INSTRUMENTATION (MICROBIOLOGY UNIT)

AUTOCLAVE

An autoclave is an apparatus which uses high temperature for sterilization under pressure. The
temperature of saturated steam at atmospheric pressure is 100 degrees Celsius approximately. When
water is boiled at increase pressure, the temperature at which it boils and at which it forms steam
increases with pressure. The temperature of saturated steam rises to 121 degrees Celsius at 15 psi.
An autoclave under this condition for 15-20 minutes is recommended for most laboratory
applications (this is usually called culture media autoclaves). But autoclaves used in sterilizing
instruments used in operation (Clinical autoclaves) are usually designed to sterilize at 134 degrees
Celsius for 10 minutes or 126 degree Celsius for 11 minutes

USES OF AUTOCLAVES

1. For sterilizing culture media and swabs for use in microbiology work

2. It is used for steaming to sterilize media that contain heat sensitive ingredients at lower
temperature (100 degrees Celsius) with the lid let loose

3. It is used for decontaminating specimen and other infectious waste prior to disposal

NOTE:

1. To obtain the correct sterilization temperature for sterilization, all air must be expelled from the
autoclave as a mixture of hot air and steam will not sterilize. Presence of air in the autoclave
reduces the temperature and hinders heat penetration.

2. It must be remembered that the higher the altitude, the lower the atmospheric pressure required
to achieving 1210 C. Therefore, the pressure must be increased to 18 psi or alternatively, use a
lower temperature (1000 C) and then a longer time (20 minutes).

3. Before commencing sterilization, first allow sufficient time (heat generation time) for the
saturated steam to penetrate or permeate the entire load and for heat transfer to occur. This will
depend on the type of autoclave and materials been sterilized.

4. Allow sufficient time after sterilization for the pressure to return to zero for the system to cool
to avoid accident and prevent injury.

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 AUTOCLAVE CONTROLS AND INDICATORS

1. Automatic Process Control:

This system carries through the whole sterilization cycle including heating or holding, cooling and
drying stages according to pre-selected scheme for the duration of temperature and pressure for
each stage.

2. Recording Thermometer:

An autoclave thermometer makes a graphic time record of the temperature change in the chamber
discharge channel and this in the absence of automatic control helps the operator to avoid errors in
timing the holding process

3. Thermocouple:

As thermometer does not give indication of the temperature of steam in the load but that in the
chamber or the drain of autoclave only, there is need for all autoclaves to be tested with a
thermocouple to measure the load temperature. This will help to know the time required (heating
time) to bring a given load to 121 degrees Celsius. The thermocouple should be placed inside the
load (in the center) where heat penetration is lowest and the thermocouple connected to a recorder
(potentiometer) to produce a graph of temperature / time condition during the sterilization cycle.

4. Chemical Indicators

In the absence of thermocouple recorders, chemical indicators can be used to monitor time and
steam. These indicators include:

a. Time Steam Temperature Control Strips (TST strips): these strips are used in autoclaves and
pressure cookers. The product is manufactured by Albert Brown and is made up of yellow chemical
incorporated in an area at the end of the strip. The control strip is usually placed in the center of the
load where heat penetration is lowest. The yellow color changes to purple when the sterilization
conditions are been meet.

b. Brown's indicator tube: this container is a chemical which turns from red to green when
sterilization is complete.

c. A pallet of sulphur in small glass tube: this show a change of shape by melting when
experimented at 120 degrees Celsius for a few minutes.

d. Adhesive Sterilization tape: this comprises of a heat-moisture sensitive ink which forms bands
on the tape and changes to dark colour after autoclaving. The tape is mainly useful in showing when
an article has been autoclaved.

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5. Spore strip

The use of spore strip or spore containing ample for testing autoclave in the laboratory is not in
common use because it only provides information retrospectively not at time of autoclaving.

CENTRIFUGE
It is an apparatus which separates substances of different densities by exerting a force greater than
that of gravity. It is usually used to spin down particles (such as cells, bacteria, casts, crystals etc)
which are suspended in fluid. Sedimentation rate and effectiveness depends on the speed of rotation
(that is revolution of the rotor per minute) and the radius of the rotation. Heavy particles settle first
followed by the lighter ones. Two types of centrifuge are required for district laboratory work:

1. General purpose bench centrifuge

 This is used to sediment cells, bacteria and parasites in body fluids e.g. urine, CSF, blood etc for
microscopic examination.

 It is used to carry out parasites concentration technique

 To obtain serum, washed red blood cells for cross matching, serum or plasma for antibody
testing and clinical chemistry.

General purpose centrifuge comes fixed for small laboratory and with variable speed time.

2. Microhaematocrit Centrifuge

 It is used to diagnose and monitor anaemia when a reliable measurement of haemoglobin is not
possible

 It is used to calculate MCHC in diagnosis of iron deficiency and other forms of anaemia

 It is used to perform microhaematocrit concentration technique to detect motile trypanosomes

and microfilaria

USE AND CARE OF CENTRIFUGE

1. Position the centrifuge well on a firm level bench away from direct sunlight. Towards the back
of bench, leave a clear area of about 30cm around the centrifuge

2. Read the manufacturer's instruction on the use, cleaning and maintenance of the centrifuge

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3. Use plastic tubes made from polystyrene or thick - walled glass tubes with round bottom
without cracks or chips to avoid breakage

4. Use tubes that are not too long when using a swing out rotor centrifuge to prevent breakage

5. Cap tubes or containers and don't over fill them

6. Do not insert loose-fitting cotton wool plug in a tube without anchor to avoid it being forced
down into the fluid during centrifugation

7. Check to ensure that the tube holds or buckets are correctly position and the content of each side
of the centrifuge are balanced

8. Do not attempt to stop the rotor by hand, if using a centrifuge that is without lid interlock, or
automatic break, to avoid injury and damage to the centrifuge bearing.

ANAEROBIC JARS AND INCUBATORS

Obligate anaerobes are defined as organisms that grow only in the absence of free oxygen. Oxygen
is required for the growth of many but not all microbes. Those that grow only in the absence of free
oxygen are called obligate or strict anaerobes example includes Clostridium tetani. Those that
grow only in the presence of free oxygen are called obligate or strict aerobes example includes
Pseudomonas aeruginosa. Those that grow in either state are called facultative anaerobes example
Staphylococcus aureus. Most organisms of medical importance fall into this large group and
generally grow more luxuriously under aerobic condition. Those organisms which grow best in an
atmosphere containing a reduced level of oxygen are termed microaerophiles. Examples include
Compylobacter species

Growth of the obligate anaerobes depends on the state of oxidation-reduction reaction in its
environment. This oxidation-reduction potential is the measure of the state of oxidation in a
solution. It is determined by immersing an electrode in a solution and measuring the electrical
potential set up between the electrodes and the solution. The more the electrical potential is higher,
the more oxidized the system is.

Strict anaerobes are unable to grow in culture media unless the electrical potential is below a certain
level.

WHILE ANAEROBES DO NOT GROW IN OXYGEN

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Many organisms form hydrogen peroxide (H2O2) when incubated in the presence of oxygen. Most
aerobic organisms produce an enzyme called catalase which catalyzes the breakdown of the
hydrogen peroxide to produce water and oxygen.

2H2O2 → H2O + O2

H2O2 → H2O + 1/2 O2

Anaerobes do not have this enzyme and are therefore destroyed by the hydrogen peroxide. When
grown in the absence of oxygen, hydrogen peroxide is not produced. Cultivation of organisms in
the absence of oxygen can be done in several ways:

1. Growth in special media. Example 0.5% glucose agar deep

2. Use of anaerobic jars

The best general method of anaerobic cultivation is the use of anaerobic jars. The jar is capable of
producing conditions sufficient for the growth of the strictest anaerobes such as Clostridium tetani.
It also allows specimens to be directly plated out on solid media. One pattern of anaerobic jar
usually used is McIntosh and Filde's jar as modified by Baird and Tatlock (BTL jar).

BAIRD AND TATLOCK ANAEROBIC JAR

This is a seamless metal or plastic vessel sufficiently large to contain Petri dishes and tubes if fitted
with a gas tight lid. The lid has inlet and outlet valves and on its inner surface a room temperature
catalyst enclosed within wire gauze envelop. The catalyst consists of alumina pellets coated with
0.5% palladium which catalyzes the combination of oxygen in the air with hydrogen to form water.
For optimum growth of strict anaerobes, 5 - 10% carbon dioxide is often incorporated in the
hydrogen supply.

GAS PARK SYSTEM

Recently, another version of the anaerobic jar has come in to common use. This is the carbon
dioxide - hydrogen anaerobic system (Gas Park). The principle employed here is the generation of
both carbon dioxide and hydrogen by adding water to pre-packed chemicals thereby eliminating the
need for many external apparatus and manipulations.

HOT AIR OVEN

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It is mostly used for drying medical instruments and medical devices in dry air. It is also used for
sterilization. Sterilization in dry air is only effective when material is exposed for 60 minutes at 160
degrees Celsius or for 40 minutes at 180 degrees Celsius. It is important to remember that timing
for sterilization is efficient when the holding period begins after the air in the oven has reached its
expected temperature.

HOW TO USE THE HOT AIR OVEN

1. Set up the thermostat at the required temperature prior to sterilization

2. If there is fan, check if it is working

3. Allow to continue heating for additional 60 minutes

4. After the temperature reaches the pre-set degree, switch off the heat. Wait until the
temperature falls to 40 degrees before opening the door.

USES OF HOT AIR OVEN

It is used to sterilize glassware, metallic instruments and some chemicals in powdered form, oil and
fat.

Materials suitable for sterilization in hot air oven are:

 Glassware: Petri dishes, pipettes, flasks, test tubes, syringes

 Glass or aluminum Petri dishes (not plastic dishes)

 Glass tubes and bottles fitted with aluminum caps or non-absorbent cotton wool plugs

 Glass flasks and cylinders

 Glass pipettes

 Metallic instruments: forceps, scalpels, scissors

 Chemicals in powdered form, oil, fat, petroleum jelly

 Pharmaceutical products: liquid paraffin, fat, grease.

NOTE: Hot air oven can also be used at lower temperatures (80 - 100 degrees Celsius) to dry
routine glassware.

PRECAUTIONS WHEN USING THE HOT AIR OVEN

1. Ensure materials kept for sterilization in hot air oven are dry
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2. Don't use rubber goods, fabrics and flammable or volatile substances inside the oven.

3. Avoid overloading the oven. Keep space for proper air circulation

4. Wrap glassware like Petri dishes and pipettes well from the outside

5. Cool the hot air oven to 60 degrees Celsius before opening the door of the oven.

ADVANTAGES OF HOT AIR OVEN

1. Sterilization can be done without water but water is compulsory to use in moist heat sterilization

2. Bacteria endotoxins are inactivated

3. Oil and powder can be sterilized only in hot air oven. They cannot be sterilized in autoclave as
clumps may form due to moisture

4. It is non-corrosive for metals and sharp objects

5. It is easy to install and have a low operating cost

DISADVANTAGES OF USING HOT AIR OVEN

1. Sometimes prions may not be killed using dry heat method of sterilization

2. Due to high temperature, glassware may become smoky

3. Plastic wares and rubbers can't be sterilized in hot air oven because these items will melt at
higher temperature

MEMBRANE FILTRATION EQUIPMENT

They are porous membranes composed of pure and biologically inert cellulose substance ranging
from 10 - 50 microns. They are two types of cellulose membrane filters:
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1. The older type (gradocol membrane) - which is made up of cellulose nitrate

2. The modern type - which is made up of cellulose acetate

Modern membrane filters

These were first developed by the Millipore membrane filter cooperation in America where they are
referred to as Millipore filters. The filters consist of cellulose acetate and are made up of two layers:
the basal layer with pores 3-5 micrometers and the upper layer of pores 0.1 - 1.0 micrometer in
diameter. This structure gives a remarkable size of porosity which ensures that bacteria are trapped
on the upper surface. They are available in various sizes and can be fitted into metal or glass
holders. Cellulose membranes filters can be used for sterilization as they can retain bacteria e.g.
Serratia marcescens. Bacteria retained on the surface of membrane filters can be cultured by
placing the filter with the retained bacteria on absorbent pad with a suitable medium such as M-
Endo broth.

The membrane filtration technique is usually applied in water bacteriology as a reliable test for the
detection of total and foecal coliforms in water.

APPLICATION

The membrane filter apparatus include 1 liter suction flask or vacuum flask, packet of membrane
filters and pad, forceps, membrane filter clamp, funnel and filter. The application is done under
aseptic condition. The membrane filter unit is aseptically assembled as follows:

Place the membrane filter (grid side up) with the filter holder base fitted into a suction with
rubber hose connected to a vacuum pump.

Place funnel on top of the membrane filter and secure it to the base with a clamp

Add the sample (about 50ml) to the funnel and evacuate using the suction pump until all liquid
has been filtered

Remove the membrane with retained bacteria from the filter unit and place it on the membrane
pad saturated with medium in a Petri dish

Incubate under a specified condition and observe for colonies that can be counted with unaided
eyes or with dissecting microscope.

MICROMETRY
It has to do with the measurement of microscopic objects using calibrated eyepiece scale called a
micrometer. There are two types of micrometers:

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 1. Ocular micrometer

2. Stage micrometer

In microbiological work, size and shape are used extensively in identification and classification of
bacterial cells. The length and diameter of prokaryotic and eukaryotic cells are commonly
expressed in micrometers. Formerly, the cells were measured in microns which stood for 1/1000
millimeter (0.001 mm). But the unit has been replaced by its equivalent, the micrometer which
stands for millions of millimeters (i.e. 1/1000000 or 10 -6). Measurement of microscopic cells or
objects is accomplished by using a scale called an ocular micrometer or in the eyepiece of the
microscope in conjunction with the stage micrometer. Because the spacing or distance between two
lines of the ocular micrometer is arbitrary hence, the exact distance between lines of graduation in
the ocular micrometer is determined by using a stage micrometer.

Ocular micrometer (eyepiece micrometer)

It consists of a special eyepiece in which a graduated scale mounted on the diaphragm of a positive
type eyepiece can be focused by means of a moveable eye lens. Ocular micrometer is simply a disc
of glass upon which are etched lines. They are usually etched 100 equally spaced divisions marked
0 - 10 upon an ocular micrometer.

When placed in the eyepiece, the ruled lines superimpose certain distance markers on the
microscope field. However, the scale on ocular micrometer does not have any standard value. The
value of this unknown scale can be determined by calibrating or matching it with a known scale.

Stage micrometer

It is a microscope glass having in its center, a known 1mm distance etched into 100 equally spaced
divisions. This 1mm (1000 micrometer) distance is encircled and then mounted on a cover glass.
Thus each division of stage micrometer equals to 10 micrometers. The distance of each division
becomes correspondingly enlarged under high power oil immersion objective of the microscope.

Ocular micrometer is calibrated under different objective lens system of microscope by determining
how many divisions of ocular micrometers superimpose a known distance on stage micrometer.

The exact value of one division of ocular micrometer in the microscope field can be used to
measure the size of various microbes in terms of length, width, breath and diameter.