DISEASE SURVEILLANCE AT DISTRICT LEVEL
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- Title
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DISEASE SURVEILLANCE
AT DISTRICT LEVEL
- extracted text
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DISEASE SURVEILLANCE
AT DISTRICT LEVEL
A LABORATORY MANUAL
NATIONAL INSTITUTE OF COMMUNICABLE DISEASES
22 SHAM NATH MARG, DELHI - 110 054
Reprinted on Riso in the Project Secretariat - 1998
am
Government of Karnataka
Department of Health & Family Welfare
Karnataka Health Systems Development Project
Disease Surveillance at
District Level
A Laboratory Manual
Office of the
Additional Director, Communicable Diseases
Karnataka Health Systems Development Project
Department of Health & Family Welfare
Bangalore.
DISEASE SURVEILLANCE
AT DISTRICT LEVEL
II
A LABORATORY MANUAL
NATIONAL INSTITUTE OF COMMUNICABLE DISEASES
22 SHAM NATH MARG, DELHI- 110 054
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Contents
' ?|l J/..".
J.
1.
2.
Role of Laboratory in Disease
Surveillance
Collection, Storage and
Transportation of Specimens
3.
Disinfection and Sterilization
4.
Microscopic Examination
5.
Serological Tests
6.
Bacteriological Analysis of Water
7.
Laboratory Diagnosis of Cholera
8.
Safety Precautions in Laboratory
9.
Common Laboratory Equipment
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CHAPTER- 1
Role of Laboratory in Disease Surveillance
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CHAPTER- 1
r
ROLE OF
»
EPIDEMIOLOGICAL surveillance of a disease is the continuing
scrutiny of all aspects of the occurrence and spread of a disease that
arc pertinent to effective control. It is a dynamic process involving the
inlectious agent, host, reservoirs, vectors and the environment as well
as a complex mechanism concerned with the spread of infection and
the extent to which spread has occurred. Surveillance of any
particular disease includes systematic collection and evaluation of
morbidity and mortality data
data, reports of investigation of epidemics,
aboratory investigations to find out the causative agent, use and
untoward effects of biologicals, insecticides and other materials used
in control, assessment of immunity status of population and other
relevant data for action. The introduction of laboratory techniques in •
epidemiological services has revolutionised the concept as well as
scope of disease surveillance. Now a days laboratory support is
considered an integral component of a sensitive system of
surveillance.
1.1
Role of Laboratory services in surveillance:
1.
Diagnosis of a syndrome
o
o
o
o
2.
Tracing the source of infection.
o
3.
Epidemiological markers
Detection of inapparent infections/carriers:
o
o
o
4.
Encephalitis
Hepatitis
Meningitis
Pyrexia of unknown origin
Japanese Encephalitis
Typhoid fever
Meningococcal meningitis
Early detection of outbreak:
o
o
Meningococcal meningitis
Hospital infections.
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5.
Retrospective diagnosis.
o
o
6.
Detection of New Disease Agents
o
o
7.
O
O
O
O
Plague
Leptospirosis
Controlled field trials.
O
O
12.
Sero-surveys
Immune status
Find out natural foci of infection
O
11.
Vaccine potency testing
Vaccine Safety studies
Prevalence studies
O
10.
Antibiogram
Sero-Therapy.
Quality Control of Biologicals
O
9.
HIV
Newer Entcropathogcns ( V. Cholerae 0139 )
Monitoring of treatment
O
8.
Rheumatic heart-disease
Subacute sclerosing panencephalitis
Newer drugs/vaccines
Newer regimens of drugs/vaccines
Key to successful laboratory based surveillance lies in :
1.
2.
3.
4. •
Right sample collection.
Right time to collect samples for disease surveillance.
Right methodology to be for transportation of sample.
Right laboratory to be chosen
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Identify right patient
Order relevent tests
Collect appropriate specimens of good quality
Label appropriately & fill proforma
Proper transport to laboratory
Proper recording in laboratory
Perform accurate and precise analysis
Document and report
Interpret
Timely action on right patient
Fig 1.1 -.Algorithm for ensuring good laboratory results
3
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1.2 Networking of laboratories
Central or National Reference Laboratory
’
Regional Laboratory
. -___ L—ZZ
Regional Laboratory
District Labs
.1.'
District Labs
—
"■*
Peripheral Lab
i
Regional Lab
District Labs
r-
Peripheral Lab
Peripheral Lab
j
Fig 1.2: Networking of laboratories
1.3
District laboratory
These laboratories are located at the point of first contact of patients,
with the health care services. In most of the developing countries
these are available only at primary health centre or community health
centre (upgraded primary health centres). These laboratories provide
technical support for preventive, curative and promotive services for
the individual as well as the community.
1.3.1 Staff
The staff in peripheral laboratories should include one technician and
one laboratory assistant/attendant.
1.3.2 Space
Space available in peripheral laboratories should include at least one
laboratory-cum-officc/record room (16 ft xlO ft ) and one store-room
combined with other services (16 ft xlO ft).
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1.3.3 Other facilities
Other necessary facilities include
□ a supply of safe water,
□ a reliable source of energy (battery, electricity,
solar or kerosene) and
□ sterilization facilities and waste disposal
There must also be transport and communication facilities between
the peripheral and intermediate laboratories for referral of samples
and patients, procurement of supplies and personal discussion.
Equipment and supplies.
Necessary equipment and supplies
include: good microscopes, centrifuge, transport media, glassware,
sterile swabs, reagents for staining (eg. Gram, Albert, Ziehl Neelsen,
Romanowsky), kits and reagents for rapid diagnostic tests, sterilized
syringes and needles, micropipettes and tips as well as
sterile
collection bottles for blood/serum and water analysis.
1.3.4 Tests to be performed
Table 1.1: Suggested microbiological tests at district laboratories
Procedure/Specimen
Disease / Organism
Microscopy for stained smears
(Gram, Albert,Ziehl Neelsen)
Nasopharynx and throat
Sputum
CSF
Stool
Culture
Serological tests
Diphtheria,
Vincent’s angina
Tuberculosis, pneumonia
Meningitis
(pyogenic & tuberculosis)
Cholera/dysentery
Cholera
Enteric fever (Widal)
RPR/VDRL
Dipstick and
Particle agglutination test
Bacteriological analysis
HBsAg, HIV
Water
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These laboratories are expected to undertake tests of public health as
well as clinical relevance.
Amongst the tests of public health
relevance, diseases of greater epidemiological importance should be
accorded priority.
Testing of environment samples (especially water) also falls into the
priorities of public health relevance. Certain serological tests may be
of use in studying epidemiological pattern of the important diseases
and the same can be performed at peripheral laboratories (Table 1.1)
As far as possible, these tests should be reliable, sensitive, specific,
rapid, easy to perform and cost effective.
i
I
)
()
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CHAPTER- 2
Collection, Storage and Transportation of
Specimens
w
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|Ti
CHAPTER2
COLLECTION, STORAGE AND TRANSPORTATION)
.■ i
OF SPECIMENS.
EFFECTIVE diagnostic microbiology depends upon the correct
collection and timing of clinical specimens and their proper transport
to the laboratory under optimal conditions. It has been observed that
most important and frequent source affecting laboratory analysis is
collection and transportation of the specimen. The guidelines for it
must be emphasized. Specimen should be in adequate quantity.
•
•
•
•
•
Specimen must be collected before the administration of
antimicrobial agents.
Contamination of specimen with externally present organisms or
normal flora of body must be prevented.
Specimen must be collected at appropriate stage of the disease.
Specimen should not get contaminated during storage.
Specimen handling should not be risky to individual
2.1 Precautions in handling specimens
o
o
o
o
o
o
o
o
o
Apply strict aseptic techniques throughout the
procedure.
Wash hands before and after the collection.
Collect the specimen at the optimum time.
Make certain that the specimen is representative of
infectious process (e.g. sputum is the specimen for
pneumonias and not saliva) and is adequate in
quantity for the desired tests to be performed.
Collect or place the specimen aseptically in an
appropriate sterile container.
Ensure that outside of specimen container is clean
and uncontaminated.
Tightly close the container so that its contents do not
leak during transportation.
Label and date the container, complete the requisition
form.
Arrange for immediate transportation of specimen to
laboratory.
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2.2
Laboratory specimens required for tests for
particular causative agents:
Suspected agent/
disease
Specimen
Arbovirus infection
Blood or brain (-70°C)
Blood or serum (+4°C)
Isolation
Serology
Cholera
Rectal swabs or stool
specimens in transport
medium, as recommended
by the laboratory.
Culture
Gastroenteritis
Stool
Viral Hepatitis
Legionellosis
Malaria
Meningococcal
meningitis
Plague
Serum (+4°C)
Blood, sputum, in
enrichment broth.
Blood smears
Spinal fluid, blood
Spinal fluid, blood,
Bubo fluid, blood
Culture,
ELISA*
ELISA*
Culture/ FA**
Typhoid fever
Blood in enrichment broth
Dysentery
Faecal specimens
or rectal swabs in
enrichment broth.
Blood/ serum
Syphilis
Test
Staining
Latex
agglutination
Staining,
Culture/ FA**
Culture;
serology
Culture/
microscopy
VDRL/ RPR
* Enzyme linked immunosorbent assay
** Fluorescent antibody test.
Because of alterations in the specimen prior to measurement, the
clinical state of the patient will not be necessarily reflected by the
result of the laboratory investigation despite the correct laboratory
performance. Some of the important specimens and their proper
collection and transportation methods are described here so as to
ensure quality.
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2.3
Blood for serological testing:
Blood is the most important and frequently collected clinical specimen
at district level.
Blood should be drawn using sterile (preferably disposable) syringes
and needles. Quantity of blood drawn should be minimum 4-5 ml.
Vial in which blood is collected should be preferably sterile, dry and
properly labeled. The needle and syringe used, as also the vial,
should be completely DRY before collecting blood. After drawing
blood, the needle should be removed from the syringe before
transferring blood from syringe to the vial. Do not shake the blood
that is collected in the vial.
Let it stand undisturbed at room
temperature for 2-4 hours.
After the blood has stood at room
temperature for 4-6 hours, it should be subjected to the process of
serum-separation. If the facilities for separation of serum are not
available, then it should be refrigerated at 4°C (NOT FROZEN).
Using a sterile Pasteur pipette, dislodge the retracted clot from the
liquid portion of blood, transfer the liquid portion into a clean sterile
centrifuge tube having a rubber cork.
Centrifuge at 500g for 5 minutes. Transfer the supernatant(serum)
using a sterile Pasteur pipette into sterile clean, dry plastic disposable
screw capped vials. Label the vials.
2.3.1 Filter paper method of collection of blood:
Transportation of liquid specimens soaked on filter paper is a simple
and safe procedure. All kinds of body fluids can be absorbed onto
filter paper strips (Whatman No.l). Cards with incorporated filter
paper strips having marked circle (usually 3 cm diameter) are
commercially available. The details of the patient are written on the
card with a ball-point pen. The blood is taken from the finger prick or
heal prick of an infant. The site is cleaned with 70% alcohol and
wiped dry with sterile gauze.
Puncture is made with a sterile
disposable lancet and the first drop of blood is wiped away with sterile
dry gauze.
Filter paper (marked area) is gently touched onto the second drop of
blood and
allow the blood to soak till the premarked area is
completely filled. Punctured site should not be squeezed to prevent
any haemolysis. Blood is applied only on one side of filter paper and
once only.
The blood specimen is dried for 3 hours in the air in a horizontal
position without letting the specimen come in direct contact with any
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surface, direct heat or sunlight.
refrigerated.
These samples should not be
The specimen is placed in a plastic bag or an envelope along with few
g anules of a desiccant and sealed hermetically before transportation
i mai ing.
f stored in a cool and dark place, such specimens give
acceptable results even upto three months.
> ,
‘
LTalters1?CtiOn Of St°O1’ CSF ’ asPirates an<* water, see relevant
2.4 Labeling of Specimens
1A
?ra .ry aS eafly aS P°ssiblecase of delay the sample
should be stored at 2-8°C before transporting to nearest laboratory.
Labels for specimen collection vial
Name'
Age
Specimen No.„
Specimen
Date
Time
2.6 Storage and Transportation:
In general, infectious materils should be kept at a low temoerature
uring storage and transport, except the CSF samples collected from
^£nP°;PyOgT meningitis which should be maintained at room
temperature. The types of refrigeration required to achieve various
temperatures are as follows:
various
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Temperature (°C)
(+)2-8
( + )4
(-)8
(-)20
(-)70
(4160
'W'-' • J
Type of refrigeration
Domestic refrigerator
Wet ice or frozen ice packs (cold bags)
Freezer of domestic refrigerator
Freezer cabinet
Deep freezer or dry ice.
Liquid nitrogen
\i
The quantity of pathogens or antibody in original clinical samples can
decline during storage or transportation which seriously affects the
diagnostic results. Hence special care should be taken before or
during transit of materials to laboratory to protect them from heating
or drying.
•
•
•
Avoid repeated thawing and freezing of specimens
Freeze the specimen only if transport is assured at -20°C.
Store and transport all specimens at 2-8°C
(Lower compartment of refrigerator) EXCEPT CSF.
For Transportation of specimen
•
•
wet ice or ice pack should be used.
Specimen containers relating to single casdnvestigation should
be placed in a plastic bag with an absorbent material surrounding
the specimen so that even if whole specimen leaks out, it will be
absorbed.
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•
•
•
The laboratory report form should be sealed within a separate
plastic bag and wrapped round the specimen or attached firmly to
the box of specimens.
The material should be packed in an insulated carton/carrier to
transport a specimen to the laboratory.
All specimens should be considered as potentiallpathogenic and
accordingly labeled with internationally accepted biohazard label.
2.7
Transportation of Virus Isolates/Specimen to
Reference Laboratory:
Specimen virus isolates to be sent to other laboratories require special
attention for packing of the material and strict guidelines for
transportation of samples should be followed.
r-Aicvtivn .i--
CHAPTER- 3
Disinfection and Sterilization
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L
CHAPTER-3
DISINFECTION AND STERILISATIONMBW
Definition:
Sterilisation implies complete destruction of all living micro-organisms
including spores. Disinfection means destruction of vegetative forms
of organisms which might cause disease or spoilage of food etc. It
does not necessarily kill spores. The two terms are not synonymous.
3.1
Disinfection of used laboratory articles
Disinfection of both reusable and disposable glassware and articles
contaminated with morbid or culture material is of utmost importance
in the laboratory. All the specimens received in the laboratory should
be considered as potentially pathogenic. The ideal method of treating
such materials is to incinerate all the disposables and decontaminate
the reusable articles by autoclaving. These facilities may not be
available in every laboratory. For purpose of disinfection, disposal
and recycling, all the articles may be divided into three categories.
o
o
o
Disposables.
Reusable articles contaminated with morbid material
such as pipettes, slides, test tubes etc.
Material containing or contaminated with bacterial
cultures.
3,1.1 Disposables:
Soak the material overnight in a strong solution of disinfectant before
disposing alongwith garbage; 1% sodium hypochlorite, 10% solution
of formalin or 3% lysol may be used as disinfectant.
3.1.2 Reusable articles contaminated with morbid
material:
Discard the articles into ajar containing solution. Let them remain in
this solution overnight.
Drain off the disinfectant. Transfer the
material to a metal pot or tray with cover. Pour water and boil for 15
minutes. Cool and drain off the water. Pass on the articles for
washing.
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3.1.3 Glassware containing culture material
Discard all the articles containing or contaminated with culture
material directly in a metal box or a bucket. Place the box with
material in the autoclave and decontaminate by autoclaving (see
sterilisation).
Drain off culture medium and pass for washing.
3.2
Disinfection of rooms
Seal all the windows, ventilators and fire places with brown paper and
adhesive tape.
Pour 500 ml of formalin and 1000 ml of water in a pan or tray and boil
with the help of a spirit lamp or a bunsen burner. Spirit in the lamp
should be just sufficient, to boil off the formalin and the lamp
extinguishes when there is a small quantity of liquid left in the pan.
Seal the door.
Open the door, next morning and spread a piece of lint soaked in
ammonia on the table. This will neutralise excess of formalin present
in the room.
3.3
Washing of laboratory glasswares:
3.3.1 New glassware
Usually new glassware are slightly alkaline, Before washing these
have to be neutralized. The method is as follows:
o
o
o
Prepare a 2% solution of hydrochloric acid in a big basin.
Soak the new glassware in this solution for one day.
Rinse twice with clean water and once with
demineralized water and dry.
3.3.2 Dirty glassware:
o
o
o
Rinse twice in lukewarm or cold water otherwise serum or
blood may stick to them and may not be washed.
Put the glassware in a bowl containing detergent solution
and scrub the inside with a brush. After scrubbing soak
the glassware in this solution for 2-3 hours.
One by one, take out the articles and rinse under running
tap water, then put all the glassware in a container
containing tap water (no trace of detergent
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should be left otherwise this may lead to false results).
o
o
o
Drain the water by putting each articles on a wall
draining rack.
Place the articles in a wire basket and dry in a hot air
oven at 60°C.
Plug each article with non-absorbent cotton
wool or
aluminium foil and store in a cupboard to avoid dust.
3.3.3 Pipettes:
o
o
o
o
Immediately rinse in running tap water to remove
blood, urine, and serum reagent, etc.
If the pipettes were used for infected materials, soak them
in cylinder full of disinfectant solution (2% dettol
or 2% phenol) for 24 hours otherwise place in a
large measuring cylinder full of water.
Soak in detergent and rinse as in case of dirty glassware.
In case the pipettes are blocked put them in
dichromate solution for 24 hours. Next day clean under
running tap water, check individually, rinse for a
number of times or use pipette washer.
3.3.4 Syringes and needles:
o
o
o
Immediately after use remove the plunger and rinse the
barrel and plunger. Syringe water through the needle
forcefully. Finally remove the needle.
If the piston is blocked, either soak the syringe for 2
hours in hot water or pipette with the syringe standing on
its end, piston down. Alternatively soak the syringe in a
container of 10 vol hydrogen peroxide.
In case of block needles use the stylet to remove the
block.
3.4 Methods of sterilisation
The common methods of sterilisation used in a microbiology
laboratory can be broadly divided into three categories depending
upon the materials to be sterilised.
O
Dry heat.
Moist heat.
O
Filtration.
O
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3.4.1 Dry heat
The two commonly used methods of sterilisation by dry heat are.
•
•
Red heat or flaming
Hot air sterilisation.
Red heat or flaming
Instruments such as inoculating loops and sea5ing1iron\a^e,X11looD
bv this technique. For sterilisation of inoculating loop, hold the loop
vertically onX blue cone of the flame for few seconds and slowly
raise upwards till whole of the wire is red hot. Move the loopholde
rapidly downwards through the flame so that several inches of the
loop holder is also heated slightly.
Hot air sterilisation
Items to be sterilised:
This is the best method for sterilisation of dry glasswares such as test
tubes, flasks, pipettes, pertidishes, assembled all giass syringes
throat swabs and other sealed materials which can withstand high
temperature and where penetration by steamis not possible.
Sterilisation by hot air can be conveniently carried out in an
electrically heated oven.
A themostat is fitted to control the
temperature. Larger units should be fitted with an air circulating fan
•to ensure uniform temperature in the different parts of the oven.
Autoclaving
This is sterilization by mpoist heat under pressure and is used for
sterilization of bacteriological media, plastic wares etc. It has been
described in details subsequently.
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CHAPTER- 4
Microscopic Examination
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CHAPTER- 4
MICROSCOPIC EXLAMINATION
1'1!
In a peripheral laboratory, microscopic examination can provide rapid
and economical presumptive diagnosis which may have significant
bearing upon control and prevention strategies.
4.1
Cleaning and storage of microscope slides
4.1.1 Cleaning of new slides
o
o
o
o
Soak the slides in a vessel containing soap water
solution for a few hours.
Place the slides either in running tap water or
several changes of clean water for few hours.
The slides should be wiped dry using a dry, clean,*
lint-free cloth.
Always handle the cleaned slides by the edges to avoid
finger marks.
4.1.2 Cleaning of used slides
o
o
o
o
o
o
Soak the slides for at least 60 minutes in 1-2% hypo
chlorite solution.
Wash in hot soap water scrubbing both the sides with the
brush, taking particular care to wash only a few slides at
a time to prevent scratching.
Clean the slides individually with gauze or cotton
wool.
Transfer the slides to a fresh detergent solution.
Wash in running tap water or several changes of clean
water.
Wipe dry with a clean lint free cotton cloth.
4.1.3 Storage of Slides
o
o
4.2
Initially, after washing and cleaning, the slides should be
kept in a dry place or a warm air cabinet.
Thereafter slides should be stored in packages of 10 which
should be wrapped in thick paper and secured with
adhesive tape or rubber bands.
Microscopy for Pyogenic Meningitis
Pyogenic meningitis is an acute bacterial infection of the meninges,
commonly caused in epidemic form by Neisseria meningitidis,
Streptococcus pneumoniae and Haemophilus influenzae.
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For the laboratory confirmation of the diagnosis, the following clinical
specimen should be collected.
4.2.1 Cerebrospinal Fluid (CSF)
CSF following lumbar puncture should be collected in 3 separate
clean sterile containers (Bijou bottles) for following investigations:
O
O
O
Biochemical analysis
Cytological examination
Microbiological tests
General guidelines for collecting CSF
o
o
O
O
CSF should be collected before the start of
chemotherapy.
Never refrigerate or expose it to sunlight, and transport
immediately to the laboratory.
In case of delay in transportation, keep CSF at 37°C.
Other clinical samples which can be collected are blood
and petechial fluids.
4.2.2 Examination of CSF
Macroscopic:
Look for the presence of turbidity, blood or coagulum.
Cytology
Cytologic examination to be done only when there is no coagulum in
the CSF.
Material Required: Neubauer's counting chamber, WBC diluting
fluid, WBC pipette, compound microscope.
The cell count should be done by the usual procedure of WBC count
using a Neubauer's chamber and count the number of leukocytes per
cmm of fluid.
The normal CSF should be absolutely clear, free of any coagulum and
should not contain more than 0-8 lymphocytic cells/cmm.
In pyogenic meningitis, appearance of CSF is turbid and contains
more than 8-10 IcukocyLes/cmm, the cells being predominantly
polymorphs in nature.
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:!
Microbiological examination:
Microscopy
Microscopic examination is required to directly visualise the causative
organism in the CSF.
Requirements
Clean slides
Coverslips
Table top Centrifuge
Centrifuge tubes
Pasteur pipettes
Clean glass vials
Reagents of Gram's staining.
Rubber teats
Discarding jar
Neubauer Counting chamber
WBC pipette.
Procedure:
Transfer about 1-2 ml of CSF in a sterile Centrifuge tube.
Centrifuge at 3000 rpm for 5 minutes.
Keep the supernatant fluid for Latex Agglutination test.
From the deposit, make smears on 3 clean glass slides and air dry.
In case of a clotted CSF, transfer 3 small pieces of clot on three
different glass slides.
• Tease the clots using needles or wooden sticks or the edge of the
slide and after spreading make the smears. Air dry.
• In case of scanty CSF, several drops of CSF should be placed at
one particular spot on the slide, each being allowed to dry before
the next is added.
• Air dry and heat fix the smear.
• Stain the smears by Gram's staining method as given in Annexure.
• Examine under microscope under oil immersion.
•
•
•
•
•
Observations:
•
•
Presence of Gram negative bean shaped diplococci, both
intracellular and extracellular suggests the presence of Neisseria
meningitidis (Meningococcus).
Other organisms which can be seen are Streptococcus pneumoniae
(Pneumococcus), which appear as gram-positive diplococci,
Haemophilus influenzae which appear as gram negative thin
filaments rods.
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4.3
Diagnosis of Pulmonary tuberculosis by sputum
examination:
Tuberculosis is a disease of great public health importance caused by
Mycobacterium tuberculosis and some other species of Mycobacteria.
The diagnosis of pulmonary tuberculosis can be established by
demonstrating the bacillus in the sputum of the patient by
microscopy.
4.3.1 Sputum collection:
O
O
o
o
o
Collect the sample preferably early in the morning.
For optimum results, 3 consecutive days samples should
be tested.
In case sputum is scanty, a 24 hour collection may be
examined.
A nebulized and heated hypertonic saline may be used to induce
sputum production in patients unable to bring out the
sputum.
Sputum should be collected in a sterile wide mouthed container
with a tight lid.
The sample should be delivered to the laboratory with minimum delay.
Specimen that cannot be delivered or processed immediately should
be refrigerated at 4-8°C for a maximum of 3-4 days.
Materials required for sputum microscopy:
o
o
o
o
o
o
o
o
o
Properly collected sputum specimen
Wooden sticks
Clean glass slides
Spirit lamp/Bunsen Burner
Petri dish
Inoculation hood
Face masks
Reagents for Zeihl-Neelsen staining
Glass Rods, Plastic clay.
Procedure:
Preparation or the smear
o
o
In an inocuk.ition hood or in an isolated room, wearing a
face mask, transfer a
portion of the sputum to a petri dish.
Using a wooden stick, tease out a small portion of caseous,
purulent or bloody material and transfer it to a clean slide.
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o
o
Using the same wooden stick or an inocuting wire loop, spread
this material uniformly over a large area, covering at least two
thirds of the slide.
Air dry the slides and flame them immediately and stain
according to the Ziehl- Neelsen staining method as given
below:
4.3.2 Ziehl Neelsen Staining (Acid fast staining)
Requirements
O
O
O
O
()
Carbol fuchsin solution
20% sulfuric Acid
25% Alcohol
Loeffler's Methylene blue
Distilled water
Staining Procedure:
o
o
o
O
O
o
0
O
O
O
C)
Put the heat fixed smears onto a platform made using two
parallel glass rods over a wash basin.
Cover the slide with carbol fuchsin and heat the slide from
below until steam uses.
Allow the stain to act for 5-8 minutes with intermittent heating,
putting fresh carbol fuchsin on the slide time to time.
Care should be taken not to allow the stain to dry on the slide.
Wash the slide preferably using distilled water. (Do not use tap
water).
Cover the slide with 20% Sulfuric Acid, Wash the slide with
water after one minute. Pour more acid and continue
decolorisation till smear is just faint pink.
Wash the slide again with water.
Cover the slide with 25% Alcohol for 2 minutes.
Wash with water.
Counterstain with Loeffler's methylene blue for 30 seconds.
Wash the smear with tap water, air dry and examine under oil
immersion.
Observations:
Mycobacteria appear as bright pink, slender, slightly curved rods,
whereas the background tissue, cells and other organisms are stained
blue.
N o Le:
o
Staining jars should never be used as with a positive stained
slide, the bacilli may get detached from the slide and float
about in staining fluid later on sticking to the negative slides
21
C:\.IS\l)SD.(loc
o
O
and may give false positive results.
After examining a positive slide, take care to wipe the lens with
a clean tissue paper before examining the next slide.
Do not record the smear to be negative unless at least 200
microscopic fields have been thoroughly examined under oil
immersion objective.
4.4 Diagnosis of Plague
Plague is an ancient scourge of mankind, which is a bacterial disease
caused by Yersinia pestis. It is endemic in rodents and fleas. In man,
plague occurs mainly in three forms, bubonic, pneumonic and
scpticaemic.
The presumptive diagnosis
microscopic examination.
of
Plague
can
be
established
by
Sample collection:
O
O
o
o
o
o
o
o
Bubo aspirate: in bubonic plague should be collected.
Under all safety precautions collect bubo aspirate by
puncturing the bubo with a sterile hypodermic syringe
and exudate is withdrawn.
Sterilize the puncture site with tincture iodine.
10 ml or 20 ml syringe, armed with 18/19 guage needle and
a few ml of sterile saline drawn into the syringe, should
be used for aspiration of bubo aspirate.
Bubo is (hen punctured and suction applied.
If aspiration does not produce fluid, then saline is injected
into the bubo again and aspirated
again.
Transfer the exudate into a sterile container.
Label the container.
Transport to the laboratory' at 2-8°C.
4.4.1 Sputum Collection in Pneumonic Plague:
o
o
o
o
o
o
Collect the sputum sample in a sterile wide mouth
screw capped container.
Label the specimen
Transport the specimen to laboratory at 2-8°C.
In the Laboratory:
Make three smears out of the same portion of
exudate/sputum taking precautions not to form aerosols.
Air dry the smear.
Stain smears cither by Methylene blue/Gram
staining/Waysons stain.
90
4.4.2 Gram Stain:
Phis is a routine laboratory procedure used for examining specimens
suspected to contain bacteriologic agents.
Direct microscopic
examination of specimens and cultures can provide a rapid
presumptive diagnosis. Gram stain results, the shape of cell (cocci,
bacilli), the type of cell arrangement (single, chained, clustered)
visualized under light microscopy, can provide a quick assessment of
what the etiologic agent may be.
Principle:
The Gram stain forms the cornerstone of microscopic bacteriology. It
was described by Hans Christian Gram over 100 years ago. Crystal
violet (gentian violet) is the primary stain that will bind to the
peptidoglycan present in the cell walls of some bacterial cells. Iodine
is added as a mordant to fix the dye. If the cell wall does not contain
peptidoglycan then crystal violet is easily washed off with acid or
alcohol (decolorizer). /A secondary dye, safranin (counterstain), is
added after the decolorization step.. If
D the primary did not bind the
cells will easily adsorb safranin. Thus gram-positive cells; are purple,
while gram-negative cells are pink/red.
Requirements:
•
•
•
•
Crystal violet (0.5%)
Gram’s Iodine (1%)
Acetone (100%) or Ethanol (95%)
Safranine (0.5%)
Procedure:
o
o
o
Cover the slide with crystal violet solution and allow to act
for about 30 seconds.
Pour off stain and holding the slide at an angle
downwards pouron the iodine solution so that it
washes away the crystal violet ; cover the slide with fresh
iodine solution and allow to act for 1 minute.
Wash off the iodine with ethanol and treat with fresh
alcohol, tilt the slide from side to side until colour
ceases to come out of the preparation. This is
easily seen by holding the slide against a white
background.
Or
Decolorize with 100% acetone. First, tip off the iodine and
hold the slide at a steep slope. Then pour acetone over the
slide from its upper end , so as to cover its whole surface.
23
C:\JS\DSD.doc
o
O
Decolorization is very rapid and is usually complete in 2-3
second. After this period of contact, wash thoroughly with
water under a running tap.
Apply the counterstain (0.5% safranine) for 30 seconds.
Wash with water and blot dry
When to use this procedure and what you expect to see
Y.pestis appears as a fat, short, gram-negative coccobacilli about Ip
by 0.5p. Gram stains are typically done on cultures/subcultures,
bubo aspirates, spleen, liver and sputum smears.
Critical value/Action to be taken:
When gram stained material reveal small coccoid gram-negative
bacilli. Material should be further worked up with culture isolation
and identification. No notification is needed at this time.
Interpretation'.
Y. pestis appears as a fat short, gram negative coccobacilli about 1 p
by 0.5 p.
4.4.3 Wayson stain for visualizing Yersinia pestis:
Wayson stain is a polychromatic differential stain used as
presumptive test for the presence of Yersinia and Pasteurella spp.
a
Principle
Basic fuschin and methylene blue in the Wayson stain bind to
bacterial cells which appear under light microscopy as bipolar, closed
safety pin-shaped cells. The differential polychromatic mrophology
can be visualized with many differenttypes of organisms therefore
Wayson stain alone is not diagnostic for Y.pestis.
Critical values/Action to be taken:
When stained unknown material has a characteristic "safety pin"
morphology, it is Wayson stain positive. Further work-up by culture
isolation and identfiication must follow. No notification is needed
unless submittor specifically requests notification.
If Wayson bi-polar organisms known to have "safety pin” morphology
cannot be visualized after staining, check reagents and check for
possible technical problems.
Repeat stain until characteristic
morphological results arc obtained with control cultures.
2'1
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Matenals needed for this test:
Wayson stain;
o
o
Dissolve 0.2 grams of basic fuchsin and 0.75 grams of
methylene blue in 20 ml of 95% ethanol. Filter solution through
Whatman #1 paper (or equivalent).
Pour'dissolved, filtered stain into 200 ml of 5% aqueous phenol.
Store at room temperature. Avoid exposure to light.
Procedure:
o
o
O
O
O
Prepare smear of tissue or culture on slide, air dry.
Heat fix smear or fix in absolute methanol for 3 minutes, air dry
slide. (Methanol fixation yields more contrasting staining than
heat fixation).
Flood smear with Wayson stain for 5-10 seconds.
Wash slide in tap water, blot gently or air dry.
Examine slide under light microscope.
Interpretation:
Consistent, striking bipolar "safety pin" morphology of small, fat bacilli
are characteristic of the Yersinia and Pasteurella spp. Other bacteria
may exhibit bipolar appearance as well, especially if the specimen is
taken from areas with a wide variety of normal flora (nasal,
pharyngeal, and fecal).
"All Y.pestis arc Wayson positive, but all Wayson positive strains
arc not Y.pestis”.
Quality control measures:
Test each lot of Wayson stain using known Yersinia/Pasteurella spp.
(positive control) and with Escherichia coli or other enteric bacteria as
negative controls. When examining tissue smears, controls slide
prepared with plague bacilli infected and uninfected tissue smears
should also be examined.
4.4.4 Methylene Blue Staining;
Ma ferial reciuired:
o
o
Air dried smear.
Methylene blue stain.
25
C:\J.S\DSI ).doc
Procedure:
o
O
O
O
O
Fix the smear by dipping the slides in a jar
containing pure Methanol for 5 minutes.
Cover the smear with methylene blue stain.
Leave the stain for 3 minutes.
Wash with tap water. Air dry .
Examine under the oil immersion of microscope.
Observation:
Characteristic bluish bacilli showing
presence of Y.pestis organisms.
4.5
bipolar
staining.
Suggests
Malaria
Malaria is a parasitic disease caused by Plasmodium species. In India,
the disease is commonly caused by P.vivax and P.falciparum. The
laboratory diagnosis is based on demonstration of different stages of
the parasite in the peripheral blood film of the patient.
4.5.1 Collection o f sample:
Peripheral blood smear:Time for taking blood:
o
o
Collect blood either during or 2-3 hours after the peak of
temperature.
Sample should be taken before administration of antimalarial
drugs.
4.5.2 Preparation of blood smear.
Both thick and thin films should be made on the same slide.
Blood sample should be collected from the tip of the ring finger of the
left hand. However in small children, sample should be collected
either from the heal or the tip of the big toe of the foot taking all
aseptic precautions using a sterile needle or a lancet ( see also page....
under “Filter paper method”).
Apply gentle pressure to the finger and collect a single small drop of
blood on to the mdidle of the slide. This is for the thin film. Apply
further pressure to express more blood and collect 2 or 3 large drops
on the slide about 1 cm from the drop intended for the thin film. Wipe
the remaining blood away from the finger with cotton wool.
Thin film: Using another clean slide as a ’spreader' and with the slide
with the blood drops resling on a Hat firm surface, touch the small
drop with the speader and allow the blood to run along its edge.
C:\JS\DSD.doc
Firmly push the spreader alongwith the slide away from the largest
drop keeping the spreader at an angle at 45oC.
Make sure the
spreader is in even contact with the surface of the slide all the time
the blood is being spread.
Thick film: Always handle slides by the edges or by a corner to make
the thick film as follows:
Using the corner of the spreader, quickly join the larger drops of blood
and spread them to make an even thick film. The blood should not 1
be excessively stirred but can be spread in a circular or rectangular
form with 3-6 movements.
Allow the thick film to dry in a flat level position protected from flies,
dust and extreme heat . Label the dry film with a pen or marker
pencil , by writing across the thicker portion of the thin film the
patient’s name , or number and the date. Do not use a ball pen to
label the slide.
Wrap the dry slide in clean paper and despatch with the patient's
record form the laboratory as soon as possible.
The slide used for spreading the blood films must be disinfected and
should then be used for the next patient, another clean slide from the
pack being used as a spreader.
4.5.3 Staining of Blood smears:
GEIMSA STAIN
Materials and Reagents
1.
2.
3.
4.
5.
6.
Geimsa stain powder/Ready Giemsa Stain solution.
Alcohol
Methanol
Marking pen
Staining jars
Boric acid Borax buffer - pH 7.2.
Preparation:
o
Dissolve the stain powder in alcohol as per the
manufacturer's instructions.
o
Prepare Borax Acid - Boric buffer as below:
a) Dissolve 12.4 gms of Boric Acid in 1 lit. of Distilled
water (1)
b) Dissolve 19.05 gm Borax in 1 lit of Distilled water (II)
27
C:\IS\DSD doe
Take 50 ml of solution I and adjust the pH to 7.2 using appropriate
volume of solution II. Then make up the volume to 200 ml with
distilled water.
Staining Technique:
o
o
o
o
o
o
o
o
Prepare thick and thin smear from malaria case on a
glass slide.
Dehaemoglobinize the thick smear by placing the film in a
vertical position in a glass Jar containing distilled water
for 5 minutes. When film becomes white, take it out
and dry in upright position.
Fix the thin smear in methanol for 15 minutes.
Dilute the Giemsa's stain solution, one part with 9 parts
of Boric buffer pH 7.2.
Immerse the smears in this stain for 1 hour.
Wash the smears in buffer solution.
Blot and dry.
Examine the slide under oil immersion of microscope.
4.5.4 J.S.B. Stain
Materials and Reagents Required:
o
o
o
o
o
o
o
o
o
Eosin yellow (water soluble)
Methylene Blue
Potassium Dichromate
Di-sodium hydrogen phosphate (dihydrate)
1% sulphuric Acid.
Round bottom flask (2 lit.)
Healing mantle
Distilled water
Staining jars.
Preparation:
J.S.B. II
Dissolve 2 gms Eosin Yellow in 1 lit. of distilled water and store in the
dark for 4 weeks before use.
J.S.B.I
•
•
•
Dissolve 1 gm of Methylene blue in 600 ml of
distilled waler and mix well.
Add 1% sulphuric acid (6.0 ml) drop by drop and shake well.
Add 1 gm of potassium dichromate and shake well till precipitation
occurs.
2<S
C:\JS\DSD.tloc
•
•
•
•
®
Dissolve the precipitate by adding 7 gms. of Di-sodium hydrogen
phosphate di hydrate.
Make up the volume to 1 lit.
Boil the stain in round bottom flask over a heating mantle for one
hour.
Cool the stain and re-adjust the volume to 1 lit by adding distilled
water.
Store in dark for 4 weeks before use.
Staining technique:
•
•
®
•
•
•
•
•
•
Prepare thin and thick smears from malaria cases on micro slides.
De-haemoglobinise the thick smear.
Rix the thin smear in methanol for few minutes.
Take3 staining jars for U.S.B. I, J.S.B.II and tap water.
Dip the smears in J.S.B. II for few seconds and immediatedly wash
in water.
Drain the slides free of excess water.
Dip the smears in J.S.B.I for 30-40 seconds.
Wash well in water and dry.
Examine the smears under oil immersion.
4.5.5 Observation:
Examine thin film first. If no parasite is found then only examine
thick film. If parasites are seen in the thick film but the identity is not
clear, the thin film should be reexamined more thoroughly so as to
determine nature of infection.
Thin film examination:
o
o
c)
Area of the film examined should be along the upper and
lower margins of tail end film as parasites are concentrated over
there.
A minimum of 100 fields should be examined in about 8-10
minutes.
The following stages of the parasite can be observed in a
peripheral blood thin smear.
1. Ring, trophozoite, schizont and the gametocytes in case of
Plasmodium vivax.
2. The infected erythrocytes is usually enlarged in P. vivax
infection.
3. However, in case of P.falciparum infection, it is mainly the
ring stages which arc seen and occasionally schizonts and
trophozoites. During the Lite stages of the disease even crescent
shaped gametocytes can be seen in the peripheral blood.
29
C.\JS\PSD.doc
Observation on thick smear:
o
O
.o
Only elements seen are leucocytes andmalarial parasites.
Morphology of malarial parasites is distorted,
species of parasites cannot be identified.
Appearance in thick film
o
o
Trophozoites appear as streaks of blue cytoplasm with
detached nuclear dots. The ring forms rarely seen.
Schizonts and gametocytes, however, retain their normal
appearances (although the pigments are seen more clearly) are
seen if present in smear.
4.6
Examination of blood for Microfilaria
Filariasis is a (disease
”
of the lymphatics caused mainly by the
nematode Wucherena bancrofti and rarely by Brugia malayi.
Laboratory diagnosis:
This is based on the demonstration of the larval stages of the parasite
in the peripheral blood of the cases.
4.6.7 Collection of blood:
The blood should be preferably collected between 10 PM and 2 AM
specially in areas where microfilaria shows nocturnal periodicity.
4.6.2 Examination of unstained preparation:
•
•
•
•
Take 2-3 drops of blood on a clean glass slide. Put a coverslip on
it.
The rim is then smeared with vaseline to prevent drying up of the
blood.
Examine the slides under low power microscope immediately or
within 24 hours of ollection of blood.
Wriggling microfilaria present in the blood can be
seen.
4.6.3 Examination o f stained smear:
Thick film:
film as per the instruction given in the
•
Prepare a thick blood
Chapter on Malaria.
•
Dehemoglobinise the smear by
containing water.
Air dry
Fix the smear with methyl alcohol.
•
•
30
putting
the
slides
in
a jar
C:US\DSI).doc
•
•
Stain with Geima's stain as described earlier.
Examine the smear under the oil immersion of the
microscope.
Thin film:
o
o
o
Prepare as described for malaria.
Fix it with Methanol by dipping the smear in ajar
containing methanol for 15 minutes.
Stain it with Giemsa stain as described earlier.
Observation:
Microfilaria of Wuchereria bancrofti are seen.
o
o
o
o
o
Size - 290 p in length and 6-7 p in breadth.
It has blunt head, pointed tail and has smooth curves.
Structureless sack called Hyaline Sheath is seen where it
projects beyond the extremities of embryo.
Somatic cells/nuclei seen as granules in central axis from head
to tail end except the terminal 5 percent area. At the anterior
end there is a space devoid of granules called cephalic space.
The granules are broken at definite space serving as the
landmarks for identification of the species.
Nerve ring, an oblique space.
Antereior V spot, represents the rudimentary excretory
system.
Posterior V spot or tail spot, represents the terminal part
of the alimentary canal.
Microfilaria of Bruqia malai.
o
o
o
o
o
Smaller than Wucheraria bancrofti (230 p x 6 p)
Possess secondary kinks instead of smooth curved.
Cephalic space is broader.
Tail tip is not free of nuclei and nuclei are blurred.
It lies folded with head close to tail.
06L813l
u
C:US\DSI).<loc
CHAPTER- 5
Serological Tests
C:\.IS\l)SD.cloc
A
SEROLOGICAL TESTS:
5.1
LATEX AGGLUTINATION TEST FOR MENINGITIS
The ideal immunological test, which is also a rapid test and easy to
perform in a district laboratory, is the latex agglutination (LA) test,
test is done to detect the bacterial antigen (Capsular polysaccharide)
in CSF samples collected from patients.
The available cornercial kits are designed to provide diagnosis for
meningitis caused by:o
o
o
o
TV. meningitidis serogroup A
TV. meningitidis serogroup C
Streptococcus pneumoniae
Haemophilus influenzae type b
The general procedure for performance of the test is givenl below,
however, the laboratory personnel are advised to go through the
instructions provided by the kit manufacturer, carefully, and strictly
adhere to the same.
5.1.1 Equipments required: (but not supplied with the kit)
o
Pasteur pipettes (sterile)
o
Rubber teats
o
Container with disinfectants (for discard)
5.1.2 Procedure
o
o
o
o
o
o
Systematically heat all CSF specimens for 5 minutes at
80-100”C.
Centrifuge the CSF samples at 2000 rpm for 10 minutes,
preserve the supernatant for further use.
Shake each latex suspension well.
In the corresponding fields of the slide, dispense one drop
of each of the latex suspension followed by one
drop of the CSF supernatant.
Mix with a stirring stick; use separate stick for each
combination of CSF and latex suspension.
Rotate the slide, and read within 2 minutes.
32
C:\JS\DSD.doc
5.1.3 Controls:
Periodically check:
a)
thatnone of the four latex reagents agglutinate in presence of
0.15 mol/1 NaCl solution.
b)
that each of the four latex reagents do agglutinate with positive
control.
5.1.4 Reading:
o
o
Negative reaction: The CSF latex suspension mixture remains a
"milky suspension" (disregard any granules that may occur with
S.pneumonae).
Positive reaction: Distinct rapid agglutination occuring within 2
minutes (normally 30 seconds).
5.1.5 Interpretation:
Aglutination with one of the latex reagents indicates presence of the
corresponding antigen in the CSF sample.
Advantages of LA test:
o
o
o
o
o
Most sensitive method available
Rapid
Good field applicability
Can diagnose the disease even in antibiotic treated
patients.
No special equipment/instrument required.
Disadvantages of LA test:
o
Commercial kits not produced in India; to be imported.
o
Expensive
o
The test does not yield any bacterial isolate; other
parameters cannot be tested.
5.2 DIAGNOSIS OF HEPATITIS B VIRAL INFECTION:
Diagnosis of Hepatitis B viral infection is very important, not only in
case of chronic hepatitis and liver cirrhosis patients, but also in the
screening of donor blood samples, to ensure safe blood transfusion
and to control or check the spread of hepatitis B infection through
unsafe blood Iransfnsion.This is achieved by detection/dernonstration
of "Hepatitis-B surface Antigen" (HBsAg) or the 'Australia Antigen' in
the patient/donor blood samples.
33
C:\.IS\l)SD.<loc
A simple latex agglutination test for rapid detection of HBsAg, which is
very much feasible in the district laboratories, is described below:
LATEX AGGLUTINATION TEST FOR RAPID DETECTION
OF HBsAg (AUSTRALIA ANTIGEN)
5.2.1 PRINCIPLE:
A distinct agglutination occurs, when serum sample containing HBsAg
is mixed with latex particles coated with purified and highly reactive
anti-HBsAg antibodies; there would be no agglutination when the
serum sample does not contain HBsAg”.
5.2.2 MATERIALS AND REAGENTS:
Commercial kits for this test are available in India, They contain the
following reagents and accessories.
Reagent 1: HBsAg Latex Reagent
Reagent 2: Positive control serum
Reagent 3: Negative control serum
Accessories:Disposable plastic slides
Disposable applicator sticks
Disposable plastic droppers
Rubber teats.
- 1 vial
- 1 vial
- 1 vial
All the reagents arc stable and active, till the expiry date mentioned,
provided they are stored in a refrigerator at 2-8(,C. Do not freeze the
reagents. •
5.2.3 SPECIMEN:
o
o
o
The test is performed on serum harvested from the
patient's/donor's blood.
Do not heat inactivate the test or the control sera
samples.
If delay in testing, store test sera samples in a refrigerator
or deep freezer, takingcare to avoid repeated freezing
and thawing of the specimens.
5.2.4 TEST PROCEDURE
o
o
Allow the reagents to attain room temperature, and shake
the vials gently to make sure that the latex reagent is
completely in suspension.
Place one drop (50 /til) of undiluted scrum in one of the
circles on the slide. More circles to be filled if more than
one test sera samples are to be tested. Use separate
droppers for each specimen.
34
C:\JS\DSD.<loc
o
o
o
Add one drop (50 /ul) of latex reagent on to each
specimen drop in circles, using a disposable dropper.
Mix the content of each circle, using separate disposable
applicator sticks for each circle, and spread the mixture
uniformly over the entire area of the circle.
Rock the slide gently, to and fro, for 5 minutes, and watch
for agglutination.
Precautions:
i.
2.
3.
To avoid contamination of reagents, make sure that the cap of
each vial is properly and promptly applied to the same vial.
Interchanging of caps and droppers lead to contamination and
erroneous results.
Improper mixing and interchange of applicator sticks also lead
to erroneous results.
Vigourous rocking of slides may lead to impaired agglutination.
Use of Controls:
Positive and negative controls are not always
required, when reagents are in continuous use. However, the
performance of the kits needs checking, occasionally, using the
controls.
5.2.5 Interpretation:
o
o
Visible agglutination < 5 minutes
No agglutination
-HBsAg Positive
-HBsAg Negative
5.2.6 LIMITATIONS:
o
o
Probability of FALSE POSITIVITY - 1% of all samples, due
to presence of other antigens (RF).
FALSE NEGATIVE results may be encountered with
specimens containing very high titres of HBsAg (Prozone
effect). In such cases the characteristic syndrome (severe
jaundice, GPT/GOT elevation) will be apparent. In that
case repeat the test after diluting the specimen 1:40, with
normal saline.
5.3 VDRL SLIDE FLOCCULATION TEST FOR SYPHILIS:
This is a test with high sensitivity and specificity and can. be used for
rapid and exact quantitative titration of the reactive sera samples.
5.3.1 PRINCIPLE
The VDRL antigen particles, which are seen as small fusiform needles
under the microscope, floculate into clumps (small, medium and
large), when (hey comcin contact with a reactive (+ve) scrum.
35
(.•.'JSXDSDxktc
5.3.2 MATERIALS:
VDRL Antigen;
It consists of a mixture of Cardiolipin, lecithin and cholesterol in
definite proportions and is commercially available. Each sealed glass
ampoule contains 0.5 ml (with sufficient excess for convenient
withdrawal). Antigen amouples should be stored in a cool, dark place.
Ampoules showing precipitate should be discarded.
Buffered Saline Solution:
10 ampoules containing 5 ml each are supplied with each package of
VDRL antigen. P
Buffered saline is required for preparing the antigen
emulsion foi' the test.
SLIDES:
Glass slides, SPyS", with 12 paraffin rings of 14 mm inner diameter are
used for the test. Slides of same size, with permanently fixed ceramic
rings arc also available commercially and may be used, 'flic following
points regarding the slides are to be noted.
o
o
o
o
New slides, as well as the used slides should be cleaned
thoroughly.
Slides should be handled by the edges only, to avoid any
greasy finger prints.
Scrum within the circles will spread evenly, within the
rings, only if the slides are absolurtely clean.
Parafin rings can be made on slides by transferring
molten paraffin on to slide using a suitable mould or
threaded wire rings.
5.3.3 PROCEDURE
A.
Preparation o f serum:.
o
o
Inactivate serum by heating at 56°C for 30 minutes.
On removal from water bath, centrifuge the serum
sample if it shows particulate debris.
Test sera sample need to be reheated (at 56°C for 10 min.),
if they are >4 hr. old since original inactivation.
0.05 ml of each sample is required for testing.
o
o
B.
PREPARATION OP ANTIGEN EMULSION:
o
Pipette out 0.4 ml of buffered saline on to the
bottom of
a 1 ox.reagent bottle with flat or concave inner bottom
su rfacc.
36
C:\.IS\l)SD.<loc
o
o
o
o
o
o
o
o
o
o
Add 0.5 ml of VDRL antigen, drawn out from an ampoule,
using a graduated pipette, directly on to saline in the
reagent bottle, while rotating the bottle on a flat surface.
The antigen should be added drop by drop, but rapidly, so
that it takes approximately 6 seconds to complete the
delivery of antigen.
Blow the last drop of the antigen and continue rotation of
the bottle for 10 more seconds.
Add 4.1 ml. of buffered saline, using a graduated 5 ml.
pipette.
Stopper the bottle and shake it vigorously for about 10
seconds.
Take care to sec that the temperature of buffered saline
solution and that of VDRL antigen is maintained within
the range of 23-29°C, during preparation of the antigen
emulsion.
Maturation of antigen is important for increased
sensitivity, maturation is complete in 15-30 minutes, after
preparation.
Store the antigen emulsion in a refrigerator, if necessary.
It should be brought to room temperature and shaken
gently before use.
5.0 ml of antigen emulsion would suffice for 250 serum
tests.
Each batch of antigen emulsion prepared must be pre
tested with known ractive and non-reactive sera samples,
in order lo confirm that exact pattern of distribution of
antigen particles, typical of reactive and non-reactive sera
samples, would result on testing.
5.3.4 TEST PROCEDURE:
o
Qualitative Test:
*
*
Pipette out 0.05ml of inactivated serum into one
paraffin/ceramic ring on the glass slide; serum should
spread.
Add one drop (1/60 ml) of antigen emulsion on the serum
within the ring.
Rotate the slide for 4 minutes, by hand on a flat surface (+
or -120 times per minute covering circle of 2"dia.)
5.3.5 READING AND REPORTING OF RESULTS:
O
o
o
Read I ho test results immediately after rotation.
Observe the slide under micrscope, using low power
objective( 100 x magnification)
Antigen particles appear as small fusiform needles, they
arc more or less evenly spread in case of a non-reactive
37
C:US\l)SI)<loc
serum sample, and aggregated into clumps (flocculation)
in the case of a reactive serum. Grade the observations as
under:
No clumps or very slight roughness
Small clumps
Medium and large clumps
NON-REACTIVE (N)
WEAKLY REACTIVE (W)
REACTIVE (R)
Zone reactions are possible; they are recognizable by irregular
clumping. The clumps arc not compact and very small and large
clumps may be seen within the same microscopic field. In such cases,
the results are reported on the basis of quantitative test done on the
same serum.
o
Quantitative test:
Quantitative test is performed on all positive (reactive) serum samples
and on samples which show weak(W) or "rough" reaction in the
qualitative tests.
■k
*
*
Prepare successive two-fold dilution (1:1, 1:2, 1:4, 1:8,
1:16,1:64 etc.) of serum sample to be tested, lising 0.9% saline.
Each serum dilution sample thus prepared is treated as an
individual sample and tested as described under "qualitative"
test.
Results are read and graded under the microscope as before.
Reporting of results:
Results are reported in terms of the highest dilution of the serum that
produces a definite positive (or Reactive, R) reaction as below. Weaklyreactive is not acceptable.
Serum dilution
1:1
1:2
1:4
1:8
1:16 1:32 1:64 Report
R
R
R
W
N
W
W
W
R
R
W
W
N
W
N
W
R
R
R
N
N
N
N
W
R
R
N
N
N
N
N
W
R
N
N
3K
N
N
N
N
N
N
N
N
N
N
N
N
N
N
R1 dil
R2 dils.
R4 dils.
R8 dils.
R16 dils
WO dils.
R1 dils.
(. :\.IS\l)SI).cloc
5.4 RAPID PLASMA REAGIN (RPR) TEST FOR
DIAGNOSIS OF SYPHILIS:
This test detects antibodies formed, in the blood of syphilitic patients,
against Cardiolipin.
These antibodies are called "Reagin".
Two
advantages of this test over the previously described VDRL Slide
flocculation test are - (a) It does not require a microscope to read the
test results; (b) The test sera/plasma sample need not be inactivated
prior to testing.
5.4.1 PRINCIPLE
"Reagin formed in the blood of syphilitic patients cause flocculation of
the antigen, which co-agglutinates with the charcoal particles, giving
small black clumps that are readily visible without a microscope".
Commercial "Rapitest" kits, designed for carrying out 50 tests per kit,
are available in India.
5.4.2 REAGENTS AND MATERIALS.
Provided in the Kit
RPR Antigen
Positive Control serum
Negative control serum
RPR Antigen dropper
Specimen droppers (disposable)
Rubber Teats
Mixing slicks (disposable)
Plastic test cards
1 vial
1 vial
1 vial
1
9
Materials required, but not provided in the kit:
Micropipette (capable of delivering 0.05 ml of test
sample)
Stop watch
HI, I /I Salipe solution (Q.9%) Only for quantitative test.
i .hHH V/1 Conjtpinerpvith c^sinfepitant (for discard)
■
I-M
H
r':
P
:
b
N Thet/PPR^tigen>{and ^pntrql;sera.will remain stable and
rYf’ til^the ^jxpiry date .printed on,,(he label, provided they are
r " '.stored in a refrigerator between 2-8bC. They should not be frozen.
i
5.4.3 THE SPECIMENS:
Serum:
o
o
Plasma:
o
o
o
o
Use fresh serum harvested from patient's blood sample.
If the test cannot be conducted immediately due to some
reason, store the serum sample between 2-8°C in a
refrigerator, BUT NOT LONGER THAN 48 hr., after
collection.
Collect patient's blood into a tube/vial containing one of
the anticoagulants (EDTA, Heparin, Oxalate, Sodium
Flouride etc.) Avoid excess of coagulant.
Centrifuge the blood sample, to separate the cells.
Use the plasma sample within 18 hr. of collection.
Inactivation of serum/plasma samples is not necessary.
PRECAUTIONS:
o
o
o
Blood samples should be collected from fasting patients,
since very lipaemic samples may give false +ve reactions.
Do not use grossly haemolysed samples.
Discard contaminated samples.
5.4.4 TEST PROCEDURE
A.
Qualitative test:
o
o
o
o
o
C)
o
Allow all reagents to attain room temperature.
Place one drop of (0.05 ml) test serum or plasma, positive
control and negative control sera on to separate circles
on the plastic test card, using disposable specimen
droppers provided.
Shake the RPR antigen suspension gently, to resuspend
the particles.
Place one drop (0.015-0.02 ml) of the antigen suspension,
on each of the circles containing test samples and the
positive and negative control sera drops, using the antigen
dropper provided.
Mix the contents of each circle, using the disposable
mixing sticks provided, and spreading the reagent mixture
over the entire area of the circle.
Grnlly rock the card, Io and fro, for 6 minutes, either
manually or on a mechanical shaker at 100 rpm, to
ensure thorough mixing.
Read the results at the end of 6 minutes, using a high
intensity light source.
40
C:\JS\DSD.<loc
Interpretation of ResuIts:
POSITIVE (REACTIVE)
Development of clearly visible clumps of
black particles, within the test circles.
NEGATIVE
No
development
of clumps,
the
charcoal particles remain in a (NON
REACTIVE) HOMOGENEOUS
GREY
SUSPENSION.
A quantitative estimation is further recommended for all samples
positive in the qualitative test.
H
B.
Quantitative testo
o
o
o
o
o
Dispense 0.05 ml (50 pl) of saline solution on to each of
the circles (No. 1-5) on the test card, using a micropipette.
Dispense 0.05 ml (50pl) of the specimen (test
serum/plasma) onto circle 1 and mix the two (saline and
test sample) thoroughly by drawing the mixture into the
micropipette, up and down several times.
Transfer 0.05 ml (50j.il) of the mixture in Circle-1, on to
the drop of saline in Circle-2. Repeat the mixing action,
several times, as explained above.
Repeat transferring and mixing actions from Circle-2
through circle-5.
Discard 0.05 ml (50 j.tl) from Circle-5, after mixing.
The dilutions of specimen obtained in different circles on
the test are as under:
CIRCLE
1
2
3
4
SALINE (ml)
0.05
0.05
0.05
0.05
0.05
5
SPECIMEN
0.05
(Serum/Plasma ml)
MIX &
TRANSFER
0.05
0.05
0.05
0.05
0.05
DILUTION
1:2
1:4
1:8
1:16
1:32
o
Using the disposable mixing sticks, spread the specimen
dilutions in the circles to cover the area of the circle.
41
C:\JS\l>SD.<loc
o
o
o
Start with circle 5 and end with Circle 1. Wipe the sticks
clean between
circles.
Gently shake the RPR antigen vial to resuspend the
particles, and add one drop (0.15-0.20 ml.) of antigen, on
to each circle, using the antigen dropper.
Gently rock the card to and fro for 6 minutes (manually or
or ;
on a mechanical shaker), to ensure thorough mixing.
Read results at the end of 6 minutes, as described above !
under Qualitative testing.
5.4.5 Interpretation:
The highest dilution of the sample, giving a definite positive reaction
is considered as the Litre of the specimen. In case the titre exceeds
1:32, continue with double dilutions beyond that point,till the titre is
obtained.
LIMITATIONS OF THE TESTS (RPR and VDRL Slide Flocculation)-.
Both these tests are considered as "non-treponemal antibody tests",
which are primarily meant as screening tests. If the tests are positive
when there is no clinical evidence of syphilis, they must be repeated; if
positivity persists, verifications by more specific tests (for antiTreponemal antibody) would be necessary to confirm syphilis. In RPR
and VDRL slide flocculation tests, false positive results may be
obtained in diseases such as leprosy, malaria, toxoplasmosis,
infectious mononucleosis and lupus erythematosUs, and also in
specimens having bacterial contamination.
5.5
WIDAL TEST FOR DIAGNOSIS OF ENTERIC FEVER:
(TYPHOID AND PARATYPHOID)
Widal test is an agglutination test for detection of antibodies against
Salmonella typhi and Salmonella paratyphi, the common causal agents
of enteric fevers.
5.5.1
PRINCIPLE:
“When serum sample containing antibodies against S.typhi and
S.paratyphi AB are mixed with respective antigens, agglutination will
take place”.
In S.typhi and S.paratyphi AB, two types of antigens are recognised as
diagnostically important:
(a) 'O' antigen or "Somatic' antigen.
(b) 'H' antigen or 'Flagellar'antigen.
42
C:\J.S\DSD.doc
O antigens of various species have components in common and '
hence only one 'O'antigcn i.e. that of S.typhi is employed; the 'H' :
antigens of Salmonella spp. are species specific, and hence the 'H'
antigens of all three, viz. S.typhi, S.paratyphi A and S.paratyphi B, are
employed in the test.
J
Commercial test kits for WIDAL test are available in India, and using
them both quantitative and quantative tests can be put up on
suspected sera samples.
5.5.2
MATERIALS AND REAGENTS:
Test kit contains the following reagents and materials
Reagent 1: S.typhi ('H')
Reagent 2: S.typhi (’O')
Reagent 3: S.paratyphi A ('H') Reagent 4: S.paratyphi B ('H') Reagent 5: Positive control
Glass slide
Product Insert
Materials required, but not supplied in the kit:
Small, dry and clean glass tubes
(for quantitative tube test)
Normal saline solution
Water bath
Micropipette/dropper
5 ml
5 ml
5 ml
5 ml
1 ml
1 No.
1 No.
8/specimen
5.5.3 SPECIMEN:
Fresh serum (patient) free from contamination should be
be used.
used. In
case of delay in testing, store the sera samples at 2-8°C in a
refrigerator.
Note:
•
•
•
Specimen is used undiluted.
Do not use haemolysed specimen.
Do not heat or inactivate the specimen.
5.5.4 TEST PROCEDURE
A.
Qualitative slide test for screening
o
Clean the glass slide provided and wipe it dry.
43
C.USMXSD.doc
o
o
o
o
o
Place a drop of undiluted serum sample to be tested in
each of the first four circles.
Add one drop of Reagent 1, Reagent-2, Reagent-3 and
Reagent 4, on to the specimen drop in Circles 1-4
respectively.
1
Mix the contents of each circle with separate mixing
sticks, and spread the mixture to cover the whole circle.
Rock the slide gently for 1 minute.
Read the results at the end of one minute.
Interpretation:
A positive reaction shows agglutination, visible to naked eye, in the i
respective circle,
Then proceed for quantitative slide test or :
quantitative tube test for the appropriate antigen.
B.
Quantitative slide test:
o
Clean the glass slide supplied in the kit and
as follows:
Circle
No.
Serum
volume
1.
2
3
4
5
0.08 ml
0.04 ml
0.02 ml
0.01 ml
0.005 ml
o
o
proceed
Appropriate
antigen
Titre
1 drop
1 drop
1 drop
1 drop
1 drop
1:20
1:40
1:80
1:160
1:320
Mix the contents of each circle, starting with circle 5 and
through Circle-1, wiping the mixing stick clean between
circles.
Rotate the slide for one minute and observe for
agglutination.
Interpretation:
Titre of the serum is the highest dilution
positive reaction.
C.
of the serum giving a
Quantitative tube test:
o
o
Take a set of 8 clean glass tubes, per specimen, per
antigen.
Prepare dilutions of serum specimen and add appropriate
antigen as below:
44
C:\JS\DSD.doc
TUBE
Serum
dilution
Normal
saline
Patient
serum
Transfer
diluted
serum
1___
1:20
2___
1:40
3___
1:80
4____
1:160
5____
1:320
6____ _
1:640
7______
1:1280
1.9ml
1.0ml
1.0ml
1.0ml
1.0ml
1.0ml
1.0 ml
1ml
1 ml
1 ml
1 ml
1 ml
1 ml
w
II
1 drop
1 drop
8______
Saline
control
1.0 ml
0;lml
II
1
(discard-
II
II
Appropr
iate
antigen
o
o
o
o
1 drop
1 drop
1 drop
1 drop
1 drop
1 drop
Mix well and incubate at 37oC for 16-20 hr. and observe
for agglutination.
Repeat steps (ii) and (iii) with all antigens which showed
agglutination in the screening test.
Note the highest dilution showing clearly visible
agglutination with naked eye.
'O’ antigen shows granular agglutination.
'H antigen shows flocular appearance.
Saline control should remain unchanged as it is a
negative control.
Interpretation:
Agglutination titre of >1:80 is suggestive
of infection.
Factors affecting WIDAL Test:
Effect of antibiotic administration:
There is evidence that early treatment with antibiotics
suppresses the antiboduy response by suppressing the
multiplication of organisms. This may result in a low titre in
WIDAL test.
Effect of past infection or typhoid vaccination:
It has been seen that th ‘H’ antibodies persist for a long time
upto many years after typhoid vaccination. Also, many years '
after recovering Irom enteric fever, any grave negative bacterial
infection can trigger a Salmonella ‘H’ antibody production,
thereby giving a false positive result in WDAL test.
45
C:\JS\DSD.doc
Time of collection of blood sample:
This is a very important parameter affecting the results of the
WIDAL test. A single blood sample collected during the first
week of the illness may give a negative WIDAL result, whereas i
in the same patient, a sample collected during the third week of ■
illness may show a very high titre. Accordingly, paired samples
should be collected; the first sample being taken as early as
possible and the second, 10-14 days later, for optimum results.
46
C\JS\DSD.doc
CHAPTER- 6
Bacteriological Analysis of Water
C:\JS\DSD.doc
CHAPTER- 6
BACTERIOLOGICAL ANALYSIS OF WATERS!
Although it is not possible to lay down fixed standards, as various
types of water are examined, from a public health point of view it is
generally sufficient to say that no faecal contamination has occured.
Coliform bacteria present in water may not be harmful, but they!
indicate that water supply is contaminated with faecal matter and:
water is, therefore, liable to contamination with more dangerous'
organisms. The coliform bacilli of human origin are the most reliable
indicators of faecal pollution.
The method of quantitative test for all coliform bacilli known as
the 'presumptive coliform count is described below.
6• 1
Collection of specimen
Collect water in presterilized bottles of 230 ml capacity with ground
glass stoppers, having an over hanging rim. Sterilise the bottles by
autoclaving.
Tap water:
When water is taken from tap, flame the mouth of the tap and. allow
the water to run for five minutes before filling the bottle.
Stream, river and lake water
Insert the bottle with its mouth closed with the stopper, a foot below
the surface of water and fill with water. Bring the bottle to the surface
and replace the stopper. Avoid the collection of surface water as it
contains organic matter.
Precautions:
o
o
o
During collection of water, avoid the contamination of the
sample.
Test the water samples as soon as possible after collection. If
delay of more than 3 hours is expected, pack the water sample
in ice for transport to laboratory.
When sampling chlorinalcd water, add a quantity of sodium
thiosulphate to the sample bottle before sterilising. This will
neutralize the chlorine present in the water.
47
C:\JS\D.SD.doc
Presumptive Coliform count:
Requirements:
•
•
•
•
•
•
Sample of water
Sterilized test tubes.
Quarter strength Ringer solution.
1 ml and 50 ml pipettes.
Double strength MacConkey's fluid medium.
Single strength MacConkey's fluid medium.
■ i
I '
i
Method:
o
o
o
o
o
o
Invert the water sample 25 times to mix.
Flame the mouth of the bottle and discard 1/3 of the
contents and mix thoroughly,
Using sterile graduated pipettes, the following amounts of
water are added.
One 50 ml quantity of water to 50 ml double strength
MacConkey medium in a flask.
Five ^10 ml quantity each to 10 ml double strength
MacConkey medium in test:
tubes.
Five 1 ]ml quantities each to 5 ml single strength
MacConkey medium.
Incubate all tubes at 37°C for 18-24 hours.
;
All tubes showing acid and gas are regarded as !
presumptive positives. Reincubate negatives for further
24 hrs.
Using McGrady's statistical tables the probable number 1
of coliform organisms present in 100 ml of sample can
be calculated.
Interpretation:
Water samples are classified based on the presumptive count in the
following way:
Class
Preumptive coliform count 100 ml.
1. Excellent
2. Satisfactory
3. Suspicious
4. Unsatisfactory
0 '
1-3
4-10
>10
Faecal Coliform Count:
From the tubes showing acid and gas in presumptive coliform count,
subculture into fresh single strength MacConkey's broth or Incubate
48
C:\JS\DSD.doc
at 44 C in a water bath. Tubes showing both acid and gas should be
taken as positive for Faecal coliform. Using McGrady's tables compute
the number of faecal coliform as in presumptive test. Water showing
even one faecal coliform is unfit for human consumption.
Most Probable Number (MPN) values/100 ml of sample, for a set of i
tests of one 50 ml, five 10 ml, and five 1 ml volumes. (McGrady’s
Statistical Table)
I
No. Of tubes giving positive reactions
1x50 ml
0_____
0_____
0_____
0_____
0_____
5x10 ml
0
0
0
_1_____
_1_____
0_____
0_____
0_____
0_____
0
0_____
0_____
_1_____
J___
j_____
_i_____
i_____
i_____
i_____
i
1
I_____
1
J___
0____
0 ___
_1____
2 ____
3 ____
0____
1 ___
2 ___
3 ____
0____
_1____
2 ____
3 ___
0____
1 ___
2 ____
3 ____
4 ____
0____
2____
__
J____
J3____
1 ___
2____
0 ___
_1____
2____
0 '___
_1____
2____
0____
2_____
2_____
2 _____
3 _____
3_____
4
0
0_____
0_____
0_____
_l_____
'1_____
1
2____
1_____
1_____
1_____
1
1______
1______
1______
1______
1______
5x1 ml
2
2_____
2 _____
2
3 _____
3_____
3_____
3_____
3 _____
4 _____
4_____
4
4_____
4
J___
2 ____
3 ____
4
49
MPN/100 ml
<1_________
1_________
2_________
1_________
2 _________
3 __________
2 __________
3 __________
4 __________
3
5 __________
5 __________
_1__________
3 __________
4 __________
6 __________
3__________
5
7 '_________
9 __________
5 __________
7 __________
10 _________
12 _________
8
____
11 _________
14_________
18___ _
21 _________
13 _________
17_________
22 _________
28_________
35
C:US\I)SI> cloc
1
4
5
5
J.
1
5
0
1
5 ■
1
1
6.1
2
3
4
5
5
5
5
43__
24__
35__
54
92__
161
>180
H?S-Strip method:
In recent years a simple, reliable and easy-to-perform (by even
untrained personell), Yes-No’ test for bacteriological quality of water
has been devised. This test, which is currently under field evaluation
and quality standardization is expected to be adopted as the field test
for water quality monitoring in the hands of peripheral health workers '
and community participants.
Principle:
Presence of coliform bacteria in drinking water is associated with
hydrogen sulphide (H2S)- producing organisms, and faecal pollution of
water can be established by demonstration of H2S production.
It has been claimed, by various workers, that the H2S-strip method
shows 80% agreement with the conventional MPN test described
above.
Description of the test device (kity
It simply consists of a pre-calibrated 20 ml glass bottle (McCartney
bottle) with a screw-cap lid, from which a strip of specially
treated/coated tissue paper hangs down, internally. The whole system
is sterile and needs to be opened at the time of water testing.
The paper strip inside the glass bottle (80 cm2,folded) is pre-soaked in j
a concenti ated medium containing peptone (20g), dipotassium i
hydrogen phosphate (1.5g), ferric ammonium citrate (0.75g), sodium
thiosulphate (1g), Teepol (1ml) and water (50 ml); 1 ml of the
concentrated medium is absorbed on to the folded tissue paper strip
and dried at 50° C under sterile conditions. It is then introduced into
the sterile bottle.
Test procedure:
o
Pour the water sample to be tested for faecal pollution
into the bottle, upto the precalibrated level (20 ml).
50
C:\J S\DSD.doc
o
o
Incubate at 37°C or allow to stand at ambient
temperature (30-37°C); no incubator is necessary under
field conditions, as the bottles can be held in the pockets
and body temperature can be made use of.
Faecal pollution is indicated if the contents of the bottle
turn black.
Advantages of FhS-Strip Test:
o
o
o
o
o
No need to measure the volume of water to be tested;
No need to dechlorinate the water sample, since it
instantaneously dechlorinates thr sample;
The end point (reading) is very clear, due to development
of black colour;
No incubator is necessary;
The test starts immediately on collection into the bottle,
unlike other methods which start after the sample is
transported to the laboratory.
’
■
LIBRARY
/
AND
OOCUMhNTATION
UNIT
C:\JS\DSD.doc
51
J)I.S-3/0
06181
CHAPTER- 7
Laboratory Diagnosis of Cholera
C:\JS\DSD.doc
CHAPTER- 7
LABORATORY DIAGNOSIS OF CHOLERA:
Cholera is characterized by sudden onset of effortless and profuse
watery diarrhoea. The watery stools with flakes of mucus and sweet
fishy odour are characteristic of cholera. These are also popularly
known as rice water stool. Cholera is caused by the organism by the
name of Vibrio cholerae which are Gram negative usually curved by
shape of coma and motile by a single polar flagellum. They are
oxidase positive.
The laboratory diagnosis is based on demonstration of Vibrio cholerae
in the stool specimen.
7.1
Collection of samples
Materials required
o
o
o
o
Wide mouth container
Swabs sticks (sterile)
Carry blair transport medium
Case investigation form.
Collection of Stool:
Voided stool
o
o
o
o
Most preferred specimen if available.
Should be collected before antibacterial therapy.
Should not be collected from bed pan so as to avoid
interference from outside bacteria or disinfectant used to
clean bed pan.
Patient may be instructed to void stool in wide mouth
container e.g. ice cream cup and transfer 3-5 gm. stool
into a sterile screw cap bottle.
Rectal swabs:
Whenever it is not possible to collect stool, a rectal swab specimen
may be collected. It is a very useful and convenient sample under
field condition and in cases of young babies. By this methodology 0.10.2 ml of liquid faeces can be collected.
o
o
Moisten the swab in sterile normal saline, if available.
Introduce the swab into 4 cm deep into rectum through
52
C:\JS\DSD.doc
anal sphancter. Rotate by 90oC and withdraw the swab.
o
7.2
Stoie the swab in stoppered container or in transport
medium e.g. Carry blair so as to avoid drying.
Storage and Transportation
O
O
Store the specimen at 2-8°C.
Transport to the laboratory at the earliest and in case of
delay use to Cary Blair transport medium and send to the
nearest laboratory.
Cary Blair transport medium:
It is a semi solid transport medium usually supplied in small bijou
bottles. It should be stored in air tight container so as to avoid
drying.
Inoculation of Cary Blair Transport Medium:
o
o
o
Insert one/two rectal swabs taken from the same patient
into the medium so that the whole swab is dipped into the
medium.
Break off the extra portion of sticks and replace the screw
cap.
Label the bottles.
PROCESSING OF SAMPLES IN THE LABORATORY:
Microscopic examination and Culture:
Materials required
o
o
O
O
O
O
Enrichment medium
- Alkaline peptone water.
Plating media
- Bile Salt agar
- Thiosulphate-citrate-Bile Salt-Sucrose (TCBS) agar
- MacConkey Agar
Inoculating wireloop
Gas supply/burner
Incubator at 37°C.
Hand lens.
After the ;specimen arrives in the laboratory, it should be given
laboratory code number and
entered iin the register before processing.
-----------------
53
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Deinoi'istrcitioii of motility by direct microscopy:
It can be done using either the direct stool sample or using 4-6 hr.
growth in alkaline peptone water.
o
o
Prepare a hanging drop using culture growth in alkaline
peptone water or direct stool suspension.
Examine under the high power of a binocular microscope.
Observation:
A darting motility is suggestive of presence of Vibno cholerae.
Culture
o
o
o
Directly streak over BSA and TCBS media and
also
inoculate alkaline peptone water (an enrichment medium).
Incubate overnight at 37° C in ordinary incubator.
Examine the plates after overnight incubation while APW
is to be examined after 4-6 hours.
Colony morphology on culture plates:
BSA (Bile Salt Agar): Small translucent raised flat colonies are
characteristic of Vibrio cholerae.
TCBS: Yellow, flat smooth colonies with pale yellow periphery are
suggestive of Vibrio cholerae.
APW: Subculture growth in alkaline peptone water after 4-6 hour onto
BSA/TCBS plates. Incubate plates and APW overnight. Look for the
characteristic colonies of V.cholerae as described earlier.
7• 5 Slide agglutination test for Confirmation of Vibrio
cholerae.
Materials required
o
o
o
o
o
o
o
Glass slides (clean)
Normal Saline
Platinum wire loop
Growth on BSA/TCBS
Discarding jars with disinfectant
Gas supply
V.cholerae 01 antisera.
Procedure:
o
Put a small drop of normal saline on a clean glass
slide.
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0
O
o
o
Using a wire loop pick a colony from a culture plate.
Emulsify the growth in a drop of Normal saline using wire
loop.
Add a loopful of cholera 01 non-differential antisera on to
the suspension mix.
Look for agglutination (i.e. immediate clumpingof
organism within 30-60 seconds.
Observation:
A positive agglutination is indicated by immediate clumping of
organism and suggests presence of Vibrio cholerae O1 and rest are
labelled as non-agglutinating vibrios.
7.6 Stock
Laboratory:
o
o
culture
and
Referral
to
Reference
Make a stab into the nutrient agar slope, the cultures
resembling Vibrio cholerae. (Agglutinating as well as non
agglutinating strains).
Tiansport the stab cultures to the reference laboratory for
further testing.
*•
C^ojnposition and source of culture media/reagents
necessary for cholera laboratory:
Bile Salt Agar (BSA)
Use:
Used for isolation and enumeration of enteric bacilli.
Readymade dehydrated media is available from Hi-Media
Laboratories Pvt. Ltd., Bombay.(Product code M-739 500 gms
pack)
Directions for use:
o
O
0
0
O
O
Suspend 43 gms of media in 1000 ml of distilled water in a
flask.
Plug flask with cotton.
Autoclave at 15 lbs pressure 121oC for 20 minutes
Cool to 60-70°C.
Pour in sterilised Petridishes.
()
Remove air bubbles using flame.
Let it solidify
O
Store in refrigerator.
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T.C.B.S. Medium:
(Thiosulphate Citrate Bile Salt Medium)
Use: Recommended for the selective isolation and culture of Vibrios
causing cholera and vibrios which cause food poisoning.
Readymade dehydrated medium available from Hi-Media Laboratories
Pvt. Ltd. Bombay (Product Code No. M-189).
Directions for use:
0
O
O
O
O
O
Suspend 89 gms of Media in 1000 ml of distilled water.
Boil tb'dissolve completely. Do not autoclave.
Cool at 50<’C.
Pour into sterilised petridishes.
Allow to solidify
Store in refrigerator.
Alkaline Peptone Water:
Use:
At pH 8.4 it is suitable for the cultivation and enrichment of Vibrio
cholerae from infected material.
Readymade dehydrated medium available from Hi-Media Laboratories
Pvt.Ltd. Bombay (Product code No: M-028 - Peptone water).
Directions for use:
o
o
o
o
o
o
Suspend 15 gms in 1000 ml distilled water.
Mix well.
Adjust pH to 8.4 with NaOH.
Dispense 2-3 in test tubes.
Plug tubes, using cotton plugs.
Autoclave at 15 lbs pressure at 121oC for 20 minutes.
Cary Blair Medium
(Transport Medium w/o Charcoal)
Use: Recommended for collection and shipment of clinical specimens.
Readymade dehydrated medium available from Hi-Media Laboratories
Pvt.Ltd., Bombay (Product Code - M-202).
Directions for use.
o
o
o
o
Suspend 12.6 gms of media in 99% ml of distilled water.
Boil to dissolve completely.
Cool to 50°C.
Add aseptically 9 ml of 1% aqueous calcium chloride solution.
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o
o
o
o
o
Adjust pl I 8.4.
Distribute 5-7 ml in screw capped small bottles.
Steam bottles for 15 minutes.
Cool, allow to solidify.
Bottles are kept at room temperature.
Nutrient Agar Stabs:
Use:
A general culture medium.
Readymade dehydrated media supplied by Hi-Media Laboratories
Pvt.Ltd., Bombay (Product Code M-012 Nutrient Agar W/ 1% peptone).
Directions for use:
O
O
o
o
o
o
Suspend 35 gms of media in 1000 ml of distileld water.
Boil to dissolve medium completely.
Pour 2-3 ml in sugar tubes.
Plug tubes with cotton plugs.
Autoclave at 15 lbs pressure at 121°C.
Cool to solidify.
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CHAPTER- 8
Safety Precautions in Laboratory
C:\JS\DSD.doc
CHAPTER- 8
SAFETY PRECAUTIONS IN THE LABORATORY:
Biosafety in a imicrobiological laboratory is very essential and basically
depends on three components:
o
O
o
Basic standard of laboratory design, operation and
equipment.
Selection and use <of essential biosafety equipment.
Safe labooratory procedures.
An exhaustive review of each component is beyond the scope of this
manual but practical and easily achievable safe laboratory rules are
listed below:
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Avoid mouth pipetting
Avoid eating, drinking, smoking and storing eatables irthe
laboratory.
Decontaminate the working area at least once a day and more
frequently after the spillage of potentially infective material.
Wash your hands after handling the infectious material.
Wear laboratory coats/Gowns in the laboratory and these should
not be taken outside.
Use gloves for all those procedures that may involve accidental,
direct contact with blood or infectious materials.
Decontaminate all liquid or solid waste before disposal.
Perform all technical procedures in a way that minimises the
aerosol formation.
Provide adequate training to the staff in laboratosjrfety
procedures.
As far as possible actively immunize the workers against the
diseases the materials of which are handled by them.
Employ only medically fit staff to work in clinical laboratories.
Report accident and illness promptly to the concerned officials.
Provide ample space and illumination for safe conduction of
laboratory rpocedures.
Design smooth easily cleanable walls, ceilings anfloors
which
should be impermeable to liquids andresistant to chemicals and
disinfectants.
Ensure a dependable and good quality water supply.
Make suitably equipped first aid' rooms readily accessible.
Provide the staff safe laboratory equipments e.g.pipetting aids,
safety cabinets, screw cap tubes and bottles, loop, incinerator if
possible, and autoclaves, etc.
Carry out periodic health and medical surveillance of the workers
to exclude the highly susceptible individuals.
sx
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•
•
•
•
Provide safety systems covering fire and electrical emergencies.
Control rodents and insects in the laboratory.
Don't permit the entry of the experimental animals which are not
to be used in the laboratory.
Immunize your staff handling blood and blood products against
Hepatitis B.
5(>
C VIS\DSD.doc
CHAPTER- 9
Common Laboratory Equipment
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CHAPTER 9
COMMON LABORATORY EQUIPMENT
9.1 Incubator
Incubator is an apparatus having a desired temperature. The heating
device used can
gas, oil or electricity. Maintenance of uniform
temperature within the incubator is essential and is achieved by fan,
blower or a waterjacket containing heated water.
Ideal temperature for most of the medically important bacteria is
35O+2°C. However for some organisms different temperatures are
necessary e.g. atypical mycobacteria (220C-45°C), fungi (22°C) etc.
Some organisms may need extra gaseous element e.g. 5-10% CO2
incubator for Brucella. The size of the incubator may vary from a
small table top to a large walkin type rooms. A proper temperature
recording thermometer and a small tray of water inside the incubator
to prevent excessive drying of air are the two other essential
requirements.
9.2
Hot Air Oven
It
a
b
c
Dry glass materials like test tubes, Petridishes, flasks, pipettes,
syringes.
Instruments like forceps, scalpels, throat swabs, etc.
Sealed materials which can stand heat and when penetration of
steam is not possible.
The instrument is electrically operated and should be equipped with a
fan to have unfirom temperature inside, the required temperature for
sterilisation is generally 160°C for 1 hour.
Operation of Hot air Oven
o
O
o
Arrange the material to be sterilised loosely and evenly on the
racks of the ovenallowing free circulation of air and thereby
even heating of the load.
Air is poor conductor of heat so do not pack the load tightly.
Switch on the power supply and control the temperature of
the oven by adjusting thermostat.
When
the
desired
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temperature is reached, note the time. Time taken for the oven
to ieach the desired temperature is called 'heating up period'.
o
O
Hold the load in the oven at this temperature for a definite
period of time. This period known as 'holding up period' is
dependent upon the temperature employed. At 160°C the
holding up period is 60 minutes, at 170°C for 18 minutes, at
180°C 7.5 minutes and at 190°C it is 90 seconds.
The most common temperature for hot air sterilisation is 160°C
for one hour. When the temperature is raised further, cotton
plugs and paper wrappings get charred.
On the expiry of holding up period, switch off the power supply
and allow the load to cool.
Open the oven door only when the temperature fall below 80°C,
otherwise it may result in breaking up of glassware and also
cause injuries to the operator.
Dry up the instruments before placing them in the hot air oven.
Do not place the heat sensitive materials inside.
9.3
Water Bath:
o
o
o
O
Water bath is a water container having an electrically operated
heating device to provide a fixed and uniform temperature,
A
theimometer is inserted inside the water bath for recording
temperature.
A mixer immersed inside water is also desired to
maintain uniform temperature throughout the water bath.
A few applications of water bath are:
37°C Water bath -
required during performance of WIDAL test;
44°C Water bath -
required in faecal coliform count (water
bacteriology) test.
for inactivating complement in the serum.
56°C Water bath -
9.4
Centrifuge
for an average laboratory a small table top centrifuge with a
maximum revolutions per minute of 6000 and capable of
accommodating 10-12 tubes of 15 ml capacity is sufficient. The tubes
should be placed exactly opposite to each other, should be of the same
weight and should contain same amount of fluid. The speed is
adjusted by a rheostat and should be allowed to rise slowly. A timer
for fixed duration of centrifugation is preferred.
r. i
f’Aicvncn
A few common uses are:
0
O
o
9.5
Sediment examination of urine - 1500 rpm for 5 minutes.
Separation of serum from clotted blood - 1500 rpm for 15
minutes.
Concentration of microfilaria from blood - 2000 rpm for 25 minutes.
pH METER
A pH meter consists of an electrode {
pair which is sensitive to hydrogen
io,n eventration
due to
.
. the development
’ x------- - of
-- an electrical gradient
winch is directly proportional to the hydrogen ion concentration. The
elcctiodes commonly used arc one of glass for the unknown and other
of
o( colomel to be used as a standard precautions while using pH meter
are:
o
o
o
o
o
o
9.6
The electrodes specially the glass ones should be handled
carefully to prevent breakage due to contact with hard surface.
Sufficient time should be given to warm up the instrument
before use.
Frequent standardizations of the pH meter should be made
using standard buffer solution.
Electrodes are to be washed with a stream of distilled water
between measurements.
The electrodes should never be removed from the solution when
the measuring circuit is closed.
When not in use, the electrodes must be kept immersed in
water or electrode solution.
Refrigerator:
Reft igferators are essential for storage of degradable laboratory
substances like media, reagents, antisera, antibiotic discs etc.
Refrigerators can vary in their capacity ranging from table top to a
large walk-in-type. The usual temperature needed is 4o+2°C which is
maintained comfortably by household use refrigerators. Substances
to be kept at frozen state like sera may be kept in the freezer units of
the same. Proper recording of the temperature is very important to
avoid deterioration of biological materials.
9.7
Microscope
o
Place a slide on 1'
the stage, specimen side up and the centre of
the section to be examined1 ;as accurately as possible over the
hole in the centre of the stage.
Adjust the mirror until it reflects the maximum amount of the
light through the specimen with the low objective in position,
lower the body tube by means of the coarse adjustment until
o
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o
o
o
o
the objective is about 1/4" from the slide.
Look through the eye piece and slowly raise the objective with
the coarse adjustment: until the specimen is in approximate
focus. Never focus downward while looking through the eye
piece.
Bring the specimen to sharp focus with the fine
adjustment. Adjust the iris diaphgram and substage. Condense
until the light intensity is optimum.
After examining the specimen with the low power objective shift
to the high dry objective by rotating the nose piece until the
objective clicks into place.
Look through the eye piece and slowly raise the body tube with
the coarse adjustment until the specimen comes into
approximate focus. Then bring the image into final accurate
focus by using the fine adjustment. Once the specimen is in
focus adjust the mirror and the iris diaphgram to give the
clearest possible image.
Focussing of the oil immersion objective:- First use
the
low
power objective to locate the portion of the specimen to be
examined. Raise carefully the body tube, and then rotate the
nose piece until the oil immersion objective clicks into the
position. Now place a drop of immersion oil on the portion of
the slide directly under the objective watching the object from
the side carefully lower it into the oil. Do not allowthe objective
to touch the slide. Look through the occular and slowly focus
upward with the fine adjustment until the age appears. Once it
appears do the fine adjustment and adjust the mirror and iris
diaphragm to obtain optimum illumination.
Maintenance
o
o
0
O
o
o
Never touch the lenses if they become dirty, wipe them gently
with lens paper.
Always remove oil from the oil immersion objective after its use.
If by accident, oil should get on either of the low power, wipe of
the objective immediately with the lens paper. If oil becomes
dry or hardened on a lens, remove it with lens paper lightly
moistened with xylol.
Keep the stage of the microscope clean and dry.
Do not tilt the microscope when working with the oil immersion
system.
When the microscope is not in use, keep it covered in a
microscope compartment. Never apply force to the microscope.
Never allow the objective lenses to touch the cover glass or the
slide. Never lower the body tube with the coarse adjustment
while looking through the microscope. Never exchange the
objective or occulars of different microscopes.
Store the microscope in its cabinet when not in use.
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9.8
Autoclave
Principle
Water boils when its vapour pressure equals the pressure of
surrounding atmosphere. The temperature at sea level is 100°C.
When water is boiled within a closed vessel at increased pressure, the
boiling point of water is increased and so is the temperature of steam
produced. This principle is employed in sterilising material by steam
at temperature higher than 100°C and the process is called
autoclaving.
For autoclaving in the laboratory, the most agreeable and commonly
used method is to use steam at 121°C for 15 to 30 minutes depending
upon the particular material to be sterilised.
Items to be sterilised:
Autoclaving is most suitable for culture media, aqueous solutions,
decontamination of discarded cultures and specimens, rubber items
such as gloves, stoppers with rubber liner, glass ware with attached
rubber tubings such as transfusion sets, glass metal syringes, etc.
Autoclaves designed for laboratory work and capable of handling
mixed loads should be used.
9.8.1 Types of autoclave
Only autoclaves designed for laboratory work and capable of dealing
with a 'mixed load' should be used. 'Porous load' and 'bottled fluid
sterilizers' are rarely satisfactory for laboratory work. There are two
varieties of laboratory autoclave
o
o
Pressure cooker types; and
Gravity displacement models with automatic air and
condensate discharge.
Pressure Cooker type laboratory autoclaves:
The most common type is a device for boiling water under pressure. It
has a vertical metal chamber with a strong metal lid which can be
fastened down and sealed with a rubber gasket. An air and steam
discharge tap, pressure gauge and safety valve are fitted in the lid.
Water in the bottom of the autoclave is heated by external gas
burners, an electric immersion heater or a steam coil.
Operating Inst nidi.ons:
c
There must be sufficient water inside the chamber. The autoclave is
loaded and the lid is fastened down with the discharge tap open. The
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safety valve is then adjusted to the required temperature and the heat
is turned on.
When the water boils, the steam will issue from the discharge tap and
carry the air from the chamber with it. The steam and air should be
allowed to escape freely until all of the air has been removed. This
may be tested by attaching one end of a length or rubber tubing to the
discharge tap and inserting the other end into a bucket or similar
large container of water. Steam condenses in the water and the air
rises as bubbles to the surface; when all of the air has been removed
from the chamber, bubbling in the bucket will cease. When this stage
has been reached, the air-stcam discharge tap is closed and the
lubber tubing removed.
The steam pressure then rises in the
chamber until the desired rpcssure, usually 15 lb/in2, is reached and
steam issues from the safety valve.
When the load has reached the required temperature the pressure is
held for 15 min.
At the end of the sterilizing period, the heater is turned off and the
autoclave allowed to cool.
Phe air and steam discahrge tap is opened very slowly after the
pressure gauge has reached zero (atmospheric pressure). If the tap is
opened too soon, while the autoclave is still under pressure, any fluid
inside (liquid media, etc.) will boil explosively and bottles containing
liquids may even burst. The contents are allowed to cool. Depending
on the nature of the materials being sterilized, the cooling (or 'run
down') period needed may be several hours for large bottles of agar to
cool to 80°C, when they are safe to handle.
Autoclaves with air discharge by gravity displacement
I hese autoclaves are usually arranged horizontally and are
lectangular in shape, thus making the chamber more convenient for
loading. A palette and trolley system can be used.
I he jacket surrounding the Gravity displacement autoclave consists of
an outer wall enclosing a narrow space around the chamber, which is
filled with steam under pressure to keep the chamber wall warm. The
steam enters the jacket from the mains supply, which is at high
pressure, through a valve that reduces this pressure to the working
level. The working pressure is measured on a separate pressure
gauge fitted to the jacket. This jacket also has a separate drain for air
and condensate to pass through.
The steam enters the chamber from the same source which supplies
steam to the jacket. It is introduced in such a way that it is deflected
AS
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»
upwards and fills the chamber from the top downwards, thus forcing
the air and condensate to flow out of the drain at the base of the
chamber by gravity displacement. The drain is fitted with strainers to
prevent blockage by debris. The drain is usually fitted with a
thermometer for registering the temperature of the issuing steam. The
temperature recorded by the drain thermometer is often lower than
that in the chamber. The difference should be found with
thermocouple tests. A 'near to steam' trap is also fitted.
The autmatic steam trap or 'near-to-stream' trap is designed to ensure
that only saturated steam is retained inside the chamber, and that air
and condensate, which are at a lower temperature than saturated
steam, are automatically discharged. It is called a 'near-to-steam' trap
because it opens if the temperature falls to about. 2oC below that of
saturated steam and closes within 2oC or near to the saturated steam
temperature. The trap operates by the expansion and contraction of a
metal bellows , which open and close a valve. The drain discharges
into a tundish in such a way that there is a complete airbreak
between the drain and the dish. This ensures that no contaminated
water can flow back from the waste-pipe into the chamber.
Operation of a gravity displacement autoclave:
If the autoclave is jacketed, the jacket must first be brought to the
operating temperature. The chamber is loaded, the door is closed and
the steam-valve is opened, allowing steam to enter the top of the
chamber. Air and condensate flow out through the drain at the
bottom. When the drain thermometer reaches the required
temperature a further period must be allowed for the load to reach
that temperature. This should be determined initially and periodically
for each autoclave. Unless this is done the load is unlikely to be
sterilized. The autoclave cycle is then continued for the holding time.
When it is completed the steam valves are closed and the autoclave
allowed to cool until the temperature dial reads less than 8O0C. Not
until then is the autoclave safe to open. It should first be 'cracked' or
opened very slightly and left in that position for several minutes to
allow steam to escape and the load to cool further.
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