The Short Textbook of Medical Laboratory for Technicians Satish Gupte
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1Medical Laboratory
2

General Introductionchapter 1

 
GUIDELINES
 
Code of Ethics for Laboratory Technicians
Following ethics are expected to be followed by laboratory technicians:
  • No assumption or guess work without proper tests of a specimen as per standard methods.
  • Accuracy and correctness in test reports should be observed.
  • Exhaustive standard method to ascertain correct report in given analysis.
  • Never postpone the work.
  • Laboratory report should only be sent to the doctor concerned. They are not to be disclosed or discussed with patients or attendants of patients.
  • Strictly follow the instruction of doctors and report only the tests they require.
 
Responsibilities of Medical Technician
 
General
  • Keep the laboratory clean.
  • Chemicals, equipment and glasswares should be kept at the places marked for them.
  • Glassware and equipment should be neat and clean.
  • Properly label the chemicals' bottles and reagents with the name.
  • Handle the microscope with care.
  • Clean and sterilize the material as required.
  • Dispose off the specimens and infected material in a safer manner.
  • Handle and process the specimens observing all safety measures.
    4
  • Maintenance of records of laboratory tests done and preparation of monthly report.
  • Indent for supplies to the laboratory through medical officer and make proper enteries to register (consumable items/stock items). Also ensure the safe storage and utilization of material/items received.
 
Laboratory Investigations
  • For examination of urine, do as under:
    • Test specific gravity and pH
    • Test for sugar
    • Test for protein (albumin)
    • Test for bile pigments and bile salts
    • Test for ketone bodies
    • Microscopic examination.
  • For stool examination:
    • Naked eye examination
    • Microscopic examination.
  • Examination of blood may be done as under:
    • Collection of blood specimen by finger-prick technique
    • Hemoglobin estimation
    • RBC count
    • WBC count (total and differential)
    • Preparation, staining and examination of thick and thin blood smears for malarial parasites, microfilariae, etc.
    • Erythrocytic sedimentation rate (ESR).
  • Sputum examination can be carried out by:
    • Preparation, staining and examination of sputum smear for Mycobacterium tuberculosis or fungi
    • Culture the specimen when required.
  • Carry out examination of skin and nasal smear:
    • Preparation, staining and examination of skin/nasal/earlobe smear for Mycobacterium leprae, fungi, etc.
  • Perform examination of the semen:
    • Naked eye examination
    • Sperm count and motility with the help of microscope.
  • Prepare throat and pus swabs, wherever indicated
  • Preparation of media and to do tests for identification of bacteria.
    5
  • In mycology, laboratory to prepare KOH preparation and put up specimen on Sabouraud's medium
  • To work in immunology or serology laboratory for performing tests like widal, pregnancy, CRP, etc.
  • In histopathology laboratory,
    • Naked eye examination of specimen
    • Processing of tissue
    • Cutting sections.
  • In blood bank to do grouping cross-matching, collection and storage of blood from donors
  • To work in biochemistry for various tests like blood sugar, blood cholesterol, blood urea, etc.
 
Instructions for Laboratory Technicians
  • Always wear apron before entering the laboratory to prevent contamination of the clothes and to avoid catching and carrying infection
  • Place the well-being and service to the sick as first priority
  • Be loyal to laboratory work and keep high standard of work
  • Respect and work in harmony with your colleagues and be courteous and considerate to the attendants of the patient
  • Never smoke, drink or dine within laboratory
  • Keep laboratory neat and clean
  • Arrange the laboratory in such a way that one knows where to find each article. Always replace chemicals and other articles in their proper places after use
  • Handle specimen with care. Wash your hands thoroughly with soap and water and rinse them with disinfectant solution after carrying out the tests
  • Avoid using pipette by mouth. Use rubber bulb teats on the pipette to suck up the material
  • Never use refrigerator in the laboratory for storing edibles, etc.
  • After finishing work, swab the working table with a disinfectant solution. Make it a point that floor is swept and swabbed with disinfectant solution daily
  • All contaminated liquid or solid material should be decontaminated and autoclaved or incinerated before disposal
  • If infected material drops on the bench, pour phenol on it, soak it with clean paper and burn the paper or keep it in disinfectant for final disposal.
    6
  • Always take care of your health by taking proper balanced diet and regular exercise to avoid catching infection in the laboratory
  • Workers must take vaccine against chlolera, typhoid, hepatitis B, tetanus, tuberculosis, etc. Always keep your nails short and clean. One should wash hands with soap and water as frequently as required.
 
Arrangement of Laboratory
An ideal laboratory (Fig. 1.1) should have adequate light which may be natural sunlight or electric light. It should be arranged in such a way so that it has:
  • A space for collection of specimens plus records in the form of requisition slips and proper entry in the register, test reports on proper forms, etc.
  • Refrigerator
  • Sink, drain
  • A space for staining
  • A space where stool and urine examinations may be done
  • A space where blood examination may be performed
    zoom view
    Fig. 1.1: Laboratory arrangement
    7
  • A space preferably near window should be reserved for keeping and using of microscope.
 
Main Requirements for Laboratory
 
Furniture
  • Laboratory table unit having:
    • Fomica top and knee space
    • Stainless steel sink with drain board
    • Storage cabinets fitted under the table
    • Reagent rack-fitted above laboratory table.
  • Writing table
  • Steel almirah
  • Chair without arms
  • Revolving chair
  • Dustbin.
 
Instruments
  • Microscope
  • Centrifuge machine
  • Refrigerator
  • Autoclave
  • Sahli's hemoglobinometer (Complete)
  • ESR stand and tubes
  • Colorimeter
  • Hot air oven
  • Laminar flow
  • Wireloops
  • Spirit lamp
  • Balance
  • pH meter
  • Incinerator
  • Gas-pack jar and envelops
  • Distillation plant
  • Needles distray
  • Colorimeter
  • Sysmex cell counter.
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Glassware
  • Beakers
  • Bottles
  • Centrifuge tubes
  • Coverslips
  • Drop bottles
  • Flasks
  • Funnels
  • Glass cylinder
  • Glass rods
  • Glass tubing
  • Measuring cylinder
  • Pipettes
  • Reagent bottles
  • Staining tray
  • Wash bottles
  • Watch glasses
  • Petri dish
  • Syringes
  • Wet chamber
 
Chemicals
  • Acetic acid, glacial
  • Acetone
  • Ammonia
  • Basic fuchsin
  • Distilled water
  • Ethanol 95% (ethyl alcohol)
  • Ether
  • Hydrochloric acid
  • Hydrogen peroxide
  • Immersion oil
  • Methylated spirit
  • Methyl alcohol
  • Petroleum jelly (vaseline)
  • Sodium bicarbonate
  • Sodium dichromate
  • Sodium nitroprusside
  • Sulfur powder
    9
  • Sulfuric acid
  • Tincture benzoin
  • Xylene
  • Glass jars
  • Lysol
 
Stationery
  • Examination request form
  • Registers
  • Report forms
  • Monthly report form
  • Gum
  • Labels for bottles
  • Office files
  • Pens or ballpoint pens
  • Glass marking pencils
  • Rubber bands
  • Ruler
  • Stapler
  • Staples
  • Thread/string
  • Plain papers
  • Paint brush
  • Brown paper
  • Adhesive tape
  • Pins
  • Notepad
 
Miscellaneous
  • Aluminium containers with screw caps (for packing specimen tubes or bottles)
  • Platinum wire
  • Aprons
  • Bottle cleaning brushes
  • Cheatle's forceps
  • Cork screw
  • Forceps
  • Hagedorn needle or lancet for taking capillary blood
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  • Loop holders
  • Slides tray
  • Scalpel
  • Scissors
  • Buckets
  • Duster
  • Scrubbing brush
  • Slide box
  • Soap dish
  • Test tube holders
  • Test tube racks
  • Thermometer
  • Towels
  • Applicator (wooden)
  • Cotton swab sticks
  • Cotton wool
  • Detergent (powder and liquid)
  • Filter paper
  • Matchbox
  • Nylon thread
  • Tongue depressor
  • Wire basket
  • Nailcutter
 
Cleaning of Glasswares
Glasswares should be cleaned in the laboratory. It is necessary to get correct results. Cleaning of glasswares is done as under:
  • Cleaning of new glasswares
    • Mix 3 liters of water and 60 ml of concentrated hydrochloric acid in bucket or basin
    • Leave the new glasswares completely dipped in this solution for 24 hours
    • Rinse twice with water and once with distilled water
    • Dry
  • Cleaning of dirty glasswares
    • Rinse the glasswares twice in cold or lukewarm water
    • Now put the glasswares in solution of water and cleaning agent like washing powder kept in a bowl
    • Brush the glasswares inside the container. Leave to soak for 2 hours
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    • Remove the articles one by one and rinse under tap water
    • Now soak them in distilled water
    • Keep glasswares on the pegs of a wall draining rack or upside down in a wire basket and allow to dry
    • Plug the containers with non-absorb cottonwool.
  • Cleaning of glass pipettes
    • Soon after use, pipette must be rinsed in the container
    • After rinsing, place the pipettes in large plastic cylinder or bowl full of water or disinfectant solution (2% phenol) for 24 hours
    • Soak in soap and water solution, brush and leave to soak for 2 to 3 hours
    • Remove pipette one by one and rinse thoroughly in tap water. Finally, rinse with distilled water. Leave the pipette to dry.
  • Cleaning of dirty glass slides
    • Slides with immersion oil are cleaned one by one by rubbing with newspaper
    • Prepare soap and water solution and soak slides in it for 24 hours.
    • Take the slide out one by one with forceps. Rinse them separately under the tap. Soak them in a bowl of water for 30 minutes
    • Now wipe and dry the slides.
  • Cleaning of coverslip
    • Soak in weak detergent solution (200 ml water + 3 ml detergent + 15 ml of bleaching powder) in a beaker
    • Keep them soaked for 2 to 3 hours
    • Rinse out the beaker with tap water atleast 4 times
    • Give a final rinse with distilled water
    • Drain the coverslips by keeping them on to a pad of gauze
    • Dry them and keep coverslips in a small Petri dish.
  • Preparation of grease-free coverslips
    • In a cylinder, mix 10 ml of 95% ethanol and 10 ml of ether
    • Pour this solution in a Petri dish
    • Place about 30 coverslips one by one in this solution
    • Shake and wait for 10 minutes
    • Bring out coverslips one by one and place in petri dish.
  • Cleaning of synringes and needles
    • Remove the part of syringe (Plunger, barrel) (Fig. 1.2)
    • Fill the barrel with water and put plunger into barrel. Now force the water out through needle several times
    • Remove the needle and rinse the hub cavity.
      12
      zoom view
      Fig. 1.2: Parts of syringe
  • Dichromate cleaning solution
    • Dissolve 25 gm of potassium dichromate in 25 ml of water
    • Slowly add with stirring 50 ml of concentrated sulfuric acid
    • When cool, store this solution in stoppered bottle
    • Always add acid to water
    • Glasswares are immersed in this solution overnight
    • Bring out glassware one by one and rinse in running tap water.
 
Disposal of Laboratory Infected Material and Clinical Specimens
  • Specimens like pus, urine, stool, etc. are usually full of disease causing germs
  • After doing test, they must be destroyed properly
  • Also germ-grown material, especially in microbiology laboratory, should be disposed of.
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Following are the methods of disposal of germ containing objects:
 
Disposable Boxes Containing Stools, Urine, Sputum, etc.
 
Incineration
Construction of an incinerator (Fig. 1.3)
  • It consists of leak proof drums
  • Fix a strong metal grating about 1/3rd of the way up the drums
  • Cut a wide opening or vent below the level of grating
  • Adjust a removable lid for the drum.
Operating of incinerator
  • At the end of each morning and afternoon's work, place all used stool, sputum, discarded culture material, etc. on the grating of incinerator
  • All material should be kept in disposable cardboard cartons or other disposable boxes along with disinfectants like lysol
  • Now close the metal drum tightly both lid and vent
  • At the time of incineration, lid and vent should be kept opened
  • Fill the bottom of the incinerator with sticks, paper, wood, coal, etc.
  • Keep the lid open and lit the fire. Keep the fire burning till germ containing material is completely burnt to ashes
  • The ash so produced is not harmful and can be thrown on the refuse heap.
zoom view
Fig. 1.3: Incinerator
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Burial of Germ Containing Material
  • Dig a pit 4 to 5 meters deep and 1 to 2 meters wide in a corner of open piece of land
  • Make a lid that fits tightly over the pit
  • Upper rim of pit should be strengthened by lining it with bricks and stones
  • Throw clinical germ containing specimens after doing tests into the pit. Replace the lid immediately (Fig. 1.4)
  • Atleast once in a week cover the refuse with layer of 10 cm of dried leaves
  • It is always better to use a layer of calcium carbonate instead of leaves.
 
Sterilization and Cleaning of Glass Containers Used for Germ Containing Materials
  • Use disinfectant like Lysol (5%). Keep disinfectant for 24 hours in the container. Clean container with detergent like soap and water.
  • Autoclave the containers for 20 to 30 minutes at 120°C at 15 lbs pressure per square inch. Cool the container and clean jars with water and soap.
    zoom view
    Fig. 1.4: Burial of germ containing material
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    1. Boil the container with detergent for 30 minutes in watering washing powder in strong solution (60 ml per liter of water) (Fig. 1.5).
    2. Alternatively, water containing sodium carbonate crystal may be used.
  • Container like tubes may be kept in 10% bleaching powder or 2% phenol for 24 hours. Rinse with water and then wash with soap and water.
  • Tubes or bottles containing fresh blood may be rinsed in cold water and then kept in detergent solution.
  • Tubes or bottles containing old blood for several days and weeks at room temperature and containing germs should be kept in 10% solution of commercial bleeching powder for 12 hours. Now rinse them with soap and water.
 
Reagents and Their Preparation
 
Acetic Acid 10% (100 gm/liter)
  • Glacial acetic acid (CH3COOH)
20 ml
  • Distilled water
qs 200 ml
zoom view
Fig. 1.5: Sterilization by boiling
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Acid Ethanol (For modified Ziehl-Neelsen Stain)
  • Hydrochloric acid
3 ml
  • Ethanol 70%
up to 100 ml
 
Barium Chloride 10% (100 gm/liter) Aqueous Solution
  • Barium chloride (BaCl2)
10 gm
  • Distilled water
qs 100 ml
 
Benedict Qualitative Solution
  • Copper sulphate (CuSO4.5H2O)
173.3 gm
  • Trisodium citrate (Na3C6H5O7.2H2O)
173.0 gm
  • Sodium carbonate (Na2CO3) anhydrous
100.0 gm
  • Distilled water:
1000 ml
    • Dissolve copper sulfate by heat in 100 ml water.
    • Dissolve the trisodium citrate and sodium carbonate in 800 ml of water.
    • Add the copper sulphate solution slowly to sodium carbonate/ trisodium citrate solution, stirring constantly throughout.
    • Make up the total volume of solution as 1000 ml with distilled water.
 
Buffered Water (For Giemsa and Leishman Stains)
  • Sodium hydrogen phosphate (Na2HPO4.2H2O)
3.76 gm
  • Potassium dihydrogen phosphate (KH2PO4) anhydrous
2.10 mg
  • Distilled water
qs 1000 ml
    • Adjust pH of solution using narrow range pH paper
pH 7.0 to 7.2
 
Buffered Water (For JSB Stain)
  • Disodium hydrogen phosphate
0.417 gm
  • Potassium acid phosphate
0.752 gm
  • Distilled water
2000 ml
  • pH required
6.2 to 6.8
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Carbol Fuchsin (For Ziehl-Neelsen Stain)
 
Solution A
  • Saturated solution of basic fuchsin
    • Basic fuchsin
3 gm
    • Ethanol 95%
100 ml
 
Solution B
  • Phenol solution 50 gm/liter (5%) aqueous
  • Phenol
10 gm
  • Distilled water
200 ml
    • Measure solution A
10 ml
    • Measure solution B
90 ml
    • Mix them and use carefully as it is highly poisonous
 
Carbol Fuchsin (For Modified Ziehl-Neelsen Stain)
  • Basic fuchsin
5 gm
  • Ethanol 95%
10 ml
  • Phenol 5% solution
90 ml
 
Cary-Blair Transport (Holding) Medium
  • Sodium thioglycolate
1.5 gm
  • Disodium hydrogen phosphate (Na3HPO4) anhydrous
1.1 gm
  • Sodium chloride
5.0 gm
  • Agar
5.0 gm
  • Distilled water:
991.0 ml
    • To be prepared in chemically clean glassware
    • Heat while mixing the contents till the solution becomes clear
    • Cool the mixture to 50°C
    • Add 9.0 ml of freshly prepared aqueous calcium chloride 1% (10 gm per liter)
    • Adjust pH to about 8.4
    • Pour 7.0 ml in properly rinsed and sterilized 9.0 ml screw-capped vials
    • Steam the vials containing media for 15 minutes, cool and tighten the cups.
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Crystal Violet
 
Solution A
  • Crystal violet
2.0 gm
  • Ethanol 95%
20 ml
 
Solution B
  • Ammonium oxalate [(NH4)C2O4.H2O]
0.8 gm
  • Distilled water
80 ml
    • Mix solution A and B
    • Store for 24 hours before use
    • Filter through filter paper into staining bottle
 
Dichromate Cleaning Solution (For Cleaning Glasswares)
  • Potassium dichromate (K2Cr2O7)
100 gm
  • Water
1000 ml
  • Pure sulphuric acid
100 ml
    • Dissolve the dichromate in water
    • Add acid very carefully and slowly
    • Stirring should be continuous
    • Always remember that acid must be added to the water
 
EDTA (Dipotassium Salt Solution) 10%
  • Dipotassium ethylene diamine tetra-acetate
20 gm
  • Distilled water
qs 200 ml
    • For use pipette 0.04 ml of solution into a small clean container marked to hold 2.5 ml blood
    • Allow anticoagulant to dry by leaving the containers overnight on warm bench or in an incubator at 37°C.
 
Eosin (2%) Solution in Saline
  • Eosin
2 gm
  • Sodium chloride (0.85%) (8.5 gm/100 liter) in aqueous solution
qs 100 ml
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Fouchet's Reagent
First prepare 10% solution of ferric chloride
  • Ferric chloride FeCl3
10 gm
  • Distilled water
qs 100 ml
Preparation of reagent
  • Ferric chloride solution as prepared above
10 ml
  • Trichloroacetic acid (CCl3 COOH)
25 gm
  • Distilled water
100 ml
    • Dissolve trichloracetic acid in 70 ml of distilled water in volumetric flask
    • Add 10 ml of the 10% ferric chloride solution
    • Make the volume 100 ml with distilled water.
 
Giemsa Stain
  • Powdered Giemsa stain
0.75 gm
  • Methanol (CH3OH)
65 ml
  • Glycerol
35 ml
    • Put the ingredients in a bottle containing glass beads and shake
    • Shake the bottle 3 times a day for 4 consecutive days
    • Filter and use.
 
Gram Iodine Solution
  • Iodine
3.3 gm
  • Potassium iodide
6.6 gm
  • Distilled water
100 ml
    • Measure 100 ml of water in cylinder.
    • First of all dissolve the potassium iodide in about 30 ml of the water
    • Now add iodine and mix until dissolved
    • Add the 70 ml water and mix well
    • Store in a brown bottle.
 
Hydrochloric Acid (0.1N) 0.1 mol/liter
  • Hydrochloric acid (HCl) concentrated
8.6 ml
  • Distilled water
qs 1000 ml
    • Measure 500 ml of water
    • Acid may be added drop by drop
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    • Add water till the volume becomes 1000 ml
    • Use it within 1 month
    • Useful only for hemoglobin estimation by the Sahli's method
 
Jaswant Singh and Bhattacharya (JSB) Stain
 
JSB Solution I
  • Methylene blue
0.5 gm
  • Sulfuric acid 1%
3.0 ml
  • Potassium dichromate
0.5 gm
  • Disodium hydrogen phosphate dihydrate
3.5 gm
  • Distilled water
500 ml
    • Dissolve methylene blue in distilled water
    • Add sulphuric acid slowly, 1 ml at a time. Stirrer it constantly and mix well
    • Add potassium dichromate. There is formation of purple precipitate
    • Now add disodium hydrogen phosphate dehydrate. After stirring, it for some time precipitates get dissolved
    • Boil the solution in a flask with a reflux condensor for 1 hour when the blue color of solution deepens.
 
JSB Solution II
  • Eosin (yellow, zinc free)
1.00 gm
  • Distilled water
500 ml
 
Leishman Stain
  • Leishman powder
1.5 gm
  • Methanol
qs 1000 ml
    • Rinse out a clean bottle with methanol
    • Add a few clean dry glass beads
    • Add staining powder and then methanol
    • Mix well to dissolve the mixture
    • The stain thus prepared is kept as such and can be used next day
    • Take precautions, during preparation and storage of this strain, not to allow moisture to enter.
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Lugol Iodine Solution
  • Iodine
1 gm
  • Potassium iodide
2 gm
  • Distilled water
100 ml
    • Dissolve potassium iodide in about 30 ml of the water
    • Add iodine and mix until it is dissolved completely
    • Add 70 ml water and mix well
    • Store the solution in brown bottle.
 
Methylene Blue Aqueous
 
Solution for Ziehl-Neelsen Stain
  • Methylene blue
0.5 gm
  • Distilled water
100 ml
    • Mix methylene blue in 100 ml water
    • Filter after dissolving and store in bottle.
 
Ziehl-Neelsen Stain
  • Methylene blue as prepared above
0.5 gm
  • Borax
5.0 gm
  • Distilled water
100 ml
 
Orthotolidine Reagent
  • Orthotolidine dihydrochloride
1.35 gm
  • Concentrated hydrochloric acid
150 ml
  • Distilled water
850 ml
    • Distilled orthotolidine dihydrochloride in 500 ml of distilled water (Solution I)
    • Make a mixture of 350 ml of distilled water and 150 ml of concentrated hydrochloric acid (Solution II)
    • Add solution I and solution II with constant stirring
    • Store the solution in amber-colored bottles.
 
RBC Diluting Fluid
  • Sodium citrate
3 gm
  • Commercial formaldehyde solution containing at least 37% formalin
1 ml
  • Distilled water
100 ml
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Safranine Solution
 
Stock Solution
  • Safranine O
2.5 gm
  • Ethanol 95%
qs 100 ml
 
Working Solution
  • Stock solution
10 ml
  • Distilled water
900 ml
 
Sodium Chloride Solution (0.85%)
  • Dissolve 0.85 gm sodium chloride in 1000 ml distilled water and store in bottle
  • It is also called isotonic saline or normal saline.
 
Sodium Thiosulfate Aqueous Solution
  • Sodium Thiosulfate anhydrous (for an equivalent quantity of Na2S2O3.5H2O)
3 gm
  • Distilled water
qs 100 ml
    • Mix and store in brown drop bottles
    • It is used to neutralize any chlorine in water samples taken for bacteriological analysis.
 
Sperm Diluting Fluid (Formalin-Bicarbonate)
  • Sodium bicarbonate
5 gm
  • Commercial formaldehyde solution (formalin)
1 gm
  • Distilled water
100 ml
 
WBC Diluting Fluid
  • Acetic acid (CH3COOH) glacial
4 ml
  • Distilled water
qs 200 ml
  • Aqueous methylene blue solution
10 drops
    • Dissolve 3.0 gm methylene blue in 100 ml of distilled water
    • Filter it
    • Add it to the acid solution (100 ml distilled water + 4 ml acetic acid).
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Willis Solution
  • Sodium chloride
125 gm
  • Distilled water
500 ml
    • Dissolve sodium chloride by heating the mixture to boiling point
    • Leave to cool and wait
    • Some of the salt powder should remain undissolved
    • If all salt is dissolved, then add 50 gm sodium chloride more
    • Filter the solution and store in corked bottle
    • This is saturated solution of sodium chloride.
 
Drabkin's Solution
It is used for hemoglobin estimation. It contains:
  • Potassium cyanide
500 mg
  • Potassium ferricyanide
200 mg
  • Potassium dehydrogen phosphate
140 mg
  • Distilled water
1 liter
  • pH
7.0–7.4
 
Indicator
Indicators in chemistry are compounds which are weak organic acids or bases. Depending upon the pH of the medium, these exist either in unionized form or ionized form. The two forms of the molecule have different colors. The color imparted to the medium by the indicators depend upon the relative proportions of unionized or ionized form of the indicator. The relative proportion of ionized or unionized form depends upon the pH of the medium and the dissociation constant (pK) of the indicator, as per the equation:
zoom view
Since pK of an acid-base is constant, the ratio of salt to acid dependents on pH. The color of the undissociated and dissociated forms are different, so the color of the medium will be dependent on the ratio of the two forms, which is, in turn, dependent on pH.
To clarify we take the example of bromocresol green. The color of the undissociated form is yellow and that of dissociated is blue. The pK of bromocresol green is 4.7. At a pH of 4.7, the ratio of salt/acid is (i.e. both dissociated and undissociated forms are equal) and hence the equation:
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zoom view
Thus the color of the medium is green (i.e. equal amount of yellow and blue).
As the pH decreases, the yellow form of the indicator increases and at pH 3.8, it is almost pure yellow. Similarly, at pH 5.4, the indicator has pure blue color. In between pH 3.8 and 5.4, there exists a mixture of yellow or blue imparting various shades.
Selection of an indicator for determination of pH or during titration is usually on the basis of the range of pH likely to be encountered. For routine titration, the following indicators serve the purpose well.
  • Strong acid versus strong base
:
Any indicator
  • Strong acid versus weak base
:
Methyl orange
  • Weak acid versus strong base
:
Phenolphthalein
  • Weak acid versus weak base
:
No indicator is satisfactory
 
Preparation of Solutions in General
 
Percent Solution
To prepare a solution of a particular percentage, dissolve that much amount in grams in the required solvent, dilute it finally to 100 ml (W/V) or take the required volume in ml of the solute in liquid form, dissolve in the solvent and make the volume 100 ml with the solvent (V/V).
Note: Solvent is a medium in which a substance is dissolved, e.g. water.
Solute: The substance to be dissolved is called solute, e.g. when salt is dissolved in water, the salt is called the solute and water the solvent.
Solution: When solute is completely dissolved in a solvent, the resultant mixture is called solution.
 
Molar Solution
All compounds have their own molecular weight depending upon the number and proportion of component elements.
When molecular weight in grams of a substance is taken and dissolved in a solvent to a final volume of 1 liter, it makes molar solution accordingly twice the molecular weight in gram per liter will be two molar 2M, etc.
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Normal Solution
Equivalent weight (molecular weight divided by valency) of a substance in grams dissolved per liter of solution is normal solution or 1 N. When the valency is one the molar solution is equal to normal solution, e.g. 40 gm of NaOH per liter of solution makes a molar or a normal solution. In case, the valency of an acid/base is two, the normal solution is half the molar solution, e.g. 74 gm of Ca(OH)2 per liter makes a molar solution but 37 gm per liter are required to make a normal solution.
 
Preparation of Subnormal Solution
Solution of lower molarity or normality can be prepared from 1M/1N solution by the formula
N1V1 = N2V2
N1 = Normality of solution I
N2 = Normality of solution II
V1 = Volume of solution 1
V2 = Final solution
Keep one side for the acid available and the other side for the solution finally to be made.
Molarity of sulphuric acid (H2SO4)
Where, W
= Weight of borax acid
T
= Volume of acid used (ml)
381.4
= Molecular weight of borax acid
Now adjust the molarity according to the equation: N1V1 = N2V2
 
Examples
N/1 sulfuric acid is prepared as under:
Molecular weight of sulphuric acid = 2.016 + 64 + 32.066 = 98.082
  • Therefore, N/1 sulfuric acid should contain 49.041 gm per liter
  • Specific gravity is 1.84, therefore volume would be theoretically = 49.041/1.84
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  • Concentrated sulphuric acid contains only 95% pure acid and so volume of acid required is
 
Method
  • Take 30 ml of concentrated sulphuric acid
  • Pour slowly with stirring into 800 ml of water
  • Cool and make upto 1000 ml
  • Place 10 ml N/1 Na2CO3 (using volumetric pipette) in a beaker or flask
  • Add a drop of methyl orange as an indicator
  • Fill a burette with sulphuric acid and titrate the sodium bicarbonate with it until the color just changes to pink.
 
Calculation
  • Suppose 9.5 ml of acid is required in the titration, then 9.5 ml acid is equivalent to N/1.Na2CO3
  • To make a normal solution, add 0.5 ml water to each 9.5 ml of sulphuric acid
 
N/10 Sulfuric Acid
  • Take 10 ml of N/1 sulfuric acid
  • Dilute to 100 ml with distilled water.
 
N/1 Hydrochloric Acid
Molecular weight = H (1.008) + Cl (35.457) = 36.465
Specific gravity = 1.18, % of hydrochloric acid = 38, i.e. 80.6 ml of hydrochloric acid made up to 1000 ml to make N/1 hydrochloric acid.
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Dilutions of Sample or Solution
Errors are usually made during calculations when samples or solutions are subjected to dilutions.
Double dilution: When one part or volume of a solution is mixed with one part or volume of diluent making a final volume of two (2 ml of blood + 2 ml saline = 4 ml).
Five times dilution: One part or volume of a sample or solution when mixed with four parts of diluent to make a final volume of five. Similarly, six times, seven times, ten times, etc.
 
Preparation of Standard Acids and Bases
 
Hydrochloric Acid (Approximately 1 mol/liter)
Titrate 90 ml of conc. HCI (sp. gr. 1.18) per liter. Titer it with sodium titraborate (Na2B4O7. 10H2O, mol wt 381.4) using methyl red indicator and adjust accordingly.
 
Sulfuric Acid 1 M
Prepare an approximate 1 M solution of H2SO4 by taking 30 ml of concentrated acid. Add slowly to about 500 ml of water. Dilute finally to 1 liter and mix well. Standaradize against borax and adjust accordingly.
 
Sodium Hydroxide 1 M
Quickly weight out 40 gm of solid sodium hydroxide and dissolve it in water and make it up to 1 liter. This makes an approximate 1 M solution. Titrate it with standardized in HCI or the H2SO4 using phenolphthalein as an indicator.
 
Standardization Against Borax
Weight out accurately about 4 mg of borax (Sodium tetraborate, Na2B4O7 .10H2O). Transfer it to a conical flask and dissolve it by warming in about 100 ml water. Add a few drops of methyl red indicator and titrate it with HCI or H2SO4 to find out the molarity of the acid, using the following equation:28
 
SI Units
To have uniformity in expression of analytical results in clinical chemistry, a system was adopted in 1966 based on SI units (System International d'Units) by International Federation of Clinical Chemistry and approved in 1967. The seven basic SI units are as follows:
Quantity
Unit
Symbol
Length
Meter
M
Mass
Kilogram
kg
Time
Second
sec
Current
Ampere
A
Thermodynamic temperature
Kelvin
K
Amount of substance
Mole
Mol
Luminous intensity
Candela
cd
These are recommended prefixes used to form decimal multiples and submultiples of SI units such as deca (da) =101, hectar (h) =102, kilo (K) =103 or milli (m) =10−3, micro (μ) =10−6, nano =(n) =10−9, etc.
The decimal sign between digits is indicated by a full stop. A zero should recede the decimal sign if numerical sign is less than 1. The use of such expressions as 0.003 mol/L is not recommended rather it should be expressed as 3 mmol/L.