The document provides information on sterilization techniques for glassware and laboratory equipment. It discusses various sterilization methods including heat sterilization using an autoclave, filtration sterilization using membrane filters, and non-heat methods like radiation and ultrasonic waves. It also covers sterilization validation tests using biological indicators and describes the rabbit pyrogen test and Limulus Amebocyte Lysate test for detecting pyrogens.

1. WASHING, DRYING, AND STERILIZATION OF GLASSWARE
BASIC CONCEPT IN LABORATORY TECHNIQUES
PGS – 504
DR. ANURAG
DEPT. OF SOIL SCIENCE
COA RAIPUR
SANJAY KUMAR
M.SC.(HORT.) PRE. YEAR 2ND SEM.
DEPT. OF FLORICULTURE

2. 1. PYREX
•
•
•
2. COREX
•
•
•

3. VYCOR
•
•
•
THE LOW ACTINIC
•
•
•
FLINT GLASS
•
•

4. 1.
2.
3.
4.
SOAP WATER:
VOLUMETRIC FLASKS
1.
2.
3.
BURETS
1.
2.
PIPETS
1.
2.
3.

5. 1.
2.
3.
4.
1.
2.
CHROMIC ACID
1.
2.
3.

6. HEMATOLOGIC
AL 1.
IMPORTANT
2.
3.
4.
5.
BLOOD PIPETTES
1.
2.
3.
CONCLUSION
1.
2.

7. 1. DRY HEAT
2. HOT AIR OVEN
•
•
3. INCINERATION
4. MOIST HEAT
•
5. BOILING
6. STEAM STERILIZATION OR TYNDALLIZATION

8. 7. THE AUTOCLAVE
8. MEMBRANE FILTERS
9.SEITZ FILTER
10.FLAMING
11.ULTRAVIOLET LIGHT
12.RADIATION
13.SUPERSONIC AND ULTRASONIC WAVES,

9. AUTOCLAVE
Autoclave:
An apparatus for
sterilization by steam under
pressure, usually at
temperatures of 250 degrees
to 270 degrees F
9

10. PRINCIPLES
• Two important factors should be controlled:
• 1- Application of pressure:
• Because it is not possible to raise the temperature of the steam above
• 100 °C under atmospheric conditions, pressure is employed to achieve
• higher temperature (it should be recognized that the temperature, not the
• pressure is destructive to the microorganism and that the application of
• pressure solely for the purpose of increasing the temperature of the
• system.
10

11. PRINCIPLES … CONT.
• 2- Application of time:
• Time is another important factor in the destruction of microorganisms by
• heat. Most modern autoclaves have gauges to indicate to the operator
• the internal conditions of temperature and pressure and timing device to
• permit the desired exposure time for the load. The usual conditions
• (pressure/temperature/time), are as follows:
•
11
Pressure Temperature Time
10 pounds 115.5 °C 30 minutes
15 pounds 121.5 °C 20 minutes
20 Pounds 126.5 °C 15 minutes

12. STANDARD TEMPERATURE AND
PRESSURE
250 °F (121 °C) and 15 p.s.i.
12

13. HOW LONG TO AUTOCLAVE
Dry goods @ 121 °C Time (min)
 Glassware, empty, inverted 15
 Instruments, wrapped 30
 Utensils, wrapped 30
13

14. Biohazardous waste bags, @ 121 °C, loosely
tied
Time (min)
• 2 or more bags 100 +
• 1 bag, full 90
• 1 bag, half full 60
Testing available for individual situations
14
How Long to Autoclave … cont.

15. Liquids, in bottles with vented caps
Size (ml) Time (minutes)
75 25
250 30
500 40
1000 45
1500 50
2000 55 15
How Long to Autoclave … cont.

16. AUTOCLAVE APPLICABLE:
• A. Applicable for pharmaceuticals preparations and materials that can
• withstand the required temperature and are penetrated but not adversely
• affected by, moisture.
• B. In sterilizing aqueous solutions, the moisture is already present, and all
• that is required is the elevation of the temperature of the solution for the
• prescribed period of time. Thus solutions packaged in sealed container as
• ampoules, are readily sterilized by this method.
• C. Also applicable to bulk solutions, glassware, surgical dressings, and
• instruments.
16

17. AUTOCLAVE NOT APPLICABLE:
A. The sterilization of oils, fats, or any oleaginous preparations.
B. Other preparations not penetrated by the moisture.
C. Sterilization of exposed powders that may be damaged by condensed
moisture.
17

18. TYPES OF STEAM STERILIZERS
• 1. Gravity displacement
• Definition: Gravity pushes air through the packages and
down through the drain. Sterilization begins when steam passes
the thermometer and reaches the desired temperature.
18

19. GRAVITY DISPLACEMENT USES
1. Metalware
2. Glassware
3. Thermoplastics
4. Linens
5. Rubber
19

20. 2. PRE-VACUUM (HIGH
TEMPERATURE)
STERILIZER
Definition: Air is completely evacuated from the chamber by a
vacuum. The steam-injector helps eliminate the air out of packages.
Steam then penetrates the packages on all surfaces.
20
Types of steam sterilizers … cont.

21. PRE-VACUUM STERILIZER USES:
Metalware
Rubber
Thermoplastics
21

22. 3. HIGH PRESSURE (FLASH)
STERILIZER
Definition: Another sterilizer that can be used in either gravity displacement or pre-vacuum. Gravity
displacement is the most common used. Steam in the jacket should be maintained at all times.
22
Types of steam sterilizers … cont.

23. HIGH PRESSURE FLASH USES
Urgent items: (dropped items for which no method of sterilization
exists) Items that are dropped, or forgotten
23

24. 4. WASHER-STERILIZER
Definition: Wash and sterilize instruments. The water floods the chamber with cold water to prevent
proteins from setting on the instrument. The water is then heated with steam to agitate and clean the
instruments.
24
Types of steam sterilizers … cont.

25. WASHER-STERILIZER USES:
to wash and terminally sterilize items which are non-heat sensitive,
immediately after operative procedures.
NO GLASS, SHARPS OR DELICATE INSTRUMENTS GO THROUGH THIS
STERILIZER. BREAKAGE COULD OCCUR!!!
25

26. BIOLOGICAL INDICATOR
CONTROLS/SPORE TESTS
Bacillus stearothermophilus: is used strictly as biological
indicator of effective heat sterilization (steam and dry heat
sterilizers) by including filter paper strips carrying
a saturated number of spores into the autoclave cycle.
The strips are then incubated to attempt to recover viable
organism. The usual autoclave cycle of 121 °C for
15 minutes is adequate to kill Bacillus stearothermophilus
with a high margin of safety.
26

27. BIOLOGICAL INDICATOR
CONTROL/SPORE TESTS…CONT.
When Used:
Upon installation of new system
After major repairs
Routine quality assurance.
With all implants
27

28. BIOLOGICAL INDICATOR
CONTROLS/SPORE TESTS
• Record results:
• Negative- no color change from original.
• Positive- color changed is usually amber in color.
• Take sterilizer out of service
• Report to supervisor
• Recall all items sterilized in sterilizer for last 24 hour
period
28

29. STERILIZATION BY FILTRATION

30. 30
- It depends upon the physical removal of
microorganisms by adsorption on the
filter medium or by sieving mechanisms.
- It is used for sterilization of heat-
sensitive solutions.
- Medicinal preparations sterilized by this
method are required to undergo severe
validation and monitoring since the
effectiveness of the filtered product can
be greatly influenced by the microbial
load in the solution being filtered.
- It removes, but does not destroy
M.O.
- Commercially available filters are
produced with a variety of pore-size
specifications (e.g. Millipore filters).
Filtration

31. 31
Most filters consist of:
1- diatomaceous earth
2- cellulose acetate or
3- nitrocellulose
0.45 µm filters removes most
bacteria
0.20 µm more inclusive,
viruses need 0.01 µm
Useful for sterilizing liquids
(enzymes, vaccines) that are
destroyed by heat
Filtration …cont.

32. 32
Factors affecting removal of M.O.:
1- Pore size of filter
2- Electrical charge of the filter
and that of the M.O.
3- pH of the solution
4- T, P, and Vacuum applied
Advantages:
1- Speed
2- Good for thermolabile
materials
3- Inexpensive
4- The complete removal of living
and dead M.O. as well as
other particulate matter from
the solution
Filtration …cont.

33. 33
Disadvantages:
1- The membrane is fragile and because
of that, it is essential to determine that
the assembly was properly made and the
membrane was not ruptured
2- Filtration of large volumes of liquids
would require more time (particularly if
the liquids were viscous)
3- Useful when heat cannot be used and
small volumes of liquids.
Filtration …cont.

34. OTHER TECHNIQUES

35. ULTRASONIC VIBRATIONS
35
– High frequency sound waves
– Know as Sonicator, untrasonic machines
– Killing by –shock waves that disintegrates cell wall
and membranes.

36. PYROGENS AND PYROGEN TESTING

37. PYROGEN
37
- A pyrogen is a material which when injected into
a patient will cause a rise in body temperature
(pyrexia).
- The lipopolysacchride that comprise a a major
part of the cell wall of gram-negative bacteria are
called endotoxins, and it is these that are the
most commonly encountered pyrogens.
- Bacterial cells may be pyrogenic even when they
are dead and when they are fragmented, and so
a solution or material that passes a test for
sterility will not necessarily pass a pyrogen test.

38. PYROGEN… CONT.
38
Two main procedures are used for the detection of pyrogens.
A. The traditional method:
It requires the administration of the sample to laboratory rabbits whose
body temperature is monitored for a period of time thereafter.

39. PYROGEN… CONT.
39
Steps:
1- Render the syringes, needles, and glasswares free form pyrogens by
heating at 250 °C for not less than 30 minutes.
2- Warm the product to be tested to 37 °C ± 2 °C
3- Inject into an ear vein of each of three rabbits 10 ml of the product per
kg of body weight.
4- Record the temperature at 30-minute intervals between 1 and 3 hours
subsequent to the injection

40. PYROGEN… CONT.
40
Steps:
5- If no rabbit shows an individual rise in temperature of 0.5 °C or more
above its respective control temperature, the product meets the
requirements for the absence of pyrogens.
6- If any rabbit shows an individual temperature rise of 0.5 °C or more,
continue the test using five other rabbits.

41. PYROGEN… CONT.
41
Steps:
7- If no more than three of the eight rabbits show individual rise in
temperature of 0.5 °C or more and if the sum of the eight individual
maximum temperatures rises does not exceed 3.3°, the material under
examination meets the requirements for the absence of pyrogens.

42. PYROGEN… CONT.
42
B. Limulus Amoebicyte Lyste Test (LAL)
In recent years, it has been shown that an extract from the blood cells of
the horseshoe crab (Limulus polyphemus) contains an enzyme and
protein system that coagulates in the presence of low levels of
lipopolysaccharides.
The discovery has led to the development of the Limulus Amoebicyte
Lyste Test (LAL) for the presence of bacterial endotoxins.

43. PYROGEN… CONT.
43
B. Limulus Amoebicyte Lyste Test (LAL) … cont.
Proclotting enzyme
Activated clotting enzyme
Endotoxin
Coagulation Clottable protein GEL
The Lysate Clotting Mechanism

44. PYROGEN… CONT.
44
B. Limulus Amoebicyte Lyste Test (LAL) … cont.
In the test procedure, the lysate is mixed with equal volume of the test
solution in a depyrogenated container, such as a glass tube. The tube is
then incubated undisturbed at 37 °C for a period of about 60 minutes.
The test is a pass or fail test. The end point is identified by gently inverting
the glass tube. A positive result is indicated by the formation of a solid
clot. The clot doesn’t disintegrate when the tube is inverted. A negative
result is indicated if no gel clot has been formed.

45. THANK YOU