CYTOLOGY

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CYTOLOGY Standard Operating Procedures Gan
esh Pd Acharya Cytotechnologist
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• INTRODUCTION
• Cytology
• It is the branch of Medical sciences, which deals
  with the study of cells which includes
• Shape and size (Morphological study).
• Chemical constituents (cyto-chemical study)
• Maturation process (Metabolic activity study)
• Specialized ultra structures of cell surface and
  their functions. (Electron Microscopic study)
• Cytogenetics (chromosomal study)
• Cancer marker (Immuno-cyto-chemical study).

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• Cyto-preparation techniques include
• Methods of specimen collection,
• Fixation and fixatives,
• Preservation of fluid specimens prior to
  processing,
• Preparation of materials for microscopic
  examination,
• Staining and mounting of the smears,
• Transportation of prepared slides to the defined
  center,
• Registration and recording of the reports,
• Distribution of reports to the respective
  department /clinic / patient,

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• Methods of collection
• In general, material for cytological examination
  is obtained either in the form of smears prepared
  by examining physician, gynecologist, surgeon or
  their assistants at the time of clinical
  examination e.g. cervical smears.
• OR
• In the form of fluid specimens which are
  forwarded to the laboratory for further
  processing e.g. Body fluids such as
• Pleural fluid
• Ascitic fluid
• Peritoneal fluid
• Pericardial fluid
• Joint fluid
• Cystic fluid (Breast/tumor)
• Sputum
• Urine
• Cerebrospinal fluid (CSF)
• Gastro-intestinal aspirates etc.

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• SMEARS
• Prior to the preparation of smears, it is
  important to secure the necessary materials and
  lay them out on a suitable, conveniently located
  surface within the reach of the operator
• Instrument(s) used to obtain smear,
• Clean, new microscopic slides,
• Suitable marker (diamond pencil) for the
  identification of slides,
• Paper clips (plastic coated or copper) used to
  separate the slides from each other if liquid
  fixatives are used,
• Fixatives,
• Laboratory form with clear identification of the
  patient and appropriate history minimum data
  required for each patient is listed on the form,
  

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• Preparation of smears
• For most diagnostic purposes, well-prepared and
  well-fixed smears are required.
• Air-drying of smears should be avoided, if
  prepared for wet fixation.
• Monolayer preparation is suitable for almost all
  processing techniques.
• Considerable skill and practice are required to
  prepare excellent smears by single swift motion
  without loss of material or air-drying.
• Excessive crushing of the material must be
  avoided.
• A competent help must be secured in advance.

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• Fixation
• Immediate fixation of smears is essential for
  the correct interpretation.
• Most of the fixatives are alcohol-based e.g. 95
  ethanol, 95 rectified spirit, 80 iso-propanol
  or propanol, absolute methanol, Ether95 Ethanol
  mixture (11), Carnoy's fixative.
• Air dried smears are required or desirable in
  special situations (special stains e.g. MGG stain)

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• Fixatives
• Two types of fixatives are commonly used.
• Fluid fixatives
• Spray fixatives
• Fluid fixatives
• Are prepared in bottles of suitable sizes,
  provided with caps or coplin jars with covers.
• Fixatives solution must be new for each batch of
  fixation.
• Filtration of fixatives should be done in case of
  repeated use.

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• 2.Spray fixatives
• Contain 2-10 carbo wax in 95 alcohol
• Protect the smears from drying by forming an
  invisible film on the surface of the slides
• May be used in lieu of fluid fixatives i.e.
  immediately after the process of smear
  preparation has been completed.
• Correct use of spray fixative calls for several
  precautions such as
• Spray must be smooth and steady
• Distance between spray nozzle and smear must be
  10-12 inches (25-30 cm)
• Smears coated with spray fixatives must be
  air-dried before mailing

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• Fluid specimens
• May be obtained from a variety of body sites
  such as
• Respiratory tract (sputum)
• Gastro-intestinal tract (endoscopic aspirates)
• Urinary tract (Urine / barbotage samples)
• Effusion fluids (body cavity fluids-pleural/
  peritoneal/pericardial) should be collected in
  anticoagulants container (1 ammonium oxalate in
  the ratio of 91 i.e. 9 parts of fluid and 1 part
  anticoagulant)

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Cytology STANDARD OPERATING PROCEDURE NPHL, Teku,
Kathmandu
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PROCEDURE Principle- The
smears of body fluids, which contain exfoliated
cells and/or cells produced by transudation,
which are then concentrated by centrifugation, so
as to make the screening process efficient.
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• Method
• Take two centrifuge tubes.
• Label the centrifuge tubes.
• Mix the fluid properly by inverting the container
  ten times.
• Put the centrifuge tubes in the centrifuge
  machine at the opposite sides so as to balance
  while centrifugation.
• Add 10 ml each of well-mixed fluid to the labeled
  centrifuge tubes.
• Set the centrifuge machine at 1500 rpm for 15-20
  minutes.

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• Remove the centrifuge tubes once the machine
  stops and discard the supernatant in the
  disinfectant container with the help of a
  Pasteur pipette.
• Label four clean slides with the help of
  diamond pencil.
• Transfer the sediment on the clean slides 2 cm
  away from the end with the help of a Pasteur
  pipette.
• Make the smears immediately by holding the slide
  with one hand and spreading with the help of the
  flat surface of another slide.

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• If the sample is haemorrhagic (reddish in
  colour), smear should be prepared from the
  buffy- coat layer of the sediment. In such
  condition FISH TAIL smear preparation is
  suggested.
• Three slides are immediately fixed (while still
  wet) in the alcoholic fixative for 20-30
  minutes.
• One slide is left to dry in open air at room
  temperature

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• Internal Quality Control Procedures
• The proportion of fluid and anticoagulant should
  be maintained (91).
• In case of delay in processing, the fluid should
  be stored in a refrigerator. (DO NOT FREEZE)
• Supernatant and remaining fluid should always be
  discarded in a disinfectant solution.
• Protective clothing such as gloves, apron and
  mask should be worn while processing.
• Smears should be prepared so as to give monolayer
  of cells for easy differentiation.
• Fixation should be done immediately after
  preparation of the smears so as to prevent
  changes in cell morphology.

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• PROCEDURE
• Principle
• Fixation prepares the samples from different
  sites of the body for the purpose of preserving
  and maintaining the existing form and structure
  of all constituent elements.
• Method
• Four slides (urine), three slides (body
  fluids/sputum), 1-2 slides (cervical smear) and
  one slide (CSF) should be immediately put in the
  coplin jar/container with 95 Ethanol (alcohol)
  or its equivalent fixative.
• Leave the slides in the fixative for a minimum of
  30 minutes.
• Take out the slides with the help of forceps and
  leave to dry in a slide rack.
• One slide for all specimens except urine and
  cervical smear is not fixed in the alcoholic
  solution but dried at room temperature.

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• Internal Quality Control Procedures
• Grease-free, clean new slides are to be used for
  all types of specimens.
• Proper fixative and time of fixation are to be
  strictly maintained.
• Use of fresh fixatives is recommended. If the
  fixative is to be re-used it should be filtered
  (Whatman filter paper) for every batch of slide
  fixation.

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• PROCEDURE
• Principle
• Properly filled request form, properly labelled
  samples and proper registration of the patient
  identification with their clinical parameters are
  the pre-requisites for Quality results.
• Method
• Match the request form and labelled sputum
  samples.
• Check the request form for patient's full name,
  age, short clinical history and other additional
  parameters.
• Enter the detail in register.
• Label the sample and the request form with the
  running number with permanent marker.
• Make them ready for further processing.

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• DOCUMENTATION
• Date of sputum sample reception.
• Name age of patient
• Name of the lab personnel who receives the smear.
• Transfer the detailed to the cytopathology
  register.

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• Internal Quality Control Procedures
• The sputum sample should be received in a wide
  mouth bottle with proper labeling.
• Accept only completely filled request form.
• Details of patient should be documented in the
  register.

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• MATERIALS REQUIRED
• Sputum sample
• New glass slides
• Wooden spatula
• Wooden racks
• Coplin jar
• 80 propanol
• Diamond Pencil

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• PROCEDURE
• Principle
• The neoplastic cells are exfoliated and are
  expectorated in sputum. The exfoliated cells are
  spread over the slide in a manner which will make
  cytological study possible. Similarly, other
  cells may exfoliate and may indicate the other
  underlying pulmonary pathology.
• Method
• Clean the working bench
• Put on protective clothing, gloves.
• Place specimen on the working bench. If specimen
  has been refrigerated, bring to room temperature.
  
• Label 4 slides with patient identification
  laboratory acquisition number.
• Examine specimen carefully. This may require that
  the specimen to be transferred to a petridish
  placed on a dark background to visualize
  suspicious areas.

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• Look for fresh or old blood stained areas,
  discolored portion of sample tissue fragments.
• Transfer suspected areas with a disposable stick
  or applicator to a glass slide. Spread material
  across surface of glass slide.
• Take a second glass slide.
• Spread them gently between the 2 slides until an
  even distribution of material is obtained.
• Fix slides in 95 ethanol for 15-30 minutes

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• DOCUMENTATION
• Date of sputum sample collected.
• Date of sputum sample received in the lab.
• Date of sputum sample processed.
• Gross appearance
• Fixatives used or not
• Total no of smears.
• Type of fixative used.
• Clinical details clinical diagnosis.
• Name of the technician handling the sample.
• Name of the pathologist who is going to report.
• Transfer the material to cytopathology register

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• Internal Quality Control Procedures
• Not to receive any broken or dirty slides.
• Accept only completely filled request form
  (Request forms must be filled by the concerned
  clinician)
• Details of documents should be registered.

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• MATERIALS REQUIRED
• Properly labeled and fixed smeared cervical
  slides.
• Request formfilled by the clinician with
  identification of both patient and smears with
  short clinical history and minimum data
  requirements.
• Permanent marker for labeling.
• Cervical cytology register.

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• PROCEDURE
• Principle
• Properly filled request form, properly labeled
  and fixed cervical smears and proper registration
  of the patient's identification with their
  clinical parameters are the pre-requisites for
  quality results.
• Method
• Match the labeled slide number with the request
  form.
• Check the request form for patient's full name,
  age, LMP (Last menstrual period), address, short
  clinical history and other additional parameters.
• Enter the details in the Cervical cytology
  register.
• Label the slides and the request form with the
  running laboratory number with a permanent
  marker.
• Store the received smears in a proper place.
• Make them ready for mailing.

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• DOCUMENTATION
• Date of smear reception.
• Numbers of slides received.
• Date of LMP.
• Name and age of patients.
• Name of the clinician who took the smear.
• Name of the Lab. personnel who received the
  smear.
• Transfer the details to the cytopathology
  register.
• Check the details on a form, which will be mailed
  with the slides for reporting.

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• Internal Quality control procedures
• Do not receive any broken or dirty slides.
• Clinician must properly fill the request form. Do
  not accept any improperly filled such form having
  no significant reason.
• Details of all documents should be register
  properly.

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• MATERIALS REQUIRED
• 95 Ethanol / 95 rectified spirit /80
  iso-propanol or propanol / absolute methanol /
  Ether95 Ethanol mixture (11) /Spray coating
  fixatives (2-10 Carbowax in 95 Ethanol)/
  Carnoy's fixative for haemorrhagic fluids.
• Grease-free, clean new slides
• Slide racks
• Forceps
• Coplin jars /suitable containerplastic coated
  paper clips
• 0.5 Sodium hypochlorite solution / 1 phenolic
  solution container
• Diamond pencil (Slide marker)
• Filter paper (Whatman)
• Funnel

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PROCEDURE Principle Fixation prepares the
samples from different sites of the body for the
purpose of preserving and maintaining the
existing form and structure of all constituent
elements.
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• Method
• Four slides (urine), three slides (body
  fluids/sputum), 1-2 slides (cervical smear) and
  one slide (CSF) should be immediately put in the
  coplin jar/container with 95 Ethanol (alcohol)
  or its equivalent fixative.
• Leave the slides in the fixative for a minimum of
  30 minutes.
• Take out the slides with the help of forceps and
  leave to dry in a slide rack.
• One slide for all specimens except urine and
  cervical smear is not fixed in the alcoholic
  solution but dried at room temperature

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• DOCUMENTATION
• Date and time of sample collection
• Date and time of sample receipt.
• Amount of fluid received.
• Gross appearance of the fluid.
• Total number of smears prepared.
• Total number of dry smears.
• Total number of clot smears (if any).
• Transfer the details to the cytopathology
  register.
• Transfer the details on a form, which will be
  forwarded with the slides for reporting.

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• Internal Quality Control Procedures
• Grease-free, clean new slides are to be used for
  all types of specimens.
• Proper fixative and time of fixation are to be
  strictly maintained.
• Use of fresh fixatives is recommended. If the
  fixative is to be re-used it should be filtered
  (Whatman filter paper) for every batch of slide
  fixation.

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• MATERIALS REQUIRED
• Cervical smears
• 95 Ethanol /80 iso-propanol / absolute methanol
  for fixation/hydration
• Coplin jars
• Staining rack
• Alcohols 50, 70, 80, 95 and absolute alcohol.
• Staining solutions
• Harris Haematoxylin
• OG-modified
• EA-modified
• Diluted 1Lithium Carbonate (30 drops of 1
  Lithium carbonate in 1000 ml of distilled water)

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• 0.5 HCl
• Tap water/Distilled water
• Xylene
• DPX Mountant
• Coverslips
• Labeling Stickers

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PROCEDURE Principle- The Papanicolaou staining
procedure was devised for optimal visualization
of cancer cells exfoliated from the epithelial
surface of the body. It is a polychrome staining
reaction, consisting of a water-based nuclear
stain and two alcohol-based cytoplasmic
counterstains, designed to display the many
variations of cellular morphology and to show the
degree of cellular maturity and metabolic
activity.
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• Method
• Papanicolaou Staining (regressive method)
• Arrange the slides in a staining rack.
• Hydrate the smears by immersing the slides in 95
  alcohol for 10-15 minutes followed by 80, 70
  and 50 alcohol (2-3 minutes each).
• Immerse the slides in distilled water/tap water
  for 5 minutes.
• Drain the excess water and put in Harris
  Haematoxylin solution for 3-5 minutes.
• Rinse in tap water for one minute.
• Differentiate in 0.5 HCl (1-2 quick dips)
• Rinse in tap water for 2-3 minutes.
• Immerse the slides in diluted Lithium carbonate
  for 1 minute.
• Rinse with tap water/distilled water for 1
  minute.
• Dehydrate by immersing in 70 ethanol for 30
  seconds.
• Next immerse in 80 ethanol for 30 seconds.

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• Immerse in two changes of 95 ethanol for 30
  seconds each.
• Stain in OG-modified solution for one minute.
• Rinse in two changes of 95 ethanol for 30
  seconds each.
• Stain in EA-modified for 5-11 minutes (depending
  on the quality of stain and frequency of
  staining).
• Immerse in three changes of absolute ethanol for
  30 seconds each.
• Immerse in three changes of Xylene for 30 seconds
  each.
• Mount the smears with DPX.
• Label the smears with stickers.
• Submit with the requisition form for microscopy.

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• DOCUMENTATION
• Date of smear preparation
• Number of smears received.
• Date of LMP
• Age of the patient.
• Name of the clinician who took the smear.
• Short clinical history.
• Name of the technician/Laboratory personnel who
  processed the smear.
• Record should be transferred to a cytopathology
  register.

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• Internal quality control Procedures
• Harris Haematoxylin should be filtered everyday
  before staining.
• Staining time can vary with each batch of new
  stains (depending on the quality of the stain and
  frequency of staining).
• Staining solutions should be prepared in small
  quantities to cover a period of three
  
  months (Stains being alcoholic
  preparations)
• 0.5 HCl and diluted Lithium carbonate should be
  prepared freshly each day.

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• MATERIALS REQUIRED
• CSF sample
• Cytocentrifuge
• Centrifuge tubes.
• New glass slides.
• Marker pencil
• Normal saline solution.

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• PROCEDURE
• Principle
• CSF sample contain either very low cell count or
  the total volume of sample is very low the cyto
  centrifugation technique is used to obtain
  comparatively higher cellular manolayer smear.
• Method
• Take a pre-coated glass slide.
• Fix the pre-coated slide into cytocentrifuge
• Take CSF sample add normal saline solution to
  make final volume of 5 ml.
• In opposite chamber counter balance is done by
  adding normal saline.
• Put the CSF solution in to the centrifuge chamber
  centrifuge 1000 rpm for 4 minutes.
• Pick up glass slides from centrifuges head.
• For wet preparation of smear fix the glass slide
  immediately into 95 ethanol.
• For dry smear leave the slide on rack.
• Send the slides for Papanicolaou stain and MGG
  stain

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• DOCUMENTATION
• Number of slides received.
• No of dry slides/wet slides
• Date of CSF sample collection
• Name of Doctor who collected the sample.
• Name of person who performed the test processing.
  
• Test result should be entering in the Cyto
  pathology Register book.

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• Internal Quality Control Procedure
• Grease free and clean slides must be used.
• C.S.F should be centrifuged at desired speed for
  desired time.
• The glass slides should be coated before smear
  preparation.

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• MATERIALS REQUIRED
• Body fluids in an anticoagulated container (1
  Ammonium Oxalate solution--Ratio of fluid to
  anticoagulant is 91)
• New, grease-free, clean slides
• Graduated centrifuge tubes (Capacity-10 ml)
• Centrifuge (Speed limit not lt1500 rpm)
• Cytocentrifuge (if available)
• Coplin jars
• 95 Ethanol/80 isopropanol/absolute methanol
• Ether/95 Ethanol mixture (11)
• Pasteur pipettes (Glass/plastic)
• 0.5 Sodium hypochlorite solution/1 phenolic
  solution container (Disinfectant)

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• PROCEDURE
• Principle
• The smears of body fluids,
  which contain exfoliated cells and/or cells
  produced by transudation, which are then
  concentrated by centrifugation, so as to make the
  screening process efficient.
• Method
• Take two centrifuge tubes.
• Label the centrifuge tubes.
• Mix the fluid properly by inverting the container
  ten times.
• Add 10 ml each of well-mixed fluid to the labeled
  centrifuge tubes.
• Put the centrifuge tubes in the centrifuge
  machine at the opposite sides so as to balance
  while centrifugation.
• Set the centrifuge machine at 1500 rpm for 15-20
  minutes.
• Remove the centrifuge tubes once the machine
  stops and discard the supernatant in the
  disinfectant container with the help of a Pasteur
  pipette.

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• Label four clean slides with the help of diamond
  pencil.
• Transfer the sediment on the clean slides 2 cm
  away from the end with the help of a Pasteur
  pipette.
• Make the smears immediately by holding the slide
  with one hand and spreading with the help of the
  flat surface of another slide.
• If the sample is haemorrhagic (reddish in
  colour), smear should be prepared from the buffy-
  coat layer of the sediment. In such condition
  FISH TAIL smear preparation is suggested.
• Three slides are immediately fixed (while still
  wet) in the alcoholic fixative for 20-30 minutes.
• One slide is left to dry in open air at room
  temperature.

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• DOCUMENTATION
• Date and time of sample collection.
• Date and time of sample receipt.
• Amount of fluid received.
• Gross appearance of the fluid.
• Total number of smears prepared.
• Total number of dry smears.
• Total number of clot smears (if any).
• Transfer the details to the cytopathology
  register.
• Transfer the details on a form, which will be
  forwarded with the slides for reporting.

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• Internal Quality Control Procedures
• The proportion of fluid and anticoagulant should
  be maintained (91).
• In case of delay in processing, the fluid should
  be stored in a refrigerator. (DO NOT FREEZE)
• Supernatant and remaining fluid should always be
  discarded in a disinfectant solution.
• Protective clothing such as gloves, apron and
  mask should be worn while processing.
• Smears should be prepared so as to give monolayer
  of cells for easy differentiation.
• Fixation should be done immediately after
  preparation of the smears so as to prevent
  changes in cell morphology.

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• MATERIALS REQUIRED
• Slide racks for air-drying the pre fixed smears
• Slide mailer kit
• Request form-identification of both patient and
  sample.
• Postal stamps
• Stickers with postal address of the center.

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• PROCEDURE
• Principle
• The smears collected for the diagnosis need to be
  sent to a defined center. For this one should
  have a thorough knowledge of mailing procedure to
  prevent the deterioration of the collected
  material, loss of smear and breakage of slides.
• Method
• After pre-fixation, slides are taken out of the
  fixative and air-dried in a slide rack.
• Slides are then arranged in an orderly form.
• Slides are arranged in the slide mailer and the
  mailer is closed properly.
• The slide mailer along with the properly filled
  requisition form is then put in the envelope used
  for transportation.
• The envelope is sealed and the address of the
  diagnostic center is pasted on it along with the
  postal stamp.
• The envelope is then taken to the post office
  promptly.

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• DOCUMENTATION
• Type of sample.
• Patient/Slide identification number.
• Date and time of sample collection.
• Date and time of sample receipt.
• Amount of fluid received.
• Gross appearance of the fluid.
• Total number of smears prepared.
• Total number of dry smears (Numbering done with a
  capital 'D' in front).
• Total number of clot smears (if any).
• Total number of smears sent.
• Transfer the details to the cytopathology
  register.
• Transfer the details on a form, which will be
  mailed with the slides for reporting.

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• Internal Quality Control Procedures
• Check the lock of the slide mailer.
• Check the seal of the envelope.
• Check the mailing address.

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• PREPARATION OF REAGENTS
• Materials required
• Measuring cylinder100 ml and 1000 ml capacity.
• Conical or flat bottom flasks100ml and 500 ml
  capacity.
• Beakers250 ml and 500 ml capacity.
• 10 ml graduated pipette or micropipette with
  disposable tips.
• Reagent bottles (Dark coloured)500 ml and 1000
  ml capacity.
• Funnels3 inches and 6 inches in diameter.
• Permanent markers for labeling.
• Analytical balance and weights.
• Heater/Stove.
• Tripod stand.
• Thermometer.
• Waterbath.

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• Preparation of Harris Haematoxylin
• Haematoxylin (Colour index No. 75290) 5 g
• Absolute methanol 50 ml
• Distilled water 1000 ml
• Mercuric Oxide (HgO) 2.5 g
• Aluminium ammonium sulphate or Potassium
• ammonium sulphate (Alum) 100 g
• Glacial acetic acid 40 ml
• Method of preparation
• Dissolve Alum in 1000 ml distilled water and
  bring it to boil.
• Dissolve haematoxylin in alcohol by warming up to
  60C.
• Add dissolved haematoxylin to Alum and bring
  again to boil.
• Remove the flask from heat.
• Immediately add Mercuric oxide.
• Stir this solution till dark purple colour
  appears (Not more than 10 seconds).
• Plunge flask into water bath (ice cold water) to
  cool.
• Store in a dark bottle.
• Filter before use.

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• . Preparation of EA modified
• a. Light green (colour index no 42095) 0.3 g
• b. Eosin Y (colour index no 45380) 4.0 g
• c. Phosphotungstic acid 2.0 g
• d. Distilled water 480 ml
• e. 95 ethanol (alcohol) 500 ml
• f. Glacial acetic acid 20 ml
• Method of preparation
• Mix above listed (abc) dyes and chemicals in
  480 ml distilled water in a flask.
• Mix well by stirring and warming to 60-80C.
• Cool the mixture to room temperature.
• Add 500 ml of 95 Ethanol and mix well by
  stirring.
• Add 20 ml glacial acetic acid to the above
  mixture.
• Store in dark coloured reagent bottle and filter
  before use.

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• Preparation of OG- modified
• a. Orange G 5.0 g
• b. Distilled water 500 ml
• c. Phosphotungstic acid 2.0 g
• d. Absolute ethanol 500 ml
• e. Glacial acetic acid 10 ml
• Method of preparation
• Dissolve abc dye and chemical in distilled
  water.
• Warm the mixture 60-80C.
• Mix by frequent stirring and cool to room
  temperature.
• Add methanol and glacial acetic acid.
• Mix by stirring.
• Store in dark bottle.
• Filter before use.

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• Preparation of 0.5 Hydrochloric acid (HCl)
• Conc. HCl 5 ml
• Distilled water 995ml
• Method of preparation
• Measure 995 ml distilled water and transfer to
  1000 ml conical flask.
• Add 5 ml of conc. HCl with the help of the
  pipette slowly with continuous mixing to the
  distilled water.
• Transfer the prepared solution in a reagent
  bottle with name and date of preparation.

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• Preparation of 1 Lithium Carbonate
• (Stock solution)
• Lithium Carbonate (LiCO3) 10 g
• Distilled water 1000 ml
• Method of preparation
• Measure 1000 ml distilled water in a conical
  flask.
• Add 10 g of LiCO3 to the distilled water and mix
  till properly dissolved.
• Transfer the mixture to a reagent bottle with
  name and date of preparation.
• Working solution is prepared by adding 30 drops
  of stock solution to 1000 ml of distilled water.

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• PREPARATION OF FIXATIVES
• MATERIALS REQUIRED
• Measuring cylinder1000 ml and 100 ml capacity.
• Conical or flat bottom flasks100 ml and 500 ml
  capacity.
• Beakers500 ml and 250 ml capacity.
• Reagent bottles500 ml and 1000 ml capacity.
• Funnels6 inches and 3 inches diameter.
• Permanent markers for labeling.

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• REAGENT PREPARATION
• 95 Ethanol
• Take 950 ml of Absolute Ethanol in 1000 ml
  capacity measuring cylinder.
• Take 50 ml of distilled or deionised water in
  100 ml capacity measuring cylinder.
• Take 1000 ml Conical/flat-bottom flask and mix
  the Ethanol and distilled water properly in it.
• Label a reagent bottle as 95 Ethanol with the
  date of preparation and transfer the above
  solution in it.

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• 95 rectified spirit
• Take 950 ml of Rectified spirit in a 1000 ml
  capacity measuring cylinder.
• Take 50 ml of distilled or deionised water in 100
  ml capacity measuring cylinder.
• Take 1000 ml Conical/flat-bottom flask and mix
  the rectified spirit and distilled water properly
  in it.
• Label a reagent bottle as 95 Rectified spirit
  with the date of preparation and transfer the
  above solution in it.

86

• 80 iso-propanol/propanol
• Take 800 ml of iso-propanol/propanol in 1000 ml
  capacity measuring cylinder.
• Take 200 ml of distilled or deionised water in
  1000 ml capacity measuring cylinder.
• Take 1000 ml Conical/flat-bottom flask and mix
  the alcohol and distilled water properly in it.
• Label a reagent bottle as 80 iso-propanol/propano
  l with the date of preparation and transfer the
  above solution in it.

87

• Ether95 Ethanol Mixture
• Take 100 ml of Ether in 100 ml capacity
  measuring cylinder.
• Take 100 ml of 95 Ethanol in 100 ml capacity
  measuring cylinder.
• Take 500 ml capacity conical/flat-bottom flask
  and mix the ether and ethanol in it properly.
• Label a reagent bottle as Ether95 Ethanol
  Mixture with the date of preparation and
  transfer the above solution in it.

88

• Carnoy's Fixative
• Take 60 ml absolute Ethanol in 100 ml capacity
  measuring cylinder.
• Take 30 ml Chloroform in another measuring
  cylinder.
• Take 10 ml Glacial acetic acid in another
  measuring cylinder.
• Mix the above solutions in a 500 ml capacity
  conical/flat-bottom flask.
• Label a reagent bottle as Cornoy's fixative with
  the date of preparation and transfer the above
  solution in it.

89
Namaste