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Module 3

Use, calibration and maintenance


of laboratory equipment

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Learning objectives
At the end of this module you will be able to:
describe briefly the operating characteristics of
biological safety cabinets, centrifuges, autoclaves,
inspissators, pH meters, incubators;
correctly use biological safety cabinets, centrifuges,
autoclaves, inspissators, pH meters, incubators;
describe the main maintenance operations for
biological safety cabinets, centrifuges, autoclaves,
inspissators, pH meters, incubators.

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Content outline

Principles, use and maintenance of:


biological safety cabinet (BSC)
centrifuge
autoclave
inspissator (coagulator)
pH meter
incubator
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Biological safety cabinet (BSC)

A ventilated contained area providing


protection for the operator and the
environment against infectious aerosols
during the handling of hazardous microorganisms

The most important equipment in all diagnostic


mycobacteriology laboratories is the BSC.
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HEPA filter

HEPA = high-efficiency particulate air


The HEPA filter traps and remove 99.97% of
airborne particles of diameter 0.3 m or
more.

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BSC class I and II
BSCs should be ducted or vented to
the outside.
No recirculation into the room is
allowed.
The BSC should be connected to a
UPS (Universal Power Supply).

BSC class I and II main differences


BSC class Minimal face Air flow (%)
velocity Exhaust system
(m/s) Re- Exhausted
circulated

I 0.38 0 100 Hard duct

IIA1 0.38 70 30 Thimble connection

IIA2 0.51 70 30 Thimble connection

IIB1 0.51 30 70 Hard duct


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IIB2 0.51 0 100 Hard duct
Class I BSC
Protection of the user and the environment
Air is ducted to the outside of the building.

HEPA filter

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Class II BSC
Protection of the user, the environment and
the product
Class IIA BSC Class IIB BSC

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Laminar flow hood for product
(not user) protection
Horizontal airflow Laminar vertical flow

Air blows towards workers.


Used only to protect product for sterile preparations
(media preparation). 9
BSC location
A , B not recommended
D, C recommended

D C

A B

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BSC operations for correct use
Switch on the BSC airflow and the UV light (if
present) 515 minutes before use.
Check the airflow velocity on the display.
Complete the logbook, recording details of operation
of the BSC.
Switch off the UV lamp.
Never work with the UV lamp on.
Disinfect the work surface, interior walls and interior
window surface with 70% ethanol or other suitable
compounds.
Do not use corrosive substances.
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BSC rules for correct use
Organize all items needed for work in the BSC to avoid
frequent displacements.
Organize the work to minimize arm movements.
Do not overcrowd the working area this disturbs the
airflow.
Conduct all manipulations within the BSC as far towards
the back of the unit as possible (at least 15cm from the
front grille).
In class II BSCs, never allow the front grille to be covered
with anything.
Do not use large open flames in the BSC.
Do not accumulate waste in the BSC; remove it when
activities are finished. 12
BSC after use

Autoclave the waste.


Wipe down the inside of the BSC and work
surface with 70% alcohol.
Switch on the UV light for 1 hour (if the lamp is
in good working order).
Switch off the BSC fan.

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Certification and maintenance

Regular certification and maintenance, with


replacement of filters, are needed and represent
a major challenge in low-income countries. If
maintenance is not carried out regularly, the
filters may become clogged and tubercle bacilli
may be blown into the face of the operator. A
BSC that is not well maintained is more
hazardous than protective.

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BSC evaluation of performance
Performance of the BSC should be evaluated by the manufacturer or a qualified professional, with specific, well-calibrated equipment.

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BSC maintenance
Modern cabinets are equipped
with airflow indicators and
warning devices.
Maintenance, with replacement
of filters, should be carried out
by professionals when the
airflow falls below the minimum
recommended level.
The BSC should not be used
Ou
until this maintenance has to
fo
rde
been carried out. r

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BSC maintenance
Airflow smoke pattern tests are
performed to determine:
whether the airflow along the
entire perimeter of the work
access opening is inward;
whether airflow within the work
area is downward with no dead
spots or refluxing;
whether ambient air passes onto
or over the work surface;
whether there is refluxing to the
outside at the window wiper
gasket and side seals.
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BSC maintenance

A cabinet leak test is carried out:


to determine whether exterior surfaces of all
plenums, welds, gaskets, and plenum
penetrations or seals are free of leaks;
before initial installation or annually on fully
installed cabinets

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BSC fumigation
Decontamination of the BSC with formaldehyde gas.
Hydrogen peroxide.
Fumigation should be performed only by professionals.

System DECONTAKIT 19
BSC fumigation
Fumigation of the BSC should be carried out:
before release of the BSC for use after a major biohazardous
spill;
before replacement of HEPA filters;
before repair work requiring access to the sealed plenum;
before service or replacement of the circulation fan or
components;
before maintenance work in contaminated areas;
before performance tests requiring entry into contaminated
areas;
before movement of the BSC to another laboratory or area of
use;
before changing work programmes, e.g. to non-TB work;
before release of the BSC for resale or salvage.
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BSC logbook

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BSC maintenance logbook

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Centrifuge selection of proper
equipment for TB culture
The centrifuge should:
Have aerosol-free (O-ring sealed) swing
buckets.
Have locking mechanism for protection to
prevent opening before rotation has ceased.
Be able to generate 3000g.
Be refrigerated.

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Centrifuge rotors

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Inadequate centrifuges and tubes

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Prevent accidents by regular
maintenance

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Centrifuge use
Inspect the centrifuge chamber for liquid or signs of
corrosion.
Check that there are shock-absorbing pads in the bottom
of the centrifuge buckets.
Balance the sample tubes and insert them in the buckets
in the appropriate position.
Never add water to a specimen to balance paired tubes
Use an empty tube filled with water as a balance .
Select the required time and speed.
Stop centrifuge immediately if any abnormal noise is
noticed.
Never operate a centrifuge with the lid open.
Open the sealed buckets in the BSC. 27
Centrifuge tubes adaptors

Centrifuge tubes must tolerate g-forces of at


least 3000g.
Centrifuge tubes must be used with
appropriate rubber or plastic cushions
matched to the tube and bucket-holder.
Use only adaptors recommended by the
manufacturer of the centrifuge in use.

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RCF, g, RPM
RCF (or g) = relative centrifugal force
rpm = revolutions per minute
RCF = 1.12 * 106 * r * v2
or
RCF (g) = 1118 x r x (v/1000)2
r = radius in mm
v = speed in rpm
rpm = 1000 *RCF/1.12 * r
r
(mm)
Always work in RCF, not rpm: new
centrifuges can convert automatically. 29
Centrifuge installation

Ensure that the centrifuge is installed on a rigid,


flat, level surface.
Because centrifuges produce vibration during
operation, they must not be installed next to
balances or other sensitive equipment.
Allow sufficient free space around the centrifuge
for ventilation to prevent overheating.
Initial calibration should be performed only by a
qualified service technician .

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Centrifuge daily maintenance

Wipe the inside bowl with disinfectant solution


and rinse thoroughly.
If a refrigerated centrifuge is turned off at
night, open the top to allow the bowl to dry.
During the day when the unit is under
refrigeration, keep the top closed to avoid
condensation and ice build-up.

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Centrifuge monthly maintenance

Clean the centrifuge housing, rotor chamber,


rotors and rotor accessories with a neutral
cleaning agent.
Clean plastic and non-metal parts with a fresh
solution of 5% sodium hypochlorite (bleach).

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Centrifuge annual maintenance

Annual service should be carried out by


a qualified service technician: centrifuge
brushes, timer, speed and electrical
leaks should all be checked.

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Centrifuge logbook

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Centrifuge maintenance logbook
PERIODICITY :
Date Maintenance operation Operator

ITEM IDENTIFICATION
Equipment CENTRI- Brand
FUGE name

Purchase Model/type
date

Location Serial no.


within lab.

Warranty expiration date


Manufacturer

Address FAILURE EVENTS


Date Event Correcti Operator
ve
action
Tel. Contact person
taken
Technical service Tel.
representative

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Autoclave

The autoclave using saturated steam under


pressure is the most efficient means of:
sterilization in a diagnostic TB laboratory;
decontamination of biological material
consisting of infectious waste.

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Autoclave

For optimum function:


All of the air in the chamber should be
replaced by steam.
The temperature must be 121 C.
Materials to be sterilized must be packed
loosely.

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Tips for loading the autoclave
Only appropriate containers should be used.
Discarded cultures should be in solid-bottomed
containers no more than 40 cm deep.
Leave a large air space around each container.
Never cover containers.
If autoclaving closed containers, add water to
generate steam.
Separate autoclaves should be used for sterilization of
solutions or glassware (clean materials) and for
decontamination of infectious materials.
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Autoclaves suitable for
laboratory use
Pressure-cooker Autoclave with air discharge by
laboratory autoclave gravity displacement

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Examples of autoclaves

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Autoclave use
Prepare material for autoclaving with thermosensitive paper.
Do not load material to be sterilized with material to be
decontaminated.
Fill the bottom of the autoclave with distilled water.
Open the air outlet valve.
Turn on the heating.
Close the outlet valve.
Do not touch the drainage tap or the outlet or the safety valve
while heating under pressure.
When the required time is up, turn off the heating completely.
When the temperature falls below 100 C, open the outlet valve
slowly.
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Autoclave use
Never unscrew the lid clamps and open the lid
until the hissing sound has stopped.
Leave the sterilized/decontaminated material to
cool before removing it from the autoclave.
Check whether the autoclave tape has turned
black and the covering paper has turned brown.
If they have not, the material is not
decontaminated. Check the autoclave for
malfunction.
Biological indicators should be used after 40
hours of use.
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Autoclave suggested protocols

Solid material for sterilization: 121 C


(appropriate pressure 115 kPa), 20 min.
Solid material for decontamination: 121 C
(appropriate pressure 115 kPa), 30 min.
Liquid material for sterilization: 15 min at
121 C if liquid cycle is available.

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Autoclave maintenance

Check door gaskets for cracks and pitting.


Check for proper operation of door clamps
and door locks.
Inspect valve discs and seats for signs of
wear or cutting.
Check pressure-release safety valves and
thermometers.
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Autoclave logbook

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Autoclave maintenance and
incidents logbook

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Inspissator
Essential when egg media are home-made (made in the
laboratory).

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Inspissator use
Carefully control the amount of heating.
Ensure the required temperature has been
reached before loading the tubes
Heat the tubes at constant temperature of
8085 C for 45 minutes.
Tubes or bottles loaded at an angle of 5
10 ( 10 ).
Always check the quality of media after
cooling. 48
Inspissator maintenance
Check temperature at each use.
Clean after preparation of each batch of culture
media.

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Inspissator logbook

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Inspissator maintenance and
incidents logbook

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pH meter

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pH meter use
Calibrate the pH meter before use at three
pH values (pH 4.0, 7.0 and 10.0), using
the calibration solutions provided by the
manufacturer.
Immerse the electrode in the test solution.
Rinse the electrode after use.
Cover the pH meter after use.

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pH meter calibration
The pH meter must be calibrated according to the
manufacturers instructions.
Calibration must be performed once daily or, in case of
infrequent use, at least on the day of pH testing. It must
be performed before the first measurement of the day.
Standardize with pH 4.0 and 7.0 buffers before each
test or series of tests.
Discard contaminated or cloudy standard buffers.
Calibration results are acceptable if the pH values of the
reference buffer solutions are within 0.1 pH units of the
expected pH.
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pH meter Maintenance
Keep the instrument clean. Cover it after each use.
Rinse the electrode after use.
Make sure that the electrode is always filled with
electrolyte according to the manufacturers instructions.
Do not touch the electrode membrane, which can be
easily damaged.
A glass electrode should be kept immersed in a standard
salt solution for long-term storage.
Calomel electrodes must be kept in a potassium chloride
(KCl) buffer solution when not in use.
Date buffer solutions and discard when unsatisfactory.55
pH-meter calibration and record
sheet
Calibration solution Solution test

Date Temp. Electrode pH 4 pH 7 pH8* Signature Solution/ pH read Signature


(C) serial no. buffer

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*pH 10 can be used if commercially purchased
pH-meter logbook
ITEM IDENTIFICATION
Equipment pHmeter Brand
name

Purchase Model/type
date

Location Serial no.


within the
lab
Warranty expiration date
Manufacturer
Address

Tel. Contact person


Technical service Tel.
representative

FAILURE EVENTS
Date Event Corrective Operator
action
taken

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Incubators
Incubators ensure optimum growth conditions for culture of
M. tuberculosis.
Incubators are available in various sizes, from small
(benchtop) models to large closets up to several hundred
liters. Walk-in incubator rooms with circulating fans to obtain
an homogeneous temperature are also frequently used.

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Incubator use
Keep door(s) closed to prevent heat loss to
the environment.
Ensure that rack positions are clearly marked.
Switch the incubator off when not in use.
Do not overload.

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Incubator maintenance

Record temperatures daily on appropriate


forms.
Check that the actual temperature
corresponds with the thermostat setting.
Clean every 14 days with 70% ethanol.
Clean immediately after any infectious spills.

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Incubator calibration

Calibrate:
before use;
after temperature changes have been
detected and rectified;
following a power failure;
after cleaning of spillages.

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Incubator calibration
Ensure that the door is closed and that the incubator is
switched on.
Set the required temperature using the temperature
control and leave the incubator to run for 1 hour.
Place a thermometer in the centre of the incubator with
the probe away from the heating element.
Take the temperature reading after 30 minutes. If the
temperature is not 36 1 C, adjust the control.
Repeat this process every 30 minutes until the required
temperature is reached.
Continue to take readings until two consecutive readings
(30 minutes apart) are 36 1 C.
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Incubator maintenance form

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Trimester : Year :

Month Temp. Visa Month Temp. Visa Month Temp. Visa

C (Initials) C (Initials) C (Initials)

1 1 1

2 2 2

3 3 3

4 4 4

5 5 5

6 6 6

7 7 7

8 8 8

9 9 9

10 10 10

Incubator
11 11 11

12 12 12

13 13 13

14 14 14

logbook 15

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15

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15

16

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18 18 18

19 19 19

20 20 20

21 21 21

22 22 22

23 23 23

24 24 24

25 25 25

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30 30 30
True and false exercise

1. The biological safety cabinet is the most important


equipment for TB culture laboratories.
2. Only class II BSCs can be used for TB culture.
3. Centrifuge speed should always be expressed in
rpm.
4. Centrifugation may produce hazardous aerosols.
5. Checking the autoclave regularly is unnecessary if it
is used only for decontamination.
6. Equipment maintenance is crucial for optimal
performance.
7. Maintenance can always be performed in-house.
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Module review: take-home messages

1. Equipment is expensive: all equipment must be treated with


care and properly maintained.
2. Quality-control checks on equipment should be run regularly
and results should be recorded on appropriate forms.
3. The biological safety cabinet is the most important item of
equipment for TB culture under safe conditions.
4. Read carefully and always follow manufacturers instructions
for routine work and maintenance.
5. Familiarize yourself with all working parts of all the
instruments.
6. Call for help when troubleshooting any equipment malfunction.
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Self-assessment

Describe the differences between the two types of BSC.


What precautions must you take when working in a
BSC?
Describe the operations to be undertaken before starting
a centrifuge.
Explain the principles of sterilization using an autoclave.

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