a review into the topic of Moist Heat Sterilization for MBBS level Microbiology presented for classroom discussion in 2008

1. Moist HeatMoist Heat
SterilizationSterilization
R.MalarvizhiR.Malarvizhi

2. TypesTypes
• Basically Moist Heat Sterilization is of 2 types, based on
the temperature.
1)Temp below 100˚C
Pasteurization ( Flash and Holder Method )
2)Temp at 100˚C
a) Boiling
b) Steam at atmospheric Pressure (Steamer)
c) Steam under Pressure (Autoclave

3. PasteurizationPasteurization
• Pasteurization (or pasteurization) is the process of
heating liquids for the purpose of destroying viruses and
harmful organisms such as bacteria, protozoa, molds, and
yeasts. The process was named after its inventor, French
scientist Louis Pasteur. The first pasteurization test was
completed by Pasteur and Claude Bernard on April 20,
1862.
• Unlike sterilization, pasteurization is not intended to kill all
micro-organisms (pathogenic) in the food. Instead,
pasteurization aims to achieve a "logarithmic reduction" in
the number of viable organisms, reducing their number so
they are unlikely to cause disease (assuming the
pasteurized product is refrigerated and consumed before its
expiration date). Commercial-scale sterilization of food is
not common, because it adversely affects the taste and
quality of the product.

4. Pasteurization ProcessPasteurization Process
• Generally Pasteurization involves Milk
• In Holder method is heated for30 min at 60˚C.
• In Flash method it is heated at 72˚C for 15-20 sec.
• Milk is not heated above its boiling point as it may form
curdles or aggregates resulting in spoiling of milk.
• By Pasteurization all non sporing bacteria like Mycobacteria,
Brucellae and Salmonellae are destroyed.
• Coxiella burnetii being heat resistant survives Holder
method.

5. That can be PasteurizedThat can be Pasteurized
• Vaccines of non sporing bacteria in special
baths at 60˚C for 1 hr.
• Serum and body fluids with congealable
proteins at 56˚C for 1hr in water bath for
several successive days.

6. • All mesophilic nonsporing bacteria
are killed on exposure to moist heat
at 60˚C for ½ hr, except a few which
need a different set of time and
temperature of sterilization.
• A temperature of 80˚C for 5-10 min
destroys all vegetative forms of
bacteria, yeasts and moulds.

7. • Staphylococcus aureus and
Stretococcus faecalis - 60˚C for 60 min.
• Spores of Clostridium botulinum – 120˚C
for 4 min or 100˚C for 330min.
• Some viruses like poliovirus & hepatitis B
at 60˚C for 30min & 10 hrs respectively.
(Unlike most viruses rap[idly destroyed at
60C)

8. • Spores of Clostridium botulinum are
destroyed at 120°C for 4min or 100°C for
330 min.
• Viruses like poliovirus & hepatitis B may
survive even for 30 min & 10 hrs
respectively at 60°C. Unlike the usual
destruction of viruses rapidly at 60°C.

9. PasteurizationPasteurization

10. Holder Method ofHolder Method of
PasteurizationPasteurization

11. PasteurizationPasteurization
Flash Method of
Pasteurization

12. InspissatorInspissator
• Inspissator is a convenient and effective system designed
to produce large batches of uniform culture medium four to
six times per day. Vessels containing culture medium are
incubated on a shallow tray which is in contact with water
held at a constant temperature of 85ºC within a tank, so
ensuring that the temperature of the vessels is constant.
• Inspissation takes 50 minutes at 85ºC. Standard
temperature: 85ºC; operating temperature range ambient
+ 5 to 90ºC
• Capacity for up to 156 test tubes (Ø16mm diameter x
150mm long) or 162 universal containers

13. Inspissator UseInspissator Use
• Sterilizing of media.
E.g.: Lowenstein-Jenson & Loeffler’s
rendered sterile at 80-85˚C for 1/2 hr on
3 successive days inspector.
• Tuberculum bacteria culture.

14. InspissatorInspissator

15. BoilingBoiling
• Boiling is the process of heating the
to be sterilized material in a liquid
(generally distilled water) at its
boiling point to kill bacteria and
other micro-organisms in it including
spores (which needs prolonged
boiling).

16. • Vegetative bacteria are killed
immediately at 90-100˚C
• In boiling the to-be-sterilised
material should be immersed in
water and boiled for 10-30 min.
• Hard water should not be used.
• During the process of boiling the lid
of the container should not be
opened

17. Steam at 100CSteam at 100C
• An inexpensive method of utilizing
free steam (i.e., at atmospheric
pressure) to sterilize culture media.
• Container and the medium are
simultaneously sterilized.

18. SteamerSteamer
• Tinned copper cabinet with lagged walls.
• Conical lid enabling drainage of condensed steam.
• Perforated tray fitted above the water level
ensuring steam to surround the material to be
sterilized.
• Usually takes 90 min.

21. TyndallizationTyndallization
• Synonym-Intermittent Sterilization
• Used for Media with sugars or gelatin which
require 100˚C for 20 min on 3 successive days.
• Principle:
1ST
exposure kills all vegetative bacteria and spores.
Since they are in favouring media will germinate
to be killed in subsequent occasions.
• May fail to destroy thermophiles and certain
anaerobic spores.

22. Steam Under PressureSteam Under Pressure
• Steam Under Pressure (AUTOCLAVE)
• Principle
Water boils when vapour pressure equals that of
surrounding. Thus increase in pressure inside closed vessel
causes increase in boiling point of the water. Saturated
steam has more penetrating power. When steam comes on
contact with a surface it give off its latent heat, thus
sterilizing the surface. The large reduction in volume sucks
in more steam to the area & the process continues till the
temperature of that surface is raised to that of the steam.
Condensed water ensures moist conditions for killing the
microbes present.

23. Instrument SterilizersInstrument Sterilizers

24. Steam Under PressureSteam Under Pressure
• Done on Temperature b/w 108˚C-147˚C
• Using appropriate time and temperature a variety of
materials ca n be sterilized.
• Aqueous solutions can be sterilized at temperatures b/w
108˚C – 126˚C.
• Types of Steam sterilizers used are
1)Lab autoclaves
2)Hospital dressing sterilizers
3)Bowl & Instrument sterilizers
4)Rapid cooling sterilizers
Even the domestic cooker can be used as a sterilizer.

29. AutoclaveAutoclave
• Types- Vertical & Horizontal
• Made of gunmetal or stainless steel in supportive sheet iron
case.
• Lid or screw fastened by screw clamps &made airtight by
suitable washer.
• Has on its upper lid a discharge tap for air and steam, a
pressure gauge & a safety valve that can be set to blow off
at any desired pressure.
• Heating is by Gas or Electricity.

30. MechanicsMechanics
• Sufficient water in cylinder
• Material to be sterilized placed on tray& autoclave heated
• Screw tight the lid with an opened discharge tap.
• The safety valve adjusted to required pressure.
• Steam air mixture allowed to freely escape till all air has
been displaced.
• Can be tested by leading the escaping steam into a pail of
water through a rubber tubing.
• When no more air bubbles come out in pail the discharge
tap is closed.
• Steam pressure rises inside &when it reaches the desired
set level, safety valve opens &the excess steam escapes.

31. Mechs contd..Mechs contd..
• The holding period is calculated
• When holding period is over, heater is turned off &
autoclave allowed to cool till pressure inside equals to
atmospheric pressure.
• Discharge tap is opened slowly & air is let into the
autoclave.
• If tap is opened when pressure inside is high, liquid media
will tend to boil & spill from container &sometimes an
explosion can occur.
• If opened when the pressure inside has fallen below
atmospheric pressure, an excessive amount of water would
have evaporated & lost from media.

32. Mechs contd…Mechs contd…
• Defects in this type of autoclave are:
1) Method of discharge is inefficient, & it is difficult to decide
when the discharge is complete. If air is not completely
removed , desired temperature will not be attained .
2) There is no facility for drying load after sterilization &
before taking it out.
Domestic pressure cooker serves as miniature
autoclave & may be used for sterilizing small articles in
clinics & similar establishments.
Wide variety of autoclave have been manufactured
incorporating various devices for overcoming these
effects & other difficulties in working.

41. Sterilizing controlSterilizing control
• A thermophilic organism, Bacillus stearothermophilus is
used in the form of spores to determine the efficacy of
moist heat sterilization.
• Its Optimum growth temperature is 55-60°C & its spores
are destroyed at an exposure of 12 min at 121°C.
• Paper strips impregnated with 10×10 spores are dried at
room temperature & placed in paper envelopes . These
envelopes are inserted in different parts of load. After
sterilization, the strips are inoculated into a suitable
recovering medium & incubated for sterility test at 55°C for
5 days.
• Chemical indicators, autoclave tapes & thermocouples are
also used.

42. Impregnated Paper StripsImpregnated Paper Strips

43. Chemical indicatorsChemical indicators

44. Autoclave tapesAutoclave tapes

45. Thermo pensThermo pens

46. Thank you!