinfection
1. Contact time: Very important, always allow enough time for proper
penetration. Longer the contact time more effective the process.
2. Temperature: Generally higher the temperature lesser the contact time.
3. Pressure: Important in autoclaves as changes can alter phase boundary of
steam and thus prevent enough latent heat release.
4. pH: Increase in pH improves the antimicrobial activity of some disinfectants
(e.g., glutaraldehyde, quaternary ammonium compounds) but decreases the
antimicrobial activity of others (e.g., phenols, hypochlorites, and iodine.
5. Relative humidity is the single most important factor influencing the activity of
gaseous disinfectants/sterilants, such as EtO, moisture also increases
effectiveness of heat

18. General factors affecting sterilization or
disinfection contd…
6. Water hardness (i.e., high concentration of divalent cations) reduces the rate of kill of certain
disinfectants because divalent cations
7. Organic matter: Neutralise disinfectant directly or form a physical barrier around microbes.
Carbohydrates reduce sensitivity to heat
8. Concentration and potency: Always use the disinfectant at the recommended ‘cidal’
concentration.
9. Biofilms
10. Intrinsic resistance

19. Cleaning( Rationale behind…)
Removal of visible dirt from surfaces
Inorganic and organic materials that remain on the surfaces of
instruments interfere with the effectiveness of sterilization and
disinfection
If soiled materials dry or bake onto the instruments, the removal
process becomes more difficult and the disinfection or sterilization
process less effective or ineffective.

20. Cleaning(methods)
Manual cleaning Friction
Fluidics
Scrubbing clean with detergent( ie.
Surface tension lowering agent)
Fluids under pressure after scrubbing
Fluids under pressure in narrow lumen tubes
where scrubbing brushes cannot reach

21. Ultrasonic Cleaners
• Disrupt by cavitations and implosion(waves of acoustic
energy are propagated in aqueous solutions) the bonds
that hold particulate matter to surfaces using ultrasonic
energy (20 – 400kHz) the bonds that hold particulate matter to
surfaces
• Ultrasound alone does not significantly inactivate bacteria, sonication can act synergistically to increase the
cidal efficacy of a disinfectant.
• Special concern – Ultrasound energy can break down cells and release endotoxins onto surgical
instruments, leading to inflammatory responses in patients.
• Usually used for delicate objects

22. Ultrasonic Cleaners

23. Sterilisation( Physical
methods)
1. Heat
2. Filtration
3. Radiation

24. A quick
glance at the
bacterial
growth curve
and the death
curve

25. Probability of killing bacteria depends on
1. The concentration of bacteria
2. The temperature
3. Time of sterilisation
Any sterilization or disinfection procedure causes 90% reduction in bacterial
population in a given time. Total eleimination of bacteria is a theoritical
concept and cannot be attained practically.

27. How long to sterilise?
Answer: The D-value
It is used to determine the time of
contact for any sterilisation process.
It is the time required to reduce the
organism number 10 times or by one log.

28. Thermal death time( concept)
Similar to D-value but it is the time taken to kill all organisms.
ie. it is a sum of successive D-values
Z-value : Measure of thermal resistance of spores.
It is the change in temp(℃) to produce
10 fold change in thermal death time of spores

29. Types of heat sterilisation
Dry heat - Kills by oxidising cellular components--- SLOW
Moist Heat - Kills by coagulating cellular proteins using combination
of heat and moisture ---- RAPID
IN GENERAL MOIST HEAT IS MORE EFFECTIVE THAN DRY HEAT.
BUT DRY HEAT IS MORE CONVENIENT

30. Dry heat sterilisation( namely the
hot air oven)
Mainly for glassware that can tolerate high temperatures
For oils, greases that are waterproof
Metallic objects and dry powders that can be damaged by moisture.
Recommended temp. and duration
180 degree celsius - 30 mins
170 degree celsius - 1 hr
160 degree celsius - 2 hrs

31. AUTOCLAVING - moist heat
sterilisation
Principle:
Saturated steam ( no admixture of air)
at more than 100 degrees celsius( usually 121 degees celsius)
at elevated pressure( usually 15lb/inch2)

32. Importance of the principle:
Recommended temperature at recommended pressure ensures steam
to stay at PHASE BOUNDARY
ie. Steam condenses on contact with items to release latent heat
Above recommended temperature(superheated steam) or low
pressure causes steam to fall out of phase boundary and does not
condense immediately on contact

33. • Steam at phase boundary condenses into water and contracts to
create a low pressure to cause ingress of more steam
-- Steam out of phase boundary does not condense to draw fresh
steam and hence temperature in autoclave falls
-- Admixture with air causes causes less ingress of fresh steam
• Air in the autoclave occupies small spaces in porous loads and
reduces steam penetration

34. Some relevant definitions:
Sterilisation holding time: Time for which the load is exposed to
recommended tempertaure and recommended pressure
Heat penetration time: Time required by the load to reach the
recommended temperature and pressure and for the steaam to
penetrate the porous spaces.

35. Types of autoclaves
Basically 2 types of autoclaves
Gravity type:
Steam being lighter
displaces air through an
exhaust below and slowly
fills up the chamber
•Complete air removal is
impossible
•Less steam penetration of
porous loads
•Does not allow active drying
of load

36. Vacuum assisted
autoclaves:
•Air pumped out before steam is
charged
•Better penetration of steam
into loads
•Allows drying after autoclave
cycle by inducing partial
vacuum

37. Uses of autoclave types:
GRAVITY TYPES - For non porous loads that do not have difficult to access areas
(eg. wide mouthed containers, objects with flat non porous surfaces)
VACUUM ASSISTED TYPES - For porous loads where steam penetration into
the smallest spaces is desired
(eg. soiled cotton,linen, narrow tubes, lumened instruments)
LIQUID CULTURE MEDIA CAN'T BE STERILIZED IN A VACUUM ASSISTED AUTOCLAVE
AS THE INITIAL VACUUM THAT IS GENERATED MIGHT VAPORISE THE MEDIA

38. Quality assurance of
autoclaves
BOWIE-DICK HEAT SENSITIVE STRIPS
- Changes colour( blue to black) at 121℃
- Checks air admixture. If air is present,
steam does not penetrate the centre of the
strip and the centre stays blue
- Does not monitor time of contact. Just
monitors temperature
- To be placed in the part of the autoclave
least accessible to steam.

39. Geobacillus stearothermophilus
ATCC 7953 spore strips
- These spores are most resistant to
autoclaving
- Test strip is exposed with the load for
the same cycle conditions
- Autoclaved spore strips are incubated
in broth at 55 ℃ to check for complete
destruction of spores( no growth)
- Keep positive control with an
unsterilized spore strip from same lot.

40. Physical methods like pressure recording devices and
temperature recording thermocouples can be placed
in the least accessible part of the load to check
whether recommended sterilization conditions are
achieved there or not

41. Low-temperature steam
formaldehyde(LTSF)
Steam at low temperature (73 degrees celsius) and sub atmospheric
pressure( 263 mm of Hg) is ineffective for bacterial spores.
But mixed with Formaldehyde it becomes strongly sporicidal due to
synergy with steam.
Hence, allows an autoclave to perform at lower
temeprature for heat sensitive items.

42. Low temperature plasma sterilisation
• Occurs at low temperatures (37 -44℃)
• Utilises gas ionised by strong electric field( PLASMA)
• Sporicidal
• Commonly ionised hydrogen peroxide gas is used to cause free radical
damage

43. Moist heat Disinfection
Boiling at 100 degrees celsius : Good for vegetative bacteria and HBV
but unsatisfactory for spores. Not
recommended for porous loads where
penetration might be an issue
Free steaming at 100 degrees celsius: Saturated steam at normal
atmospheric pressure. Mainly for heat sensitive media such as XLD,
TCBS, DCA.

44. Pasteurization( a form of heat
disinfection)
Process of decontamination of milk
Kills only vegetative forms and not spores
Low temperature holding method(LTH) - 145°F or 62.8℃ for 30 mins
High temperature short time method(HTST) - 161°F or 71.7℃ for 15 secs
THE PRODUCT MUST THEN BE KEPT REFRIGERATED TO PREVENT SPORES FROM
GERMINATING

45. Pasteurization( concepts behind.....)
• Every particle of milk must be heated to the
recommended temperature for the recommended period.
• Why a temperature of 145°F for 30 minutes for LTH ?
Milk transmitted pathogen C.burnetti can
survive upto 143°F for 30 mins.

46. Radiation Sterilisation
Ultraviolet light - Most effective at around 265nm wavelength( ie. UV-C)
Forms pyrimidine dimers in bacterial DNA
Very poor penetration, hence used only for surface
sterilisation ( decontamination of surfaces in food
industry, decontamination of air in biosafety cabinets)
Gamma rays - Ionising radiation damages vital cellular molecules
Immense penetration power
Can be used to sterilise objects of considerable thickness
( packaged food, medical devices, thick plastic syringes)

47. UV-A( 315-400nm) and UV-B(280-315nm) are not germicidal

49. Filtration
• Used for sterilisation gases(eg. air) or liquids( eg. serum)
• Filtration depends upon pore size and
charge carried on both filter material and organism surface
Namely
The asbestos pad Seitz filter
The diatomaceous earth Berkefeld filter
The porcelain Chamberland-Pasteur filter
The sintered glass filter

50. Nitrocellulose membrane filters with varying pore sizes
0.22 microns -- for Pseudomonas diminuta
0.45 microns -- for coliforms
0.80 microns -- for general airborne pathogens
High efficiency particulate air (HEPA) filters
Air filters for very small particles mainly in a laminar airflow
Consists of a dense meshwork of filter fibres

51. HEPA FILTER PRINCIPLE
Inertial Impaction:
Inertia works on large, heavy
particles suspended in the flow
stream.
These particles are heavier than
the fluid surrounding them. As
the fluid changes direction to
enter the fibre space, the particle
continues in a
straight line and collides with the
media fibres where it is trapped
and held

52. HEPA FILTER PRINCIPLE
Interception:
Direct interception works on
particles
in the mid-range size that are not
quite large enough to have inertia
and not
small enough to diffuse within the
flow stream. These mid-sized
particles follow the flow stream as it
bends through the fibre spaces.
Particles are intercepted or captured
when they touch a fibre.

53. HEPA FILTER PRINCIPLE
Diffusion:
Diffusion works on the smallest
particles. Small particles are not
held in place by the viscous fluid
and exhibit random(Brownian
motion) within the flow stream. As
the particles traverse the flow
stream, they collide with the fibre
and are collected.

54. Thus, particle
size of 0.3
microns are the
least filtered in
an HEPA filter
So, the effciciency of any HEPA filter is assessed by its
capability to filter particles of around 0.3 microns

55. Transmission prevention
"However stringent source control might be, some notorious bug
at some point of time will surely find some route of entry
into some individual to cause some disease"

56. SUMMARY
• Microbial control involves both killing microbes and preventing thir
transmission.
• Sterilization and disinfection differs by the fact that sterilisation kills
spores while disinfection does not.
• Cleaning is essential before sterilization.
• Physical methods of sterilization include primarily heat, filtration and
radiation.
• Choice of sterilization method depends on the object to be sterilized
(whether solid, liquid or gas, heat susceptibility, susceptibility to
moisture )
• Transmission prevention is equally important in microbial control as
sterilization and disinfection.

57. THANK YOU

58. References:
1. Guideline for Disinfection and Sterilization in Healthcare Facilities, 2008, Centres
for Disease Control and Prevention
2. Microbiology, 5th edition, Pelczar, Michael J.
3. Mackie and McCartney, Practical medical microbiology, 14th edition, Collee, J.G
4. Guidance for Filtration and Air-Cleaning System to Protect Building
Environments from Airborne Chemical, Biological, or Radiological Attacks,
Department of Health and Human Services Centers for Disease Control and
Prevention ,National Institute for Occupational Safety and Health (NIOSH)