Sterilization Protocol

for Lab Equipment and

Glassware
Engr. Muhammad Junaid Khalid
Equipment Used in Microbiology
Laboratory

 Microscopes:

Microscopes are used for visual observation of morphology, motility, staining and
fluorescent reactions of bacteria.

 Hot Air Oven for Sterilization

It is used for sterilization of glassware’s, such as test tubes, pipettes and

petri dishes. Such dry sterilization is done only for glassware’s.
Equipment Used in Microbiology
Laboratory

Microscope Hot Air Oven

Equipment Used in Microbiology
Laboratory
 Drying Oven:
For preparation of certain reagents, the glassware’s, after proper cleaning and
rinsing with distilled water, are required to be dried.

 Autoclave:
 Autoclave is the nucleus of a microbiology laboratory. It is used not only to
sterilize liquid substances such as prepared media and saline (diluents) solutions,
but also to sterilize glassware’s, when required.

 A temperature of 180 oC can be obtained in an autoclave.

Equipment Used in Microbiology
Laboratory
 The maximum temperature that can be obtained by boiling water in an open
container is 100°C (boiling point of water).

 This temperature is sufficient to kill most of the bacteria, but some bacteria like
Bacillus (aerobic) and Clostridium (anaerobic) are resistant to harsh conditions and it
is difficult to kill them at this temperature.

 In a closed container, due to increased pressure inside it, the boiling point elevates
and steam temperature much beyond 100°C can be obtained.

 This high temperature is required to kill all the bacteria including the heat resistant
spore-formers.
Equipment Used in Microbiology
Laboratory

Drying Oven Autoclaves

Equipment Used in Microbiology
Laboratory
 Incubator:
It is used in cultivating, multiplying and in the characterization tests of
microorganisms.

 It provides the necessary heat for the growth of microorganisms.

 The ideal temperature for the microbes is 37 oC which can be obtained

in incubator.

 Moisture is supplied by placing a beaker of water in the incubator during

the growth period. A moist environment retards the growth of bacteria.
Equipment Used in Microbiology
Laboratory
 Shaker Incubator:

 In the microbiology laboratories it is among the leading devices which are

based on the principle of shaking at different temperatures according to the

purpose and the work load of the laboratory.

 This device provides the heat as well as mixing necessary for the growth of

microorganisms.
Equipment Used in Microbiology
Laboratory

Incubator Shaker Incubator

Equipment Used in Microbiology
Laboratory
 Refrigerator:

It is used for the storage of stock solutions, chemicals, and nutrient media that should be stored at
low temperature (0-4 oC).

 Deep Freezer (-86 oC):

It is used to store the materials that should be kept at very low temperature (cells, tissues,
enzymes, proteins etc.)

 Electronic Analytical Balance:

They are used in precise weighing of small amounts (up to milligrams) of samples and chemicals
used for preparing media and stock solutions.
Equipment Used in Microbiology
Laboratory

Analytical Balance

Deep freezer (-86 oC)

Equipment Used in Microbiology
Laboratory

 Biosafety Cabinets:
It is used in microbial inoculation and isolation studies.

 In addition, it is utilized for protection of user, samples and the environment

from hazardous contamination.

 Magnetic Stirrer:
Magnetic stirrer is a device which provides mixing and keeping the chemical
solutions and mixtures at a certain time and temperature by the help of a
magnetic bar.
Equipment Used in Microbiology
Laboratory

Magnetic Stirrer

Biosafety Cabinet
Equipment Used in Microbiology
Laboratory
 pH Meter:
 It is used to determine the pH of the media prior

to experiments and to monitor pH value during

experiments.

 The device is used especially in the preparation

of stock solutions and the culture media used for the growth of microorganisms.
Equipment Used in Microbiology
Laboratory

 Distilled Water Plant:

It is used in the preparation of distilled water.

 Water is used in the preparation of media and solutions. If the media are
prepared using tap water, the chemical impurities present in it may interfere
with the growth of the microorganisms in the media.

 It is also used in the preparation of reagents, because the chemical impurities

present in tap water may interfere with the proper functioning of the reagent
chemicals.
Equipment Used in Microbiology
Laboratory

 Ultrapure Water Purification System:

 For precision analytical works, now-a-days, instead of using distilled water,
micro- filtered water is used.

 In case of distilled water, there is chance that, few volatile substances

present in the water get volatilized during heating of the water and
subsequently get condensed into the distilled water collected.

 To overcome this, ultrapure water is used.

Equipment Used in Microbiology
Laboratory
 Here, water is allowed to pass through very fine microscopic pores or filters,
which retain the microscopic suspended particle including the microbes.

 Then, the water passes through two columns of ion exchange resins.

 The anion exchange resin adsorbs the cations present in the water, while the
cation exchange resin adsorbs the anions. The water that comes out is
extremely pure.
Equipment Used in Microbiology
Laboratory

Ultrapure Water purification Systems

Equipment Used in Microbiology
Laboratory
 Hot Plate:
Hot plate is used to heat chemicals and reagents. The hot plate is made of an
iron plate, which gets heated by an electric heating element from below. The
required degree of heating is obtained by a regulator.
 Colony Counter:
A colony counter is an instrument used to count colonies of bacteria or other
microorganisms growing on an media plate.
 It is assumed that a single bacterium gives rise to a single visible colony,
when grown on a plate of solidified nutrient medium.
Equipment Used in Microbiology
Laboratory

Hot Plate Colony Counter

Equipment Used in Microbiology
Laboratory
Thus, by counting the number of colonies, the number of bacteria in a sample
can be estimated.

 Electronic Cell Counter:

It is used to directly count the number of bacteria in a given liquid sample.

 Sonicator:
It is used to rupture cells using high frequency waves.
Equipment Used in Microbiology
Laboratory

Electronic Cell Counters

Equipment Used in Microbiology
Laboratory

Sonicator
Equipment Used in Microbiology
Laboratory
 Spectrophotometer:
Every chemical compound absorbs, transmits, or reflects light (electromagnetic
radiation) over a certain range of wavelength.

 Spectrophotometry is a measurement of how much a chemical substance

absorbs or transmits by measuring the intensity of light as a beam of light
passes through sample solution.

 This measurement can also be used to measure the amount of a known

chemical substance.
Equipment Used in Microbiology
Laboratory

Spectrophotometer
CLEANING, DISINFECTION AND
STERILIZATION

Microscope
 The microscope is an essential instrument for the diagnosis of diseases.

 It requires careful maintenance to prevent damage to mechanical and

ocular parts and also to stop fungi from obscuring the lenses.

Cleaning and Maintenance

 Microscopes must be installed in well ventilated environment, away from
chemicals.
CLEANING, DISINFECTION AND
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Cleaning the Optical Surfaces

The optical surfaces (condenser, objectives, eye pieces) must be kept free of dust

with a soft camel hair brush.

If dust is found inside the eyepiece, unscrew the upper lens and clean the inside.
Oil residue on the lenses should be removed with special lens tissue paper,

absorbent paper or medical grade cotton wool.

The optical surfaces may be finally cleaned with a special solution consisting of

80% Petroleum Ether & 20% 2-Propanol

CLEANING, DISINFECTION AND
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 NOTE: Do not use 95% ethanol, xylene or toluene for cleaning the lenses,
since these substances dissolve the cement of lens.

Cleaning the instrument

 Heavy contamination can be removed with mild soapy solutions. Grease and
oil can be removed with the special cleaning solution as described above.

 The instrument should then be cleaned with an 1:1 mixture of distilled water
and 95% ethanol. This mixture is not suitable for clearing the optical surfaces
CLEANING, DISINFECTION AND
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Precautions
 Never use ordinary paper to clean the lenses.

 Never touch the lenses with your fingers.

 Never clean the support or the stage with Xylene or Acetone.

 Never clean the inside lenses of the eye pieces and objectives with cloth or paper
(this might remove the anti-reflective coating); use a soft camel-hair brush.
CLEANING, DISINFECTION AND
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 Never press the objective on to the slide, since both the slide and the

objective may break.

 When changing a bulb, avoid touching the glass with your fingers, as finger

prints reduce the intensity of illumination.

 If the mains voltage fluctuates excessively, use a voltage stabilizer.

 Keep the microscope under an air tight plastic cover when not in use.
CLEANING, DISINFECTION AND
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Glassware
 New Glassware: Glassware that has never been used may be slightly
alkaline.
In order to neutralize it: use 1-2% solution of hydrochloric acid (Add 60 ml
concentrated HCL in 3 liters of water.).

 Immerse new glassware and leave for 24 hr. Rinse twice with ordinary water
and once with demineralized water. Dry the glassware.
CLEANING, DISINFECTION AND
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 Dirty Glassware: Preliminary rinsing - Rinse twice in cold or lukewarm
water.

 Soak for 2-3 hrs. in a container of water mixed with washing powder or liquid
detergent. Brush the inside of glassware with a test tube brush.

 Remove articles one by one and rinse under running tap water, then soak them
all in ordinary water for 30 minutes and then finally rinsed with distilled
water.

 Place containers (beakers, flasks, measuring cylinders) on a draining rack. Test

tubes are placed upside down in a wire basket for draining.
CLEANING, DISINFECTION AND
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 For drying, place the baskets either in hot air oven at 60°C or in a sunny spot
and cover with a fine cloth.

 Place in a cupboard to protect it from dust.

Pipettes
 Once a pipette has been used, rinse it immediately in a stream of cold water
to remove blood, urine, serum, reagents etc.
CLEANING, DISINFECTION AND
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 Next place the pipettes in a large plastic cylinder full of water. If pipettes
have been used for infectious material place them in disinfectant solution for
4 hrs. (e.g. 1% sodium hypochlorite).

 Then clean the glassware in detergent solution and water as above.

Blocked Pipette
 Place blocked pipettes in a cylinder filled with dichromate cleaning solution
and leave for 24 hours.
CLEANING, DISINFECTION AND
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 Next pour the dichromate solution into another cylinder (can be used 4 times)
and rinse the pipette in the cylinder thoroughly under tap water.

 Remove the pipettes from cylinder one at a time, check thoroughly for any
obstruction and rinse again.

 Soak in ordinary water for 30 minutes, change water and soak again for 30
minutes. Finally rinsed with distilled water.

 Dry in hot air oven at 60°C or air dry

CLEANING, DISINFECTION AND
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Microscopes Slides
New Slides
 Place slides overnight in a bowl of water mixed with washing powder or liquid
detergent.

 Rinse each slide under tap water and the with distilled water.

 Wipe the slides, one at a time with a soft cloth. Place on blotting paper or
filter paper and allow to dry.

 Pack the slides in stacks of 10 or 20 groups in paper

CLEANING, DISINFECTION AND
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Dirty slides

 Oil is removed by rubbing vigorously with newspaper.

 Coverslips are removed using a needle or forceps.

 Now soak in detergent solution for 24 hrs.

 If slide have been used for infected specimen (e.g. urine stool), they should

be placed in disinfectant solution before cleaning.

CLEANING, DISINFECTION AND
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Sterilization
 Sterilization is the process of freeing an article from all living organisms, including
bacteria and their spores.

 Disinfection is the destruction of pathogenic bacteria but not necessarily of the resistant
spores.

Sterilization can be done by:

1) Physical methods (heat, radiation, filtration)
2) Chemical method (disinfectants)
CLEANING, DISINFECTION AND
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Sterilization by Heat
 Heat is the most commonly used and most reliable method of sterilization.

Heat can be

1) Dry heat
2) Moist heat

Dry heat
 Dry heat is suitable for glassware, instruments, articles not spoiled by very high
temperatures.
CLEANING, DISINFECTION AND
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Types of Dry Heat

1. Red heat (For sterilization of inoculating loops etc.

Sterilization is done on the flame of Bunsen burner

or spirit lamp)

2. Flaming (For sterilization of forceps and scalpels)

3. Hot Air Ovens (For sterilization of glassware like tubes, flasks,

measuring cylinders, glass pipettes, all glass syringes and glass

petri dishes.
CLEANING, DISINFECTION AND
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Precautions (Hot Air Oven)

 Glassware should be perfectly dry before being placed in the sterilizing oven
(wet-glassware will crack).

 The oven should not be overloaded and space must be left for circulation of
air through the load.

 The holding time should be calculated only when the temperature indicator
reaches the specified temperature (160°C or 180°C).
CLEANING, DISINFECTION AND
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 The door of the oven should not be opened in between heating as the

temperature is not maintained and improper sterilization occurs.

 After the completion of sterilization, oven should be allowed to cool to 40°C

before the door is opened to remove any articles (takes approximately 2

hours), otherwise cracking of glassware may occur on contact with cold air.
CLEANING, DISINFECTION AND
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Moist heat

 Steam kills microorganism by the irreversible denaturation or heat coagulation of

cellular protein.

 Microorganisms and their spores are killed at lower temperature by moist heat than by

dry heat.

 Used for sterilization of culture media and other liquid to retain their water

content.
CLEANING, DISINFECTION AND
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Moist Heat

1. Steaming at 100°C

2. Steaming above 100°C (Most commonly used)

Steaming Above 100oC

Autoclaving

 It provides moist heat at temperature above 100°C and at pressure greater than

atmospheric pressure.
CLEANING, DISINFECTION AND
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 Autoclaving is the most reliable method and most widely used method for

sterilization of culture media and surgical supplies.

 It is also used for sterilization of infectious waste before discarding.

CLEANING, DISINFECTION AND
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Autoclaving Procedure

Preparation of material for autoclaving

Glassware

 Specimen tubes, petri dishes etc. should be wrapped in autoclavable polythene bags and
tied with string.

 If autoclavable polythene bags are not available then use kraft paper, cotton or gauze
for wrapping the tubes/ petri dishes etc.

Pipettes

 These should be placed in a large tube which are then plugged.

 Or use autoclavable polythene bags.

CLEANING, DISINFECTION AND
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Sterilization procedure
 Fill the bottom of the autoclave with water but the water should not touch
the basket.

 Put the basket containing the material to be sterilized in the autoclave.

 Close the lid, making sure the rubber washer is in its groove. Screw down the
lid clamps evenly and firmly but not too tightly.

 Open the air outlet valve and begin heating the autoclave.
CLEANING, DISINFECTION AND
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 When a jet of steam appears at the air outlet wait for 3-4 minutes till the jet
of steam becomes uniform and continuous (shows that all air has been
removed) close the air outlet valve, tighten the lid clamps and reduce the
heat slightly.

 Watch the pressure gauge-when it reaches the 15 pounds mark- then regulate
the heat to maintain at this pressure.

 Start timing now when the pressure is stable at 15 pounds- this is the holding
time.
CLEANING, DISINFECTION AND
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Removing load from autoclave
 Turn off the heat after holding period is finished. When pressure gauge shows

that the autoclave is at atmospheric pressure, open the discharge tap very slowly to

allow air to enter as cooling of steam proceeds.

 Unscrew the lid clamps and take off the lid and remove the baskets of sterile

equipment.

 During cooling process, sterilized items should never be placed on cold metal

surfaces as moisture will condense onto the items and contaminant them.

 The sterile load should be kept in wire mesh racks or baskets till it gets cooled.
CLEANING, DISINFECTION AND
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Precautions
 All the air must be removed from the autoclave chamber and articles of the
load, so that the load is exposed to pure steam during sterilization.

 For sterilization, exposure of microbes to 121°C for 15 minutes at 15 pounds

pressure is required.

 Avoid autoclaving small and large volumes in the same load because with the
same holding period (e.g. 20 min) small amounts (e.g. 10 ml) may be damaged
by overheating and large amounts (e.g. 2000 ml) may not be sterilized.
CLEANING, DISINFECTION AND
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 Never touch the drainage tap, outlet valve or safety valve while the autoclave
is under pressure.

 Never leave the autoclave unattended while the pressure is rising.

 Never open the lid before the pressure has dropped to normal. During
sterilization, make sure that the lid is secured and no steam escapes.
CLEANING, DISINFECTION AND
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Autoclaving is Used for:

1. Culture media 2. Glass test tubes/slides/containers
3. Enamel metal trays 4. Filters

5. Plastic autoclavable petri dishes/ 6. Reuseable specimen containers

test tubes

7. Cotton 8. Rubber Gloves /stoppers/tubing

CLEANING, DISINFECTION AND
STERILIZATION

Hot air oven

 Glassware- Beakers
- Flasks - Petri-dishes
- Pipettes - Slides
- All glass syringes - Test Tubes

 Powder, oils, grease, paraffin

 Swab sticks

 Filter paper discs for making antibiotic discs

CLEANING, DISINFECTION AND
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Disinfectants
 10% bleach or 70% ethanol solution (To wipe down benches and work areas or spray on instruments)

 1% sodium hypochlorite solution (For pipettes and test tubes)

Some Other Disinfectants

 Phenol

 Isopropanol 70%

 Methanol 37%

 Hydrogen Peroxide 6%

 Ammonium Hydroxide 2.5%

Thank
You
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