Prof: R.

Shinde

INDEX

SR. NO

NAME OF TOPIC

PAGE NO.

1.

Human health & medical care in India

2.

Organization of the clinical laboratory and role of medical lab technician

3.

First aid, safety regulation and lab record system

4.

Basic laboratory equipment

5.

Reagent preparation

6.

Quality control of laboratory findings

7.

Syllabus copy

8.

Model Question Paper

Prof: R. Shinde

CHAPTER 1 HUMAN HEALTH AND MEDICAL CARE IN INDIA Q1. What is Homeostasis & how does it differ from the pathogenic state of the body? Ans: 1. Health is defined as state of complete physical, mental, or social well being and not merely the absence of disease or infirmity. 2. Health is of two types good health and bad health and they are in a delicate balance this is called as Homeostasis. 1. 3.This delicate internal balance under normal condition is disturbed by various factors that lead to pathogenic state or abnormal condition. 3. Some of the notable factors for pathogenic state of the body are genetic disorders, infection through pathogens, trauma caused by accident, circulatory disorder causing disturbance in the blood flow, metabolic abnormalities and neoplasia. 4. E.g. Neoplasia or the abnormal growth may be controlled growth or spreading in nature, which leads to uncontrolled growth or reproduction of cells a condition often referred as cancer. 5. Hence to maintain Homeostasis and good health is not only an individuals responsibility but is also dependent on the interaction with the community, government and other nations of the world. 6. Hence a person should not smoke not only for the sake of his own health but also for the control of air pollution and this will ultimately used for maintaining homeostasis to good health. Q2. What is diagnosis? How does the laboratory help in the process of diagnosis? Ans: 1. The pathophysiological condition of body found in patient brought to normal in two steps. 2. First step is diagnosis and second step is treatment. 3. Diagnosis means to identify or determine the nature of disease and its causative factor. 4. Without correct diagnosis the treatment to eliminate the condition or the causative agent cannot be effective. 5. Diagnosis begins with the physical examination of the patients by the physician. 6. Then it also includes the laboratory investigation of the patient sample such as blood, urine, ..stool etc. 7. This helps or supports the physical examinations done by the physician. 8. After the laboratory results submitted these will either confirmed or change the provisional diagnosis, hence laboratories plays major role in the early well being of the patient. 9. It analyzes the specimen of the patient, which gives particular reading that reflect the state of the person.

Prof: R. Shinde

Q3. In what ways do the medical care in India differ from that of USA or USSR? Ans: 1. Medical care in India is combination of old and modern medicines. 2. Old or traditional system of medicine includes Ayurveda, Homeopthy Unani which are now described as unscientific, medicine. 3. The choice of medicine depends upon the socio-economic study of the individual. 4. India is unique in respect to healthcare. 5. It cannot be compare either with underdeveloped countries or with advanced countries. 6. Government of India cannot provide free treatment to all its citizens neither there is enough awareness or money to buy health insurance policy from the people side. 7. The doctor patient ratio in India is 1: 2000 (1985 Study). 8. Doctors are scares in rural areas money is looking, also the facility for drugs, equipments etc. 9. In USA and USSR modern system of medicine is adopted where stress is given on. Modern equipments as well as facilities are provided even in rural areas. 10. Care of ill is taken by government as they have sufficient fund for the same. Q4. What is health insurance? How does it differ from governmental health care plan? Ans: 1. Health insurance is the system where person adopts a health policy according to his needs. 2. In this customer pays a fixed amount of premium to service provider takes care of his medical bills, during his illness period as per the mutual agreement. 3. Government provides a various types of health care centers ranging from primary health care centers to tertiary hospitals, where the diagnosis as well as treatment facilities are made available to the patient either free or at the low cost. 4. In health insurance plan, service provider gives list of hospitals from where patient can avail cashless facility. Q5. What is competency based education? How does it differ from classical (fundamental) education? Cab be competency based education be combined with classical education? Ans: 1. Competency based education is more of practical oriented nature where it teaches technician the practical problems while handling machinery and specimen. 2. Classical and fundamental education gives importance to causes rather than specifically handling of the instrument. 3. The modern approach should be the give competency based technical education. 4. Registry should be made compulsory for qualifying technicians /technologist. 5. The registry examination should be based on work knowledge and not merely on good to know information. 6. The competency based education can be combine with classical education or fundamental education in a manner that clinical laboratory technician should be made to understand the basic of anatomy and physiology of body under healthy and diseased conditions.

Prof: R. Shinde

7. Mastery in performing various diagnostic tests practically, calculation of results, adopting new quality control measures also teaching about communicating results to the physician. 8. Principal of the test should also be known to the technicians for trouble shooting. Q6. Identify the problems of the medical laboratories in India what are your suggestion to improve the situation? Ans: Medical laboratories in India lack the following i. Competent and well educated staff ii. ii. Automation is not provided at many places. iii. Rural places lack clinical laboratory services. iv. At many places technician doesnt know that he lacks the knowledge. v. minimum required standards are not laid down or followed. Suggestions: Minimum standards should be adopted and followed. Automation is must Laboratory services to rural areas should be made available at responsible rates. Laboratory staff should be well trained practically as well as theoretically. An independent body should be formed which will a) Do periodic examinations of laboratory and grade it accordingly. b) Conduct continues education classed for laboratory staff and attendance is compulsory. c) Testify the knowledge of technician for exams, practical as well as theoretical. d) Safety measures and increase in pay should be provided to technicians. e) Improvement in facility as well as quality of technician combined with modern automation will change the complete scenario in India of the medical laboratories as mainly technicians are bench workers in laboratory. Q7. What are the difference between MLTs and MT in terms of education training and job performance? Ans: 1. Medical laboratory technicians are bench workers who receive 1-2 yrs. Of certificated training after higher secondary school. 2. Medical technology on other hand has bachelor of science Degree. Medical laboratory technician and technologist both are bench workers. 3. M.L.T is having more workable knowledge rather than the theoretical one. Due to lack of knowledge he may fail to give require quality of the test, which in turn may affect the result. 4. Medical technologist along with workable knowledge also have the theoretical knowledge, can understand the results better having knowledge of principle test have better reasoning also adapt to modern automation easily. 5. M.T. having more knowledge and experience will naturally having better job, opportunities compare to MLTs having more number of M.Ts in laboratory means maintaining good students of lab.

Prof: R. Shinde

Q8. What is the significance of containing education for the clinical laboratory personal? Ans: 1. Continuing education is importance as specific achievements and advances are constantly growing and our knowledge should be never stagnated. 2. The constant update of knowledge is necessary in every field. 3. As the technician works in laboratory he does not have link with the education institute and his old knowledge become absolete. 4. Hence to meet the challenge of constant up gradation in medical laboratory science, professional bodies. Should offer work ship for technicians. 5. There is increasing demand for medical laboratory technicians hence all the employees of laboratory should be better equipped with current information. 6. MLTs should have awareness of personal responsibility and continues drive for self improvement in personal skill and knowledge. Q9. What is quality control? Why it is important to introduce quality control measures in the clinical laboratory operations? Ans: 1. Quality controls means laiding down certain standards of regulations which are supposed to be strictly implemented on the laboratory in respect of minimum qualification of staff, working condition, environment and quality of equipments. 2. The laboratory and hospital are not beyond legal restraint. Its standard of performance is based on moral obligations towards the fellow human beings. 3. Such rules of conduct both personal and professional are called ethics. 1. 4.The most important consideration of professional ethics is honesty, the date provided by technician should be reliable. 4. The tragedy lies in the fact that many technicians do not know that they do not know. 5. Hence running of quality control measures is crucial and so the use of revised procedure. 6. Confidentiality of the patient must be maintained, the place of work must be neat and clean, equipment is properly checked at intervals and in case of breakdown. It must be reported to supervisor. Q10. Justify or refute the following statements indiscriminate training of laboratory wi thout appropriate educational background as resulted in poor quality of laboratory results in developing countries? Ans: 1. At many places non qualified staff is trained to work in laboratory, who has only work had knowledge. 2. They lack the recurred theoretical knowledge, like having no knowledge about anatomy and physiology of the body. 3. Also they do not have knowledge about cause of disease and disease process. 4. They failed to understand the principle of the test. 5. This all things get reflected into the total quality of the laboratory which can get reflected from behavior of the staff to the results of the tests. 6. Also lack of knowledge makes laboratory workers fail to handle the modern equipments. 7. All this factors results in poor quality of results in developing countries. *******

Prof: R. Shinde

CHAPTER 2 ORGANIZATION OF THE CLINICAL LABORATORY AND ROLE OF MEDICAL LABORATORY TECHNICIAN. Q1. What are the responsibilities of a pathologist medical technologist and medical laboratory technician? Ans. 1. Pathologist is a medical science graduate with specialization in medical science and is in charge of the clinical laboratory. 2. Pathologist check the work done by the technician and technologist. 3. All external communication bears the signature of pathologist. 4. Medical lab technician and technologist are trained to do diagnostic test practically. 5. Internal communication (internal result) bears the signature of technicians or technologist. 6. The technician complies with the physician result for investigation. The results are sending to physician. 7. The pathologist screens all the reports before they are dispatched to the physician. 8. The technician should be enough smart to understand what physician is looking for the technician should confirm his with other section of pathology laboratory to help physician reaching diagnosis. 9. Sometimes he even can suggest other possible test for confirmation for diagnosis. 10. The medical laboratory technicians and technologies play a vital role in lab diagnosis. 11. the pathologist is responsible for their performance bench work is done by technician. Q2. What are the educational backgrounds of a technician and a technologist? Ans : 1. The medical laboratory technologist is a graduate with BSC Degree with receives a specialized training which pertains to clinical laboratory procedures. 2. The medical lab technician holds a higher secondary school certificate and receives clinical lab training. Q3. List the important section of the clinical laboratory and state each of their functions. Ans: 1. Clinical laboratory has the following sections. 2. Hematology 3. Blood bank a) Microbiology b) Bacteriology c) Mycology d) Parasitology 4. Serology or immunology 5. Clinical pathology 6. Clinical biochemistry 7. Histopathology and cytology

Prof: R. Shinde

HAEMATOLOGY: Haematology is study of blood and its components. Haematology section of laboratory is primarily involved in the microscopic examination of blood with along with physical examination of whole blood and determination of HB. It includes the test like CBC, ESR, PCV, BT, CT, PT, PTT etc. BLOOD BANK: The primary function of blood bank is to provide appropriate blood to the patient for transfusion. Tests involved in blood bank is like blood grouping, coombs test, cross matching and blood transfusion and blood components separation and processing. MICROBIOLOGY: The microbiology section conducts microscopic examination of the specimen. Its culture and identification of the offending Microorganism. Tests involved in microbiology are identification of organisms, culture and antibiotic sensitivity test etc. SEROLOGY: Serology helps in detection of antibodies in the serum which are produced by infections agents. Tests involved in this like WIDAL, VDRL, RA, CRP, ASO HIV etc. CLINICAL PATHOLOGY: It is a branch of science which deals with study of body fluid except blood. Clinical pathology helps to examine specimen like urine, CSF, stool and semen. CLINICAL BIOCHEMISTRY: Clinical biochemistry helps in identify organ related disorders like liver, kidneys pancreas etc. Tests procedures involved like estimation of glucose, BUN, cholesterol, triglyceride, bilirubin, SGOT, SGPT etc. HISTOLOGY AND CYTOLOGY: Histological examination involves microscopic study of tissue in order to find out any abnormality. Q4. What are the desirable qualities of medical technologist? Ans: 1. Medical technologist should have towards patients. Dedication, Co-operation neatness are essential on the part of the technical. 2. He should have knowledge of the test and about the condition of the patients. 3. Technologist should be honest, reliable and accurate and should have organizational skills.

Prof: R. Shinde

4. He should understands the priority of the test and be courteous with patients while collecting specimen. 5. Lab technician should behave technically. 6. Technician should keep the information of the patient very confidentially. 7. Medical lab good relation with superiors, patients and their families. Q5. How would you counsel a high school for becoming a laboratory technologists? Ans: 1. There are the jobs of trained and knowledgeable to lab technologist. 2. This job has fixed hrs of duties especially in day time which is an advantage to married females. 3. Medical technologist can also do the job of phlebotomist. 4. Job in this field is available in all places like bigger cities as well as in small towns. 5. There is a job satisfaction in this filed as medical technologist assist physician to reach diagnosis and patients. Q6. What is the goal of professional organization of medical laboratory technologist and technician? Ans: The primary goal of such organization is to provide common platform for its members to exchange information and voile their needs to the governments and public. Q7. After five years in the job of laboratory technologist one begins to feel that training of the school is becoming absolute. What would you suggest for updating the information while on the job. Ans: 1. Through the organization of technician continue education should be arranged for its members periodically and which will help them to updates their knowledge, professional bodies should offer workshops for technician and periodic tests should be conducted for the technicians. 2. Medical laboratory technologists and technicians should join to form a professional organization like all India medical laboratory platform for its member to exchange information.

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Prof: R. Shinde

CHAPTER 3 FIRST AID AND SAFETY REGULATION Q1. What are the basic of primary causes of accident in a clinical laboratory and how can these be prevented? Ans: 1. Basic causes of laboratory accidents are given below 2. Use of unexperience or unskilled people. 3. Failure to maintain discipline. 4. Failure to obey safety rules and regulations. 5. Failure of the person in change to give adequate information. 6. Failure to provide proper equipment and protective device. 7. Failure to follow instruction or rules. 8. Physical condition (i.e. illness, fatigue, to intoxicate) 9. Lack of use of equipment with undiscovered defects. Prevention: We can prevent or minimize laboratory accident by: 1. 2. 3. 4. 5. Use of experienced and skilled people. To obey safety rules and regulations. The person which is In charge has adequate information. By providing proper equipment and protective devices. Follow instructions or rules as per the given by senior or manual.

Q2. State the use of each thing kept in the first aid box. Ans. First aid box should at least contain following things:1. Sterile cotton 2. Medical adhesive tapes 3. Sterile gauze piece 4. Roller bandage of various width 5. A pair of scissors 6. Ammonia 7. Safety pins 8. Sodium carbonates (5%) 9. Iodine and mercurochrome solution 10. Baric acid solution 11. Soap water 12. Burnol. Use: 1. Sterile cotton: It is used for cleaning and wipe out the blood from the damage are of the body. It is also used for cleaning the containers. 2. Medical Adhesive Tape: for dressing purpose

Prof: R. Shinde

3. Sterile gauze piece: It is used for cover the wound area. 4. Roller Bandage: It is used for dressing the damage site. 5. A pair of Scissors: It is use to cut the bandage, while cleaning the site of damage it is also used for holding the cotton. 6. Ammonia: It is used in fainting case. 7. Safety Pins: It is used to packing the bandage. 8. Sodium Carbonate (5%): It is used in when acid splash in the case of eye accident cause due to acid. 9. Iodine and Mercurochrome: It is used as antiseptic. 10. Boric Acid Soln : It is used in alkali splash in case of eye accident cause due to alkali. 11. Soap water: It is used as emetic when any chemical gets swallowed. 12. Burnol: It is used in case of Burns. Q3. How would you handle a person suffering from asphyxia? Ans: Asphyxia is different inbreathing which leads to fainting. Fainting usually results from slight injuries, exposure to overheated rooms and a want of food exhaustion and fatigue. The person about to faint feels dizzy and weak, turns pale and falls unconscious on the floor or on the chair. If you notice that a person to faint, you can revive him by taking the following steps in sequence. 1. Bend his dead down between his knees, it he is sitting on the chair. 2. If he on the floor laid him flat on his back and lower his head by shoving of folded coat or blanket under his hips or by raising his feet and legs. In either condition, loosen all clothing around the neck and waist. Open the window or fan see that he gets plenty of fresh cool air. Hold the handkerchief containing a few drops of ammonia under his nose every minute or two. Q4. How should ether handled in the laboratory? Would you recommend a refrigerator for storing ether? Ans: 1. Ether is highly flammable and volatile in nature. It has flash point -450c. So never store the ether in refrigerators as this could lead to explosion. 2. Store ether in metal containers. Small quantities should be placed in amber colour bottles. Ether is toxic so on the bottle, this should be written as position. 3. Ether always kept away from the flame. Q5. What steps should be taken to protect the laboratory from biological hazards? Ans: 1. Laboratory specimens are very dangerous and may cause serious lab acquired infections like hepatitis, T.B, AIDS, etc. 2. Various steps should be taken to protect laboratory from biological hazards in following processes. 1) Specimen collection:1. Wash the hands thoroughly after the collection of specimen. 2. Label the specimen clearly if it is suspected to contain dangerous organisms.

Prof: R. Shinde

3. The outside of the container should be wiped clean with alcohol swab. 2) 1. 2. 3. Transportation of specimens: Clearly labeled the container, whenever possible put the specimen in a plastic bag. Wear disposable gloves while removing the specimen from transport bags. If a leaky or spilled specimen arrives in the microbiology lab, place the container on several layers of waste paper or newspaper and thoroughly the outer surface. After processing the specimen for laboratory testing, thoroughly clean the work area, wash hands with phenolic soap.

3) Specimen processing in the laboratory: 1. Always use gloves in handling infectious material highly infectious agents. E.g. AFB must be handled in fume hood which is kept under negative pressure. 2. While working in the microbiological lab, discard all cotton swabs and small containers with unused specimens in 5% phenol or bleach. 3. Aerosolization during flame sterilization of loops and needles is a source of biological hazard. 4) Disposal of specimens: 1. Most unused specimens after treating with 5% phenol for two hours can be discarded in the drain. 2. The container can be recovered for re-use after autoclaving for 30 minutes at 1210c. 3. Never throw the specimen in the sink. They can be disposed of in the sewer and flushed thoroughly. 4. This is standard procedure for discarding urine, stool, stomach, contents, body fluids etc. 5. Highly infective specimens cab be covered with water and autoclaved before discarding. 6. The same procedure can be followed in case of liq.Media. 7. Solid wastes, that can burn, and incinerated. 8. Sputum specimen suspected from T.B must be autoclaved for 40 minutes. Q6. What is the significance of technicians record book? Ans: 1. It keeps all the raw date, calculations, and results reported along with the accession number given by the laboratory. 2. The technicians record book should also indicate the result of quality control and only maintenance record. 3. When the technician communicates the results to the central desk though the result reporting slip. The results are entered into the central registration book and the receiving clerk sends the requisition slip with the report of pathologist signature. 4. This is then dispatched to the physician who requested the tests. 5. If the patient needs a copy, he gets 1 from the physician; the laboratory does not send the result directly to the patient, unless it is asked to do so.

Prof: R. Shinde

Q7. What preventive measures would you take against laboratory fire? Ans: The following preventive measure against fire are recommended 1. Turn off the flame before leaving the laboratory ay time. Never keep an open flame near flammable liquids. Work with the latter under a hood. If the flammable liquid is on fire inside the container, quickly cover the container with non inflammable material. 2. Put NO SMOKING signs in areas where flammable compound are handled. 3. Do not throw burning matches into a waste basket, through them into a non flammable metal container. Q8. What steps should be taken to remove an object loaded in the ear? Ans: First aid for an object loaded in the air involves addition of oil into the ear with the object. Later when the head is titled the object may flow out with the oil. Q9. What is the difference between qualitative, quantitative, and semi quantitative? Ans: a) Qualitative: qualitative reports give a general impression without any numerical value. Example of such reports is, Positive or negative. Present or absent. b) Semi Quantitative: this kind of report is halfway between qualitative and quantitative reports. The actual numerical value is considerably subjective and various from technician \to technician and yet the physician is able to some extent assess the severity of the situation. For example : negative Doubtful Mild +/+

Moderate ++ Severe Gross +++ ++++

Above notations are used to communicate different things. In blood banking it may indicate the degree of agglutination; in protein test for CSF by the pandy method, it show degree of precipitation in parasitology it is useful in giving an idea of the number of parasites seen under microscope. Such expression as few, many, abundant are also semi quantitative measures. Some quantitative results are given below. ML FR MO SR : : : : Mild Fair moderate Severe AD SI IN LO : : : : adequate Slightly increased Increase Low

Prof: R. Shinde

OC AF

: :

Occasional A Few

VL

Very low

c) Quantitative : results are reported in quantitative terms. The unit are more often in mg/100ml, but some are in g/100ml and electrolytes are in mmol / L or mEq/L, theclerance rate is expressed in ml/min. all quantitative results are compared against a standard. Quality control serum must be analyzed at the beginning of the day. Q.10 How would you take the daily inventory so that you are not interrupted during the laboratory testing? Ans: The inventory is taken during evening hour so that all supplies are ready before new specimen arrives on the following mornings. The drawer under bench should well organize with proper compartments for pipettes, rubbing tubes, glass rods and others. Number the drawer and list the materials as they are kept in these drawers. Take a daily inventory and replenish the items used up during the days work An effective system of inventory controls provides economic laboratory management and avoids unnecessary delays in obtaining test results. This inventory can include items such as glass ware, reagents solution, and culture media (in the bacteriology laboratory) etc. the daily records of supplies serves as an important aid in measuring the work load of the laboratory and is a source of information for requisition and for more general inventories of stock rooms. *************************************************************************** EXTRA QUESTIONS Q1. Write a short note on the common types of Laboratory accidents. Ans: There are number of common types o lab accidents some of them are given below: Physical Injury: 1. Physical injury may be due to number of causes like falling on a wet floor or cutting ones self with broken glassware. 2. To avoid such injuries we would to be keeping the floor dry, wear shoes, tie up long hair, and wear a protective covering over the dress. 3. Broken glassware on the floor must be cleaned and discard all broken glassware in a separate container so that if can be handled carefully. 4. In case of would caused by broken glass, first remove any piece of glass wash and clean the area apply antiseptic like tincture iodine (1% iodine in 95% alcohol) then apply bandage. 5. Another cause of physical injury is improper lifting of heavy objects. 6. Keep your back straight and bent at the knees. Do not bend your back at this may lead to disc dislocation.

Prof: R. Shinde

Electric Shock: 1. Electric stock is mostly due to improper maintenance of electrical equipment. 2. Check the laboratory wiring regularly. Use rubber-sheaths, waterproof cords and connecting wires on all equipment. 3. Extension cords should not be used except as a temporary measure. 4. Equipment should be properly grounded. Do not attach a two or three way outlet to single outlet. This may overload the wire and may result into fire. 5. In case of sparks, immediately disconnect the apparatus and notify the maintenance department on the dealer. 6. In case of electric fire use only CO2 extinguisher never throw water. Exposure to dangerous chemicals: 1. The most common accidents due to chemicals is by physical contact, ingestion or inhalation of toxic fumes. 2. Never stored ether or flammable chemicals in the refrigerator that may lead to explosion. 3. All flammable chemicals must be marked with Red ink as FLAMMABLE. 4. Some imp. Safety regulations in handling chemical are; Follow the manufacturers instructions for unpacking and storing chemicals. Use laboratory carts for moving big containers and gas tanks. Especially chemicals in the inventory register or card as soon as they are received. When handling small containers, do not carry too much in hands of arms instead we plastic buckets. Place flammable in mental containers while working inside the laboratory. Do not touch chemicals with your hands, use a spatula or spoon. Wear apron and rubber gloves while handling corrosive chemicals. Dispose of chemicals only by recommended cyanide are discarded during and after use into the sink followed by a sufficient amount of running water. Avoid mouth pipetting use autopipette available in the market. Exposure to radioactive chemicals: 1. Radioactive chemicals are used in radio immunoassays. 2. Pregnant women must not work with radioactive substances. Handling of these chemicals is potentially hazardous. 3. Technicians must always wear lead apron while handling radioactive chemicals and these should be screened at regular interval. Fire and Exposition: 1. Open flames are on the sources of fire hazards in a laboratory never leave them unattended. 2. There are number of flammable objects inside the laboratory (e.g. paper, cotton, flammable chemicals etc.) 3. In case of fire accident, close all doors and window and prevent draft. 4. For small blaze, use sand, water and fire blanket to put out the fire. A fire extinguish is use for larger blaze. Do not use water on an electric fire or fire cause by oil. 5. Turn off electric current and use only CO2 extinguisher which does not allow to pass electricity.

Prof: R. Shinde

The following preventive measures against the fire are recommended. a) Turn off the flame before leaving the laboratory any time. Never keep an open flame near flammable liquids. b) Do not throw burning matches in to a water basket, throw them into a non flammable metal container. Q2. Explain first aid procedure in following conditions. Ans: 1) Bleeding 1. Bleeding can be external which is visible or may be internal. 2. Try to stop bleeding by applying pressure directly over the external bleeding spot. This is done by a pressure dressing. 3. Place a thick pad of gauze or other clean cloth over the wound and bandage it. 4. Apply the hand pressure if necessary over the pad. If nothing is quickly available, use your hand to apply pressure over the wound until a pressure dressing cab be obtained. 5. If the bleeding if from an arm or leg, elevation of the limb will help to control bleeding. 6. Severe bleeding and serious wounds must be attended by the doctors. 7. In case of bleeding remove the foreign bodies like glass then clean the wound and disinfect it before putting on the bandage. 8. If bleeding is caused by the sharp object which is contaminated, allow the blood to flow out then apply disinfectant to the wound that is tincture iodine. This is followed by washing thoroughly with soap water, and then applies dressing. Q3. How will you handled the patient suffering from poisoning or explain first aid in poisoning. Ans: 1. The signs and symptoms of poisoning vary with the poison taken. 2. Nausea, vomiting, pain in the stomach, diarrhea are some possible immediate effects. 3. If a toxic substance is inhaled, place the victim in the open air while waiting for the physician. 4. If the poisoning id due to swallowing of the toxic substance induce vomiting in order to wash out the poison from the stomach. 5. This is accomplished by giving a substance that causes vomiting i.e. emetic. 6. Some of the readily available emetics are warm salt water and warm soapy water. 7. Do not induce vomiting in case of poisoning due to strong acids or alkalis. Poisoning with strong acids and alkalis like HCI, H2SO4, HNO3, NaOH, ammonia give appropriate antidote to neutralize the poison. Q4. Write a short note first aid in case of Burns. Ans: 1. The most common burns are caused by flames, hot liquids, splashing of inflammable solvents, explosions etc. 2. In case of minor burns, only the surface of the skin is affected and in case of an extensive burn the deeper tissues will be affected.

Prof: R. Shinde

3. Immediately after the minor burn put the affected part into cold water to soothe the pain. 4. After this apply sterile burn ointment, olive oil, or mineral oil and cove the burned area loosely with a dry gauze dressing. 5. If wound is exposed, apply mercurochrome. 6. Severe burns require immediate medical aid. If the victim is on fire, roll him in a fire blanket to smother the flames. 7. Until the emergency care is available, lay the victim on the ground. 8. Do not remove this clothes, tie a piece of gauze or any clean cloth over the mouth and nose to serve as a mask. 9. Contamination with germs from the mouth and nose is responsible for most infections of serious burns. Q5. Explain the first aid procedure in case of Eye accidents. Ans: 1. If the chemical enters the eye, immediately wash out the chemical with lots of water. 2. There are two ways to wash, spray from a wash bottle or rubber bulb put the water drops in the corner of the eye near the nose. 3. Alternatively, hold the eye under the running tap. After through washing, put a few drops of 2% sodium bicarbonate into the eye is case of acid splash or several drops or satuboric acid soln in case of alkali splash. 4. Do not use water if there has been any delay in giving first aid get medical help at once. 5. If a foreign body like dust is lodged in the eye do not try to remove the speck if it is on the iris (dark portion of eye). This should be done only by the eye doctor. 6. First aid is confined to removing particles on the white portion of the eye. Be very careful while doing this. 7. Instruct the victim not to rub the eye, put the lower lid down gently and look for the dirt. If visible then remove the speck with sterile cotton swab or clean handkerchief. 8. If the speck is on the inner lining of the upper eyelid, grasp the lashes of the upper eyelid gently, have the victim look up, and pull the eyelid forward and down over the lower lid. 9. If these fail, flush the eye with sterile water or solution of 2% aq. NaHCO 3 dropped from an eye dropper. 10. In case of persistent irritation, refer the victim to doctor. Q6. Write a short note on following 1. REQUISITION: 1. A test request is made by a physician following the physical examination of the patient. 2. The requisition slip bears the identification of the patient and the name of the referring doctor. 3. A list of diagnostic tests offered by the laboratory is listed in the request slip for the physician the check. 4. A properly filled request slip may be sent to the ward for collection of the appropriate specimen or the specimen may be collected in the laboratory itself. 5. The latter is often done for outdoor patients.

Prof: R. Shinde

6. The physician must communicate properly with the technician through the request slip by providing the provisional or presumptive diagnosis based on physical examination and clinical history of the patient. 7. This will help the technician to be aware of the physicians expectations and choose a certain course of testing (e.g. media for the culture of specific infectious agent). 8. The requisition slip is also used for reporting. 9. It provides the normal values a proficient technician may circle or underline the abnormal findings. 2) LABORATORY REGISTERS: 1. The central laboratory register provides an accession number to the requisition slip which is sequentially recorded in a register or log book. 2. The log book may keep the record of urgent (technically referred as stat) specimen separately. 3. The log book must show clearly the identification of the patient, type of patient (indoor or outdoor), referring doctor, date and time, type of specimen received and the tests requested. 4. Specimens with the corresponding request slips are distributed to the various sections of the laboratory for the completion of the tests. 5. It may take a single day or several days (e.g. in microbiology) to complete a test and after the completion of the tests which may take a single day or several days (e.g. in microbiology laboratory), the results are recorded in the laboratory register with the date and time of reporting the results to be physician through the request slip. 6. The register also records any information sent by phone. Thus any discrepancy is easily caught on the day-to-day basis. 3. LABORATORY RECORD BOOK 1. Diagnostic tests are carried out in the different sections and these sections keep their own record of the test done and its results. 2. They use the same accession number as shown in the main register. 3. Wide variation exists between the various sections of the laboratory (histology, microbiology, haematology and others) regarding keeping of records. 4. For example, the histology laboratory maintains the accession register but no detailed observation report. The latter is maintained by the pathologist. 5. The technician of the microbiology laboratory, on the other hand, Dust makes sequential observations though direct examination, laboratory culture and biochemical testing. 6. The physician should be posted with the preliminary information (direct examination) within 24 hours and later after 24 to 48 hours, unless the culture takes a longer time to grow (e.g. mycotic agents) 7. Under emergency condition (e.g. positive findings of budding yeast in CSF), the physician must be informed immediately. 4. TECHNICIANS RECORD BOOK 1. This is the diary of the technician. 2. It keeps all the raw data, calculations and results reported, along with the accession number given by the laboratory.

Prof: R. Shinde

3. The technicians record book should also indicate the result of the quantity control and any maintenance record. 4. When the technician communicates the results to the central desk through the result/reporting slip, the results are entered into the central. 5. Registration book and the receiving clerk sends the requisition slip with the report to the pathologist for signatures. 6. This is then dispatched to the physician who requested the test. If the patient needs a copy, he gets it from the physician. 7. The laboratory does not send the result directly to the patient, unless it is asked to do so. 8. Note: Urgent specimens (stat), like spinal fluid, are handled specially and are given priority over other specimens and written as stat with red ink. 5. STOCK REGISTER 1. The accuracy of the work performed in a laboratory can usually be estimated from the orderliness of the laboratory usually be estimated from the orderliness of the laboratory, 2. Laboratory procedures require a sequence of actions which must be conducted correctly and methodically. 3. The laboratory worker must be sure that all the materials required to run his tests will be available when and where needed. An effective system of inventory control provides economical laboratory management and avoids unnecessary delays in obtaining test results. 4. The materials used in the laboratory are broadly classified as expendable items (e.g. cotton swabs, matches) and non-expendable items (e.g. instruments, inoculating needles, bottles). The expendable Items need to be replenished. 5. Thus a supply will be needed. The store room of the laboratory holds the bulk materials and materials for daily use are checked out as needed. A smart technician always plans ahead of time. 6. WORKBENCH INVENTORY 1. Workbench inventory is done daily. 2. The inventory is taken in the evening hours so that all supplies are ready before new specimens arrives on the following morning. 3. The drawers under the bench should e well organized with proper compartments for pipettes, rubber tubing, glass roads, and others. 4. Number the drawers and list the materials as they are kept in these drawers. 5. Take a daily inventory and replenish the items used up during the days work. 6. Modify the inventory according to your needs. Remember, and affective system of inventory control provides economic laboratory management and avoids unnecessary delays in obtaining test results. 7. This inventory can include items such as glassware, reagents solutions, culture medias (in the bacteriology laboratory) etc. 8. The daily record of supplied serves as an important aid in measuring the work load of the laboratory and is a source of information for requisitions and for more general inventories of stock rooms.

Prof: R. Shinde

7. STORE ROOM INVENTORY: 1. At least one person of the laboratory staff should be assigned to care for the store room, prepare order or requisitions, check orders received, distribute supplies are needed, and keep a running inventory. 2. An easy way to maintain an inventory list is to prepare an index card for each item (10 cm x 15cm) 3. Each card keeps an account of the number of items in hand and orders are placed before it is exhausted. 4. An example of the inventory card is when materials are received, the storekeeper notes on the card the date, the amount of materials received, and the total amount in hand. 5. When materials are distributed, he adds to the card the date, the amount of materials distributed, and the balance in hand. Periodic review of these cards will indicate those regulatory used items which must be ordered. 6. It is well to have a notebook close at hand in the store room in which to makes notes concerning items to be ordered, 7. Do not depend on your memory. When it is evident that an items needs to be reordered and you cannot take time to check the inventory card, make a note immediately in the notebook. 8. While ordering chemical care must be taken to order the proper grade and proper quantity. 9. Some of the chemicals may not stay good for ever. Similarly, proper storage condition must be provided for the material arriving. 10. For example some of the materials need to be refrigerated. 11. The store must have a large size refrigerator and if possible, a freezer, to store the goods 12. Although large quantities are economical, an excessive amount may be wasteful as it may spoiled or may get contaminated when taken out in small quantities. 13. In addition, bulky container is difficult to handle. 14. If material is issued in small containers, make sure that the empty container is first labeled before it is filled. 15. Bottles of highly poisonous solution or reagent should be labeled POSION. 16. Large container of chemical that are opened from time to time to dispense a portion of the content should be dated when the container is first opened. 17. It is most important that every container be correctly labeled as to its contents. *****************************************************************************

Prof: R. Shinde

CHAPTER 4 BASIC LABORATORY EQUIPMENTS Laboratory glassware is usually manufactured from borosilicate glass. It is resistant to the action of chemicals with the exception of hydrofluoric acid. It is made to withstand mechanical breakage and sudden change in temperature. Difference between Borosilicate and Soda lime glass Borosilicate glass 1. They are heat resistance and chemical resistance. 2. It can stand good for mechanical stress. 3. It will not break due to sudden change in the temperature. 4. It contains Boron and silica atoms join to from tight network of Boron and silicon atoms. 5. It is more costly. 6. It is used for the heating the reagents purposes. Soda lime glass 1. They are less heat and chemical resistance. 2. It cannot stand well for mechanical stress. 3. It will break due to sudden change in temperature. 4. It has high alkali composition and free soda. 5. It is cheaper than borosilicate glass. 6. It is used for storage the reagents and making lab items such as bending the tubes, making posture pipettes.

Prof: R. Shinde

Glass wares used in the laboratory BREAKERS: These have capacities from 5 to 5000 ml. they are generally in a square form which is cylindrical and has a spout. These are used for heating liquid and for preparing reagent solution.

FLASKS:

FLASKS: These have capacities of 25-5000 ml. Different types of flasks used as follows: 1. Conical flask (Erlenmeyer type): these are used for performing titration and for boiling the solutions since evaporation is minimum because of the conical shape. 2. Flat bottomed round flask: these are mainly used for heating liquids. 3. Round bottom flask: These can withstand higher temperature. They may be heated in a naked flame. 4. Volumetric flasks: They are flat bottomed pear shaped vessels with long narrow necks with volume marks and fitted with a stopper. 5. These are mainly used to make final volume of the reagent accurately. 6. Measuring cylinders: They are available in 10-2000ml capacities. They are used to measure quantity of the liquid. A high degree of accuracy is not possible because of there wide bore.

BOTTLES: 1. The general types of bottles are described below: 2. Reagent bottles: They are available in 25-5000 ml capacities. They are cylindrical, have narrow necks and fitted with stoppers made up a plain glass or amber coloured glass. (Amber coloured bottles are useful to store certain reagents like silver nitrate which are light sensitive)

Prof: R. Shinde

3. Screw caped bottles: These are available in 5-1000ml capacities and may be round or flat. The caps may be made up of metal or plastics 4. Whichester Quart Bottles: They are of 2000ml capacities and are available in white or brown glass. They are fitted with glass stoppers. They are useful for storing stock solutions, reagents and also for storing specimen like urine (24 hrs collection) 5. Drop bottles: These are about 50-100 ml capacities and made in white or brown glass, with a narrow neck and slotted glass stopper. They are used for delivery of drops of solutions, such as stains.

1.

BURETTES: These are used for measuring variables quantities of liquid. They are available in capacities of 1100 ml. They are graduated tubes of uniformed bore and they are closed at lowered end means of glass stopcocks. These are used for titrations.

CONDENSER : These are available in variable size and used mainly for distillation and for reflux operation.

Prof: R. Shinde

DESSICATORS: (Used for keeping the reagents in dehydrated condition) These are available in variable sizes. Chemicals like sulphuric acid, phosphorus pentoxide, calcium chloride and silica gel can be used to desiccate chemicals, used for the preparation of accurate normal solutions and standards.

FUNNELS : These are available in variety of range of the separation of Solids from liquids Liquids from liquids For pouring liquids chemicals Or solutions into a container. They commonly used funnels are Of diameter of 50, 60, 75 and 100 mm

TUBES: Test tubes are used to performed different types of experiments in a clinical laboratory 1. Test tubes (with or without rim): These are uniformed thickness and withstands mechanical and thermal shocks. Tubes with rim are preferred when reagent in a test tube is directly heated on the flame with test tube holder. 2. Centrifuge tubes: These are either graduated or plane and are available in the conical shape. The commonly used tubes are of the size 17 x 120 mm. 3. Folin-wus tubes: These tubes are mainly use for the determination of blood sugar by folin-wus method. These are engraved at 12.5 to 25ml. 4. Digestion tubes: These are calibrated at 35 and 50 ml mainly used for conversion of organic matter into inorganic matter by heating it in the presence of digestion mixture. (50 % sulphuric acid and selenium dioxide).

Prof: R. Shinde

PIPETTES: These are used for digestion controlled quantities of liquids are classified as follows: 1. 2. 3. 4. Graduated pipette Serological pipette Mohr pipette Volumetric pipette

1. Graduated pipette These are available from 0.1 to 10 ml capacities. The graduations are durable, resistant to chemical attack and normal washing. They are available in class A and class B accuracies. Class A Graduated pipettes are very accurate and used for quantitative determinations. 2. Serological pipette These are graduated pipette marked up to the tip. These are mainly used for pipetting water and reagents. However 0.1 to 0.1ml pipette specimen like blood, serum, plasma, standard solutions etc. These are mainly used for performing serological tests. 3. Mohr pipette These are graduated above the tip so that even if the tip is broken, these can be used for their full capacities. These are used to performed serological tests. 4. Volumetric pipette These pipettes are not granted but designed specially with a central bulb to deliver a specific quality of the specimen. 5. Pasture pipettes: These pipettes can be prepared in the laboratory by using glass tubing. These are used by attaching rubber for the separation of serum and plasma from the cells. The drops of specimens can be conveniently added in a reagent by using a pasture pipette.

Prof: R. Shinde

OTHER REQUIREMENTS: 1. Test tube rack: it holds the test tubes in the upright position. These are made up of metal or plastic. 2. Test tube Holder: are used during heating of the test tubes. 3. Cuvette: They are special kinds of test tubes or of a rectangular shape which hold the solution intended for photometric readings in a colorimeter. 4. Volumetric glasswares: includes graduated cylinders, pipettes, burettes and volumetric flasks. Volumetric glass wares are used in measuring accruing accurately the volume of a liquid. 5. Stripping rod. Is used for the dissolving solute in preparing solutions. 6. Tongs: are made up of metal and used to hold objects hold of cold. 7. Petridishes: petridishes are made of glass and used in the aerobic culture of microbes. These are round in shape and having 2 halves which fits on the one another.

Prof: R. Shinde

Prof: R. Shinde

Basic laboratory Requirements

Prof: R. Shinde

Prof: R. Shinde

VOLUMETRIC GLASSWARES EXAMPLES WITH USE 1. Volumetric glassware included graduated cylinders, pipettes, burettes and volumetric flasks. They are most famous for measuring the accurate quantity of They volumes held are indicated by permanently etched line called calibration mark. Example: Graduated cylinders: 1. It is a long cylindrical piece of glassware with calibration markings on it in a ascending order (zero at bottom) 2. It is used to measure volume of liquids. 3. They are available in various sizes and they may be made up of glass or plastics. Pipettes: 1) They are used for measuring liquid with high accuracy and for transferring liquids. 2) The tip of the pipette is usually tapered and has an opening that is critical in controlling draining time called Drainage time. 3) Pipetter are broadly classified as (i) (JD) To Deliver (ii) TC Pipette is to is To- Contain pipette. 4) T.D. Pipetter calibrated to deliver small or fractional volume of fluid. 5) Volumetric pipette are calibrated to deliver definite volume of liquids. The process is liquid must be allowed to drain freely from the calibration mark to the tip and the last drop should not blown out. 6) T.C. pipettes use to delivery very small quantity of volume (10-100) and is non volumetric pipette. Burette : 1. It is long, cylindrical, graduation pipette type glassware having stopcock at the lower end. 2. Graduation markings are etched on it with zero marking is at top. 3. Before dispensing the liquid, bring the level to the zero mark then dispensed by opening the stopcock. 4. It is used for titration purpose. Volumetric flasks: 1. 2. 3. 4. They are flat bottom shapes vessels with long narrow neck has a single calibration mark. They are fitted with glass or plastic stopper,. They contain only the specified volume of fluid as marked on flask. The commonly used size 35 ml to 1000 ml they are used for preparing solutions. liquids.

Prof: R. Shinde

DIFFERENT TYPES OF THE PIPETTES USED WITH ITS USE 1. They are used for measuring liquid with high accuracy and for transferring liquids. 2. The tip of the pipette is usually tapered and has an opening that is critical in controlling draining time called drainage time. 3. Pipettes are broadly classified as T.D.i.e. To deliver Pipettes T.C.i.e. To contain Pipettes 4. 4. TD Pipettes calibrated to deliver very small or fractional volume of fluid. 5. T.C. Pipettes used to delivered very small quantity (10 0 100l) and is non volumetric Pipette. 6. Sahil Pipette is type of TC Pipettes that hold 20 l blood and after use it should always washed with diluents. 7. Graduated Pipettes: They have advantage of delivering variable quantity fluid. 8. Mohr Pipette is special graduated Pipette where the last calibration mark is far from the tip. 9. Serological Pipette requires blowing out residual fluid after dispensing. It is usually graduated and graduation marking continue up to the tip. The serological Pipette is identified by the etched double band at the suction end. 10. The primary use of serological Pipette is to obtain serial dilution of serum in determining titre. 11. Ostwald Pipette: It is looks like volumetric Pipette where delivery tip is closer to the bulb. This Pipettes should blown out. No residual fluid should be left in the pipette or in its inside walls. The usual volume of Ostwald pipette is between 1-2 ml. 12. Thoma pipette: It is blood diluting pipette using in counting blood cells. The marking does not indicate volume they only indicate propertionate dilutions. 13. Automated pipettes: They are often blown out pipettes with mechanical devices. They can be use disposable tips or tips can be wash and dried and then also used. These pipettes can deliver multiple volume or single volume. It is highly accurate device.

Prof: R. Shinde

GLASS WORKING IN THE LABORATORY 1. Some glass equipment needs to be constructed in the laboratory. Only soft glass can be melted and manipulated in the laboratory with the use of Bunsen burner. 2. Be careful when breaking the glass tubing. If the tubing has been scratched properly very little pressure will be required to break the tubing. 3. The cut edges of the glass tubes are sharp and should be smoothed by fire polishing. 4. This is easily done by holding the cut end in the hottest part of the blue gas burner flame at an angle of 450, while rotating the glass slowly to ensure even heating. 5. The tube should be heated until the glass just begins to colour and soften. Prolong heating reduces the inner diameter of glass tubing. 6. To make stirring rods use a solid rod of about 5 mm diameter. Cut the rod to the desired length and fire polished the end. Bending the glass tubing: Place the flame spreader on the Bunsen burner and light the gas. Select the piece of glass tubing to be bent. Hold the tubing over the flame by constantly rotating. Make the desired angle of the glass tubing and lay it on a wire gauge. If the glass has been heated correctly the bend will smooth and even throughout the bend. Making of posture pipette: Use an open burner flame Cut the glass tubing to the required size and fire polish each end. Hold the piece of glass tubing in horizontal position with the centre of tubing on blue flame. When it becomes reddish, soft then flame turn yellow. Remove the tubing from the flame and pull the two ends apart slowly in parallel position. Hold the tubing until it hardens cut the tip to desired size and fire polish the end.

Inserting glass tubing into stopper: Always use glass tuning with fire polish end. The diameter of the tubing should be slightly larger than the hole for good fitting. Lubricate the hole with water or glycerol wrap the hands with towel. Hold the stopper in one hand grasp the tubing immediately behind the end of the tubing to be inserted into stopper. Carefully insert the end of the tubing into the stopper with gentle pressure and with twisting motion. CLEANING OF THE DIFFERENT LAB GLASSWARE 1. Cleaning of the lab glasswares is very important part. 2. New glasswares should be neatly cleaned. Soft glasswares (sodalime) should be treated overnight with 5% HCI, this will neutralize free alkali found on the surgace.

Prof: R. Shinde

3. Acid treated glasswares must be first rinsed with tap water followed by rinsing with D/W. 4. In General cleaning of glassware place glasswares into 2% detergent solution. Soak for one hour. 5. Scrub thoroughly with brush and thoroughly clean with hot soap with solution. Wash with tap water and then finally rinse with D/W. 6. If the glasswares are too dirty to coagulate of organic matter or other substances it must be cleaned with chromic acid solution and special cleaning procedure should be adopted as below. 7. Rinse the glassware in tap water and then dip it in the cleaning solution for one hour. Then wash under tap water and then rinse with distilled water. 8. The cleaner used as 50% HCL remove iron strains. Nitric acid for remove Iodine stain Sodium carbonate, acetone, other ordinary grease stain. 1. CLEANING of PIPETTES: 1. Discard the pipettes in metal or plastic container which is half full with water after use. 2. After the days work the pipettes are drained and then transferred to a container of detergent solution. 3. If pipettes are too much dirty then put in chromic acid solution. 4. After soaking for several hours drain the pipette and run tap water over it until all dirt and acid are removed. 5. Finally was thoroughly with fresh tap water and rinse with distilled water. 6. Dry the pipette in hot-air oven for quicker drying, rinse with acetone and blow off with air. 2. CLEANING OF BURETTE: 2. Take out the stopcock or open the stopcock and scrub with hot detergent and with long brush. 3. If the burette is too dirty put it in the cleaning solution overnight. 4. After the detergent or acid cleaning was thoroughly with tap water and then rinse with distilled water. 5. Dry the burette by clamping it on stand in inverted position. 6. Lubricate the stopcock before it is put back. 3. BLOOD PIPETTE OR SAHLI PIPETTES: 1. 2. 3. 4. 5. Immediately after use, put the pipette in mild detergent solution ( 2%) The pipette is then cleaned by suction with the help of filter pump. Was with tap water under force. Rinse with tap water and then with distilled water. Dry in oven, or dry by rinsing with 1:1 acetone alcohol solution by air suction use suction bulb for suction.

Prof: R. Shinde

4. HAEMOCYTOMETER OR BEUBAURS CHAMBER: It is cleaned with 2% mild detergent followed by rinsing with cold water. Dried with soft cloth. Do not rub the ruled portion of chamber and do not touch with hand. 5. WINTROBE TUBE: 1. After use immerse the wintrobe tubes in a upright position in a beaker and with the help of Pasteur pipette fill the tubes with a mild detergent. 2. R.B.C will be immediately hemolysed. 3. While handling the tubes wear gloves to avoid biohazard (micro organism) 4. After immersing in the detergent for a while hold the breaker under running tap water. 5. The tubes will be fairly clean. Use a Pasteur pipette to empty the contents. 6. Finally wash the tube with D/W and then dry in hot air over. 6. MICROSCOPE SLIDES AND COVERSLIPS: 1. The microscope slides must be perfectly cleaned and should be free from scratch. 2. If the slides are used in bacteriology or for stool examination soaks in 5% phenol or in lysoi solution or place the slides in glacial acetic acid for 10 minutes rinse with distilled water and wipe dry with clean paper or cloth. 3. Use xylene or detergent to remove oil immersion oil. 4. For cover slip first they dipped in Lysol solution then dipped in xylene and left in contact with mild detergent. 5. Wash them in running tap water and finally rinse with D/W. 6. Dry them with soft cloth or paper. 7. GLASS SYRINGES: 1. 2. 3. 4. They should be cleaned after removing barrel. Keep it in mild detergent solution. Wash under tap water and then with D/W Wrap in paper and sterilized in the autoclave.

CARE AND MAINTENANCE OF LABORATORY GLASSWARES Heating and Cooling: Never leave the vessels unattended when heating work is being carried out. The vessel may be crack when dryness condition approaches. Do not place hot glasswares on a damp surface. Cool the hot glass wares slowly to prevent thermal breakage. Heat all the liquids slowly and always used metal gauze or water bath to diffuse hears. USE AND CARE OF COMMON LABORATORY INSTRUMENT

Prof: R. Shinde

MICROSCOPE

Fig: Monocular and binocular microscope Commonly two types of light microscopes are used in the laboratory. a) Monocular b) Binocular

Basically they are the same except that the binocular microscopes have two eye pieces which allows the user to keep both eyes. Open while viewing through the microscope. Both the microscope has three major systems. 1. Support system 2. illumination system 3. Magnification system COMPONENTS OF LIGHT MICROSCOPE: 1. OCCULARS: The oculars or eye pieces located at the top of the microscope and attached to the microscope arm. The oculars through which the object is viewed, contains lenses that magnify object. The usual magnification is ten times (10 x ) 2. OBJECTIVES: The underside of the are contains a revolving nose piece to which the objectives are attached. Most microscope have at least three objectives, low power objective (10 x), high power (45 x) and oil immersion objective (100 x) To determine the degree of magnification multiply the magnification listed on the objective used. The ability of a microscope to distinguish between two separate but closely packed details in the object being viewed is called its resolving power and it is determined by the quality of the objective lenses. 3. MICROSCOPE BASE: The arm of the microscope connects the objective and eye piece to the microscope base. Which support the microscopes.

Prof: R. Shinde

4. LIGHT SOURCE: The base also contain the light or mirror which supply light to the objective viewed. 5. CONDENSER AND DIPHRAGM: The light or mirror has a movable condenser and iris diaphragm located above it. The condenser focused the available light into the objectives as it is raised or lowered. Lowering the condenser will increase the contract of unstained specimens. The iris diaphragm, located in the condenser unit, regulates the amount of condenser light much like the shutter of camera. This iris diaphragm may be adjusted by movable lever. 6. COURSE AND FINE ADJUSTMENT: The two focusing knobs may also be located just above the base. The course adjustment is used to focus with low power objective only. The course adjustment should not be when using the higher magnification. This is to prevent the objective from accidentally striking the slide and become damage. The fine adjustment is used to give a sharper image after the object is brought into viewed with the coarse adjustment knobs located just below the state. These move the stage left and right or backward and forward. Other stage are fixed so the slide must be moved manually to view different area. USE OF OIL IMMERSION OBJECTIVE (STEPS) First screen with low power and high power. Centre the object under the high power objective. Swing the objective to one side and put a drop of immersion oil on the object. Bring the oil immersion objective in the line. The objective will probably touch the immersion oil and will come under the focus. HOT AIR OVEN Principle: Hot air oven contain heating coil and thermostat the principle belong it is conversion of Electrical energy into heat energy. Thermostat is used for adjusting the temperature when temperature is on the rise it is indicated by pilot. The actual temperature of the oven is shown by thermometer. Use: It is used for dry heat sterilization process and applied for the sterilization or articles made of glass or metal. It cannot use for liquid media. Sterilization procedure: 1. Wrap the glassware like Petri dishes, pipettes, tubes, container for urine collection. 2. Put the metal container with metal lid kept loose during sterilization. 3. Pasture pipettes are put in large size test tubes or glass container then plugged with cotton. Needle must be put in the tubes or put in a paper or cloth wrap and placed in metal tray. 4. Switch on the oven, the indicator light will be on. Set the thermostat at 175 0C.

Prof: R. Shinde

5. Leave the materials for dry heat sterilization inside the oven for one hour. 6. Switch off the oven and wait until the temperature falls to 400 C.

Prof: R. Shinde

COLORIMETERS AND DIFFERENCE BETWEEN COLORIMETER AND SPECTROPHOTOMETER

Principle: It work on the principle of Bees-Lambert low it states that when beam of light passes through a transparent medium the rate of decrease of intensity of light w.r.t. thick and concentration is directly proportional to the intentisity of incidence light. Mathematically: A = Ebc Where ,

A B C D

Absorbance or Optical density Thickness of cell Conc. Of sample Molar extinction coefficient.

Uses: It is used to measure the optical density of given solution.

Basic procedure of Colorimeter: 1. Pipetting of reagent in three test tubes labeled as Test, Standard, and Blank. 2. Addition of specimen and standard in the respective tubes and distilled water in the blank. 3. Keeping the tubes for incubation at specified temperature. 4. Reading the intensities of test and standard against blank at the specific wavelength. 5. Calculating unknown concentration by Beers law. In analytical procedures comparison of blank, std, and test (by developed colour) is measure. The concentration of unknown is calculated by a formula O.D. OF TEST Conc. Of unknown = -------------------------- X CONCENTRATION OF STD. O.D. OF STD.

Prof: R. Shinde

Care and maintenance: 1. Always put a plastic cover on the photometer, when it is not in use. 2. Before putting the photometer on put a filter and cuvette filled with distilled water in their respective position 3. Most of the biochemistry experiments are based on the photometry hence use the photometer very carefully. 4. Check-the sensitivity of galvanometer occasionally by using a standard dichromate solution. . Colorimeter It is used for measuring absorption in visible range. It involve filter as monochromator. It is cheaper. It is used to analysis of coloured compound. Spectrophotometer It is used for measuring absorption in both V.V. Visible region It involve prism as monochromator. It is expensive. It is used in analysis of coloured as well as colourless compounds.

Prof: R. Shinde

CENTRIFUGE Introduction: Centrifuges are design to accelerate the sedimentation process by using centrifugal force. The two general type of centrifuges used in the laboratory. a. The swing out head type (horizontal rotor) b. The angle head type( fixed angle rotor) A rotor is the device used to hold centrifuge cubs and tubes. RPM is only relative number which gives an idea that how much regimenting force is being applied to a sample to be centrifuge. Important component of the centrifuge: 1. The centrifuge head (rotor) with the tubes or cubs with cushions (Rubber pads) at the bottom inside the cups or tubes. 2. Rotor device assembly. 3. The chamber which encloses above part. 4. A power switch. 5. Speed control and the additional parts. 6. A timer. 7. A tachometer and graphite brushes. Principle : The working of centrifuge is based on the centrifugal force, which acts on the substance in the circular motion towards the periphery. The factors which govern the speed of centrifugation are, 1. The revolution per minute (RPM) 2. The length of the radius the radius is measured from the centre of the rotation to the inside bottom of the tube., 3. Shape and size of the particles. 4. The viscosity and the specific gravity of the fluid under centrifugation. 5. Gravitational force acting on the particles. Uses: The centrifuge is mainly used in the laboratory for. 1. The separation of serum or plasma from red blood cells. 2. The separation of the sediment in urine. 3. The separation of protein free filtrates. 4. Washing of the red blood cells by normal saline. Care and maintenance: 1. Place the centrifuge on firm base. It should not be placed near any electronic instrument. It should not run near any combustible fluid.

Prof: R. Shinde

2. Before centrifugation, the centrifuge tubes and cups or tubes of the centrifuge should be properly balanced. Add equal amount of sample to be centrifuge in the tubes and place facing each other. 3. The chamber should be kept clean, because some chemicals are biohazardous. 4. Always make sure that the lid is closed while centrifuge is on. 5. Observe the normal operation noise during operation. Noise change is observed Change is observed during misbalancing of tubes, cracking of tube inside the cup or tube (holder). Leaking of contents etc. 6. The rotor will not function if the graphite piece wears out completely. So in such case it is necessary to replace it along with the graphite brush. Brush the cups or tubes regularly. 7. Place a plastic cover on the centrifuge, when not in use.

Prof: R. Shinde

AUTOCLAVE Principle: It works on the principle that steam put under pressure will reach temperature above the temperature of boiling point of water. While steam under 15 Ibs pressure will be 1210C and this will penetrate the. Wrapping and sterilized the desired object Use: Syringes, needles, media, glasswares surgical instrument can be satisfactory sterilizes with this technique.

Preparation of material for Autoclaving: Materials must be properly wrapped before putting in the autoclave. Liquid media are kept in conical flasks, round bottom flasks or in a test tubes or in a breaker. Cotton plugs, metal caps. Plastic cops or aluminum foil are used wrap the vessel. Do not fill the liquid to the top, leave th space for the liquid to expand or boil during the sterilization. Difference between vertical and horizontal autoclave and sterilization procedure for it. The vertical autoclave are in a standing position and materials to be sterilized are inserted from the top. While Horizontal autoclave have the door on side and materials are placed at eye level. The vertical autoclave is heated by gas burner while horizontal autoclave is heated by electric elements. Sterilization procedure with vertical autoclave: 1. Fill the bottom of the boils with water. 2. Put the materials to be sterilized in chamber anchclose the lid. 3. Screw down the lid clamps firmly not too tight. 4. Open the air outlet valve and turn on the burner to heat the water inside the boiler. 5. Continue heating until a jet of steam appears through the air outlet valve. Wait for 5 minutes. 6. Close the air outlet valve. 7. Tight the temperature and reduced the heat slightly. 8. Watch the temperature gauge until 1210C is reached. Start timing when temperature reaches to 1210C.

Prof: R. Shinde

9. Continue sterilization of materials for 20 minutes. 10. Turn off the heat as soon as required time is up. 11. Open the air outlet valve when temperature falls below 1000C. This will equalize the pressure inside and outside the chamber., 12. Unscrew the clamps when hissing sound stops, indicating drop of the steam pressure to zero. 13. Take off the lid and leave the materials to cool. 14. Remove the basket when it is cooled. CALIBRATION OF PIPETTE After receiving a new batch of pipettes that must be calibrated (at least a few at random) This is particularly true for micropipettes, automated or manual. This is because the volume dispensed by the micropipette (e.g. specimen volume) will multiply the error when the final value is expressed to be mg/100 ml or per. Litre. For calibration purposes, 20 is considered to be the standard temperature but the laboratory temperature is often above 20 C in the developing countries. Hence, adjustments have to be made during calibration. There are two methods of calibration: (a) Gravimetric or weight method (b) Photometric Method. Only the gravimetric method will be descried here because of its simplicity. Photometric method, however, is recommended for TC Pipettes. Gravimetric Method: Principle: One gram of water occupies one millilitre (1 mI) Volume at 200 C The weight of water varies with the temperature and for the same volume of one millilitre; it weighs less at higher temperature than 200 C and lower temperature. Procedure:
1. 2.

3. 4.

Take the weight of a weighing vial (W1) Take the pipette to be calibrated and draw distilled water upto the calibration mark. Wipe the outside surface and dispense the distilled water held by the pipette into the pre-weighed vial. Weigh the weighing vial with distilled water dispensed by the pipette (W2) The difference of weights (W2-W1) is the weight of water dispensed the pipette.

Prof: R. Shinde

Note : Measure the volume of water dispensed by the pipette at least three times and takes the average for the following calculations. Each time the water is dispensed note the increase of weight. Va = Wt/F Where Va = Wt = F = Actual volume of water held by the pipette at 200 C (W2-w1) Weight of 1ml of water at the existing room temperature

Calculation of Error The error inherent in the volume stated by the manufacture (Vs) is calculated as follows: Error (1) D/Vs x 100 Where D Average difference between the corrected volume Va and Started volume Vs. Vs Stated volume of the pipette.

Prof: R. Shinde

TITRATION TECHNIQUE 1. Fill the burette with titrant or solution of known strength. Fix the burette to the stand and remove the air bubble and bring the liquid level in the burette to the O mark. 2. Transfer a known volume of titrating fluid and a few drops of indicator in an Conical flask and then proceed with titration by letting the titrant to flow out of burette into the flask. 3. Swirl the flask during titration. 4. Stop adding the titrant into the flask when the colour of the indicator changes. 5. Read the level of the fluid in the burette at the meniscus. 6. A white paper on the back is helpful in taking the burette reading. 7. Wash the burette after use.

STORAGE OF REAGENTS 1. Store acids in glass- stoppered bottles preferably in a drop tray. 2. Store alcohols under lock and key and all details of their use should be recorded. 3. Label all poisonous chemicals (POISON) with red ink and keep them locked. Maintain a record of the amount issued and to whom it is issued. 4. Store flammables securely in metal containers if they are in larger quantities. 5. Small quantities for daily laboratory use are kept in glass bottles with wax-linked or corklined Bakelite screw caps. 6. Never store flammable liquids and solvents on the bench tops or shelves over benches. 7. They should be placed away from the flame, heat, sunlight and electrical switches, the cupboard under the bench is quite safe. Most organic solvents attack rubber and rubber bungs, and these should not be used. 8. All containers with flammables must be clearly marked as FLAMMABLE with red ink. 9. Never store flammables in an ordinary refrigerator. 10. Some of the chemicals like picric acid and perchloric acid can become serious explosion hazard if they are allowed to dehydrate. The quantity of these chemicals kept in the laboratory should be limited in order to use the chemicals within six months. 11. Hygroscopic chemicals like phenol, trichloracetic acid, ferric chloride, sodium carbonate, sodium hydroxide, potassium hydroxide and others must be stored in air tight containers or in a desiccators. 12. Desiccator helps to keep the reagent dry. It contains desiccant in the lower section (e.g. calcium chloride, anhydrous calcium sulphate). The lid must be greased and tightly fitting. 13. Some desiccators are provided and tightly fitting. 14. The desiccant must be in active state and in some cases (e.g. calcium sulphate) the desiccant can be revived by heating at 1000 C for several hours. 15. Pressure of an indicator, like copper sulphate crystals, in the desiccant helps to determine the hydrated (pink) and dehydrated (blue) states. Replace the desiccant when it is too old. 16. Storing in an incubator (370 C) may help in the rainy season but some chemicals may deteriorate.

Prof: R. Shinde

17. Reagents like trichloroacetic acid and ferric choride, which are extremely hegroscopic, might have already absorbed water when they arrive in the laboratory. 18. Fnotosensitive chemicals (iodine, silver nitrate, potassium permanganate and sodium nitroprusside) must be stored in a dark, glass- stoppered bottle, as they decompose with exposure to light. Iodine has an additional problem, it attacks rubber, hence do not use rubber stoppers in case of bottles cantaining iodine. 19. Solutions of sodium hydroxide and potassium hydroxide should be stored in plastic bottles. 20. The alkali attacks glass and long storage in a glass bottles will corrode the glass and the chemicals composition of the solution will change. In addition, glass stoppers may get fixed due to the formation of salt when the alkali present around the stoppers reacts with the carbon dioxide present in the air. 21. Alkali solution in daily use can be stored in an aspirator which is connected to a sodalime guard tube that will absorb and prevent any carbon dioxide from entering the aspirator. 22. If the solution is stored in the refrigerator, it should be brought to room temperature before use. 23. It is good practice to take out only the amount needed for the day and leave the stock solution in the refrigerator on a regular basis.

Prof: R. Shinde

Chapter 5. REGENT PREPARATION INTRODUCTION The metric system is the system of measurements used in clinical laboratories. Most common laboratory units are used for the measurement of weight, lengths, time of conc, of solutions. As different units were used in different parts of the world, scientific communication become difficult as a result an international system of units was introduced in 1971 and was called SIU (System international D Units.) The metric system is named because it is based on the fundamental unit of distance, the meter. The metric system, the meter (M) is the basic unit used to measure distance. The gram (g) is the basic unit used to measure volume. Mole is weight measurement in chemical language. The conc. Of solution of expressed in molarity. SI Base Units

Quantity Length Weight Volume Temperature Amount of substance Solution Weight Volume Length : : :

Name Meter Gram Liter Celsius Mole Morality

Symbol m gm or g l or l C mol M

1 gm = 103 mg = 106 g = 109ng =1012 pg = 1015 fg 1 L = 103 ml = 106 l = 109nl =1012 pl = 1015 fl 1 m = 103 mm = 106 m = 109nm =1012 pm = 1015 fm New ST equivalent of some older terms

Order terms Micron () Cubic Micron (3) Micro-micro gram ( g) Micro gram (mcg) Milli micron (m) Lambed () Cubic millimeter (mm3) Percent (%)

New ST Equivalent Micrometer (m : 10-6m) Femtolitre (fl : 10-15) Picogram (pg: 10-2 g) Microgram (g : 10-6 gm) Nonometer (ng : 10-9m) Microlitre (l : 10-6 L) Microlitre (l : 10-6 L) gl dl (w/v) or ml/dl (v/v)

Prof: R. Shinde

FEW OTHER PREFIXES OF THE METRIC SYSTEM COMMONLY USED 1 decilitre (dl) 1 centimeter (cm) 1 cubic centimeters (cc) : : : 100 ml 10 mm 1ml OTHER MORE CONVERSIONS 1 inch (in) 1 pound (lb) 1 quart (qt) 1 fluid ounce (fl oz) : : : : 2.54 cm 454 gms 0.95 L 30 ml SOME INPORTANT TERMS A solution : It is a solid substance dissolved in a liquid or it is combination of solute and solvent. The substance which is present in larger quantity in the solution. The substance which is present in minor quantity in the solution. TYPES OF REAGENT SOLUTIONS Stock Reagent Solution: this is a concentrated reagent solution which is diluted to prepare a working solution. The stock solution has a longer shelf life than the working solution and occupies less space in storage. Working Reagent solution: The working reagent solution can be diluted from the stock solution or prepared directly with reagent chemical following the recommended procedure. Saturated Solution : It is a solution in which further solute cannot be dissolved. A saturated solution must have the deposits of the solute at the bottom of the reagent bottle. SOME IMPORTANT DEFINITIONS 1. Percent Solution (%) : One gram of a solute in 100 ml of solution is one percent. It is expressed in different ways; percent, percent, %, gm/dl, g/100 ml. 2. Molar Solution (M) : 1 Mole of solute in 1000 ml of solution is equivalent to Molar solution. It is expressed in different ways 1M, 1mol/I, 1mol/L, 1mole /L. 3. Normal Solution (N) : 1 equivalent weight of substance in 1000 ml of solution is equivalent to 1N.

Solvent Solute

: :

Prof: R. Shinde

EXERISE UNDER TEST Q1. What is the basic metric units of length, weight and volume? Ans: The basic unit of Length Weight Volume = = = meter = gram = Liter = m gm L

Q2. Convert the following units a) 0.001 gm into mg 1 gm = 103 mg 0.01 gm = mg 1x = = = 0.001 x 103 10-3x 103 1 mg

0.001 gm = 1 mg b) 300 mg into Gms 103 mg 300 mg X 103 300 mg = 0.3 gms c) 750 g into mg 106 g 750 g X 106 750 g = = = = = = = = 103 mg mg 750 x 103 750 x 103 106 750 x 103 x 10-6 750 x 10-3 x mg 0.750 mg 0.750 mg = = = = = 1 gm gm 300 300 x 10-3 0.3 gms

Prof: R. Shinde

4. 4000 mg into gms 103 mg 4000 mg 103 X = 4000 1000 = 4 gms 4000 mg 5. 280 mg into g 103 mg 280 mg 103 X 280 mg 6. 10 mg into pg 103 mg 10 mg 103 X 10 mg = = = = = = = 106 g g 280 x 106 280 x 103 280000 g 280000 g 1012 pg pg 1012 x 10 1012 x 10 x 10-3 109 pg = 4gms = = = 1 gms gms 1 x 4000

= = = = =

1010 pg

Q3. convert 98.6 0F to Celsius Data :- 0C = ? 0 F = 98.6 Solution : C = F-32 5 9 C = 98.6 - 32 5 9

Prof: R. Shinde

C = 370C Hence : 98.6 0F = 370C

Q4. Prepare 250 ml of 2% solution of acetic acid using 10% acetic acid solution. Ans: Data :- C1 = 2% V1 = 250ml C2 = 10% V2 = Solution: C1 V1 = C2 V2 2 x 250 = 10 x = 500 10

= 50 ml So when we dilute 50ml of 10% acetic acid solution Q5. To prepare 100 ml of 10% formalin from the commercial grads of 37% strength, how much the formalin would you measure ? Ans: Data :- C1 = 10% V1 = 100ml C2 = 37% V2 = ml Solution: C1 V1 = C2 V2 10 x 100 = 37 x

= 10 x 100 37 = 27.03 When we dilute 27.03ml of 37% formalin up to 100 ml we get 10% of Formalin Q6 To prepare 1L of 2% acetic acid from conc. Acetic acid how much of the formalin would you measure ? Ans: Data :- C1 = 2% V1 = 1000ml C2 = 100% V2 = ml Solution: C1 V1 = C2 V2 2 x 1000 = 100 x V2 = 20 ml

Prof: R. Shinde

Q7. You have 1.15 N HCI solution (too concentrated). How much water would you mix in order to obtain 1L of 1N HCI solution? Ans Data : Sca Vca Sda Vda Sca Vca Sda Vda = 1.15 N =? = 1N = 1000 Ml

strength of too conc. Acid Val. Of too conc. Acid Strength of diluted acid Volume of diluted acid. Sca x Vca 1.15 x Vca Vca = 1000 1.15 Vca = 869.56 Ml = = Sda X Vda 1 x 1000

Solution:

So volume of water required is 1000 869.56 = 130.44mL Q8. 1 L of 70% alcohol is needed. How much 95% alcohol is required? Ans: Data: C1 = 70% V1 = 1000Ml C2 = 95% V2 =? Solution : C1V1 = C2V2 70 X 000 = 95 X V2 V2 = 70 X 1000 95 V2 = 736.8 Ml Hence when we dilute 738.6 Ml of 95% alcohol upto 1000 ml with distilled water we get 70% of 1L alchol.

Prof: R. Shinde

Q9. How much of 80% alcohol should be mixed with 30 ml. of 95% alcohol in order to prepare 90% alcohol. Ans: C1V1 + C2V2 = C3 (V1+ V2) (80 X V1) + (95 X 30) = 90 (V1 + 30) 80 + 2850 = 90 + 2700 2850 2700 = 90 80 150 = 10 = 15m

Q10. The direction ask you to use 25 gm of anhydrous cu So4 of where as yours lab hashydrated cu So4 how much hydrated from would you weight in order to meet the requirement of direction. Ans: Anhydrous / hydrated:Mol. Wt. of hydrated Mol. Wt of anhydrated Hydrated cu So4 = = = = = Weight of hydrated Weight of an hydrated 159.5 + 7 (18) 159.5 + 126 285.5

Mol. Of anhy. cu So4 285.5 = 159.5 25 x 285.5 158.5 = 44.7 gms 95 =

= 159.5

Prof: R. Shinde

Chapter 7: QUALITY CONTROL OF LABORATORY FINDINGS INTRODUCTION The object of quality control is to identified and eliminate laboratory errors. As laboratory also depends on external agencies an effective quality control programme will not only monitor internal laboratory procedure but will also communicate the needs and requirements of clinical laboratory to other departments of the hospital. Strict enforcement of laboratory policies will produce reliable and high quality results. GENERAL APPROACH TO QUALITY CONTROL 1. Laboratory test request 2. Specimen collection and processing 3. Laboratory records 4. Errors in procedure 5. Proficiency testing 6. Laboratory personnel 7. Quality control in individual laboratory 8. Heamatology laboratory 9. Blood bank laboratory 10. Microbiology laboratory 11. Parasitology 12. Serology laboratory 13. Clinical pathology laboratory 14. Clinical biochemistry laboratory COMMONLY USED TERMS IN QUALITY CONTROL Control: The control is comparable to the specimen but is composition has been pre-determined. It is different from standard which is artificially made. The same control is used daily in order to find out whether the results are within the tolerance range. Tolerance range: It is the acceptable range of variation in quality control. This is equivalent to 2 S.D. (Standard deviation) Standard Solution: It is a carefully made solution of the test substance and whose concentration is known.

Prof: R. Shinde

Precision: Close findings of repeated analysis. It may not be accurate but is reproducible. Accuracy: Close to actual or true value. E.G. Control serum. The results are comparable when methods are identical. Dependability: A combination of precision and accuracy that implies reproducibility and accuracy (close to true value). Mean: Mean is measure of central tendency and is expressed as X. Formula Where X X/N X Is mean X is individual observation X is summation sigh X is no. of observation

Standard deviation: It is the variability from mean. Manual method X X N = = = = = summation sigh individual observation difference mean no. of observation

Calculator method X2 = ( X) 2 = N = sum of square of individual value. square of sum of all valued. no. of observation.

Coefficient of variation (C.V): Or relative standard deviation (RSD) standard deviation is expressed as the percentage of the mean value. CV% = 100 X (SD/mean)

Prof: R. Shinde

A CV value of less than 5% is desirable. Significence of C.V.: It tells how good technician is in his laboratory precision. Following are the steps involved in preparation of quality control chart. Step 1 : Repeated analysis of control serum. Step 2: Calculation of standard deviation (S.D.) and coefficient of variation (C.V.) Step 3: Establishing the tolerance range. Step 4: Drawing the quality control chart. (QC chart) Step 5: Daily plotting (Use of QC Chart)

Prof: R. Shinde

GENERAL APPROACH TO QUALITY CONTROL 1. LABORATORY TEST REQUEST: All laboratory test request should be made in writing on the appropriate request from their timings must be maintained. When request was issued. When specimen was obtained. When results were reported. 2. SPECIMEN COLLECTION AND PROCESSING Proper patient preparation before collection of sample is of maximum importance. Laboratory results are as good as the specimen bad specimen cannot give good results. Identification of patient and checking the name on requisition slip is very important. Specimen should be collected in appropriate container and container should be labeled before collection. Transportation of specimen and processing of specimen should be done on time. 3. LABORATORY RECORDS: Laboratory accession number should be given to each specimen on arrival to laboratory. It should be recorded in the laboratory register (log book). Technician record must refer to accession number; the results must be noted in accession register with time of report and name of technician, who reported results. 4. ERROR IN PROCEDURE: Laboratory procedures adopted for performing tests must be documented for physicians information. The laboratory must maintain a procedure manual for each test performed. Reagents must be stored properly & out dated reagents must be discarded instruments must be properly maintained and checked at regular intervals like wavelength of colorimeter, efficiency of autoclave, water bath temperature, oven, freeze, clod room temp etc. Calculation should be done properly after noting down actual readings calculations should be done twice before sending reports. 5. PROFICIENCY TESTING: The technician must maintain daily plotting of quality control chart, participate in proficiency testing programme. 6. LABORATORY PERSONNEL: Only properly trained personal should be appointed by the laboratory, he should attend continuing education every year.

Prof: R. Shinde

7. QUALITY CONTROL IN INDIVIDUAL LABORATORY: Each laboratory must adopt their own quality control approach with special attention to specimen collection, identification, processing, records and maintenance of instruments, reagents and procedures. 8. HEMATOLOGY LABORATORY: Hemolysed blood is of little value, test must run within specified time. ESR Within 2hrs WBC Count Within 4 hrs RBC/PVC Within 8-10 hrs Check diluters. Temperature of water bath, centrifuge machine at regular intervals. Correlation of results. Normal hematocrit should have a normal hemoglobin. Stained smear must correlate with count. Slide with Hypochromic red cells must have low Hb. Presence of macrocytes and Megaloblasts should correspond with increased MCV. Correlation of no. of leucocytes under high power (40x) and WBC count. No. of WBC High power No. of WBC High power Estimated WBC Count per 14 mm 2-4 4-7 x 103 4-6 7-10 x 103 6-10 10- 13 x 103 10-20 13-18 x 103

Correlation of average no. of platelets to oil immersion (100 x ) and actual platelet count. No. of platelet oil immersion field Less that 1 6-15 (several platelets ) More than 25 Comparable platelets count Less than 2 x 104 (thrombocytopenia) 3 x 105 (normal) 5 x 10 5

Platelet count per cu.mm = Average count of 10 fields to oil immersion x 20000/9. BLOOD BANK LABORATORY: Check all reagents frequently. Check coombs regards with coombs control (sensitized cells). Precaution should be taken to avoid wrong tranfustion. A complete record of every unit of blood received by the blood bank must be kept in a long book thats indicates date of receiving donors blood source, code no,. b lood type, result of compatibility testing, name of recipient, date of tranfustion.

Prof: R. Shinde

Before match blood leaves laboratory, it should be rechecked and countersigned by the technician. 10. MICROBIOLOGY LABORATORY Proper collection of specimen and transportation to the laboratory are important. Performance of equipment and reliability of reagent are first step of quality control followed by appropriate culture technique and direct microscopic examination. Testing of media and stains against type cultures is very important. Sensitivity testing must also be checked with appropriate organisms and zone formed must tally with supplier specification. TEST OF PERFORMANCE OF COMMON MEDIA AND BIOCHEMICAL REACTION Media Blood agar Chocolate agar Mc conkey S- S agar Ureas agar Test organism Streptococcus pyogens N. gonorrhea E- coli E- coli E- coli proteus Result Beta hemolysis Growth in 5% Co2 Red colonies Pink colonies Negative Positive

Parasitology : Fresh, properly collected and uncontaminated stool specimen can yield a proper diagnosis. A parasitology reference book must be available in laboratory for identifying eggs. Every tenth negative report must be checked by supervisor and positive specimen be tallied by supervisor. A set of prepared slide should be kept in laboratory for reference. Keep all type culture at 50c in refrigerator 11. SEROLOGY LABORATORY: All serological should have positive and a negative control. Technician should recognize the positive reaction and should grade reaction (1 + to 4+) the border line reaction needs personal judgement. 12. CLINICAL PATHOLOGY LABORATORY: Urine specimen must be collected properly, morning, mid stream in a sterile urine container. The specimen should be proceed within 2 hrs. Urine culture must be done with well mixed specimens and not with the sediment. If bacterial population is less than 1 x 105 the specimen is rejected.

Prof: R. Shinde

13. HISTOLOGY AND CYTOLOGY: All steps of tissue processing should be carried out correctly. Stains must be checked with known sectors. Permanent slide should be kept in laboratory for reference. 14. CLINICAL BIOCHEMISTRY LABORATORY Hemolysed specimen should be rejected. Specimen for bilirubin should not be exposed to strong light for blood gas analysis specimen must be in ice and under anaerobic conditions. Each morning analysis of control serum should be done. At regular intervals prep 37 0C /18 hr prepare a new calibration curve. Preparation of quality control chart Step 1 : Repeated analysis of control serum. Step 2: Calculation of standard deviation and coefficient of variation. Step 3: Establishing the tolerance range. Step 4: Drawing the quality control chart. Step 5: Daily plotting (use of QC chart) Step 6: Interpretation of quality control chart. Step 1: The goal of repeated analysis with control serum is to establish accuracy (close to true value) and degree of variation (CV) which depends on type of test and on the technical skill. The control serum can be preserved in freezing compartment for daily analysis of control and plotting QC chart. The control serum can be obtained by pooling normal specimen or purchase from market. The latter gives true value (manufactures analysis ) and helps to establish accuracy of procedure, in laboratory. Step 2: Calculation of standard deviation and coefficient of variation. X = 72.08 / 10 = 7.2

= * ( X X) 2] ---------------N- 1 = 1.63/ 9 = 0.18 S.D. = 0.425 S.D.

Prof: R. Shinde

C.V% = = =

100 X (S.D./mean) 100 x (0.425 /7.2) 5.90%

(This examination of CV tells the technician how good he is in his laboratory precision. A CV value of less than 5% is desirable) Tolerance range is equivalent to, 2 S.D. + 2 S D., is, 7.2 + 2 (0.4) = 7.2 + 0.8 = 8.0 = -2 S.D. is 7.2 2 (0.4) = 7.2-0.8 = 6.4 (Range of 6.4 to 8 is tolerance range) ***************