Internship Report
By
Qaisar Abbas
2014-GCUF-03811
Report submitted in partial fulfillment of
the requirement for the degree of
BACHELOR OF SCIENCE
IN
PHYSIOLOGY
DEPARTMENT OF PHYSIOLOGY
G.C UNIVERSITY FAISALABAD
?
Dedication
This report is dedicated to my parents support me throughout my studies. I also want to dedicate this report to my teachers who has been a source of knowledge, inspiration taught me during my bachelor studies. My colleagues are also deserving for this dedication because I also learned those things which I can not learn from my teachers or supervisor.
Certificate from Supervisor
I certify that the contents and form of report submitted by Mr./Miss/Mrs.………………………., Registration No…………………. has been found satisfactory and in accordance with the prescribed format.
Signature of Supervisor ………………………
Name: …………………………….
Designation with Stamp…………………………..
Chairperson
Signature with Stamp………………………………
?
Acknowledgement
In the name of Allah who is the most beneficent and more merciful.
I am very thankful to Almighty Allah for giving me the strength to complete the internship successfully.
I would like to express my gratitude to Dr. Mubashir Razzaq Head of Pathology Department Faisalabad institute of Cardiology, Faisalabad for allowing me to work in lab.
I am also thankful to Dr. Kashif Sarfaraz for supporting me throughout my internship with proper guideline. I am thankful to Dr. Aamir Alam for giving me healthy tips about preparing internship report.
I am also thankful to Dr. Haseeb Anwar Head of Physiology Department, Government College University Faisalabad for providing me an opportunity to work in highly professional and established lab to polish my theoretical skills and knowledge with practical exposure.
Objectives of Internship:
Following are the objectives of internship;
• An internship gives fruitful outcomes for young workers.
• Internships helps in building the resume.
• Do not become pessimist in the period of internship.
• Take advantage of the many benefits of holding an internship.
• Finding or being placed in an internship and receiving credit for the experience are privileges, not rights.
Introduction to Hospital:
• In November 13, 2007 Faisalabad institute of cardiology, Faisalabad start working.t
• This is a special institute for cardiac disease.
• It consists of 202 beds and situated near Chenab Club, Faisalabad.
• Cardiac patients from Faisalabad, Toba Tek Singh, Sargodha, Chiniot, Jhange and beyond areas are getting services.
• This is also a teaching hospital providing post-graduation in Cardiac Surgery, Cardiology and BS in Cardiac Perfusion.
Mission statement:
• Faisalabad Institute of Cardiology Faisalabad is striving to play a leading role in providing the excellent healthcare services to cardiac patients and to prepare professionals for the challenges of future through academic activities.
Objective Statement:
Following are the objectives of this institute;
• To provide the best cardiac diagnostic and treatment services to the patients.
• Improve cardiac health care services.
• Provide facilities of highly specialized nature.
• It is also a referral center for the central Punjab.
• Train medical, nurses, and paramedical personals in this field.
• Provide research services in the field of cardiac surgery and cardiology.
Lab ethics:
Every student and researcher should follow the following SOPs strictly;
• Must wear a lab coat.
• Wash separately from other clothes with detergent and bleach.
• No personal effects are permitted in the lab, only essential lab supplies.
• Long hair must be tied.
• Do not touch your hair.
• Wash hands with detergent soap for minimum of 30 sec.
• Remove gloves using finger of opposite hand.
• Do not eat and drink in the lab
• Never mouth pipette.
• Always use a pro-pipette.
• Cover any cuts with a bandage.
• Students should wear closed shoes.
• Treat animal tissue as a biohazard.
• All biohazard disposable containers must be labelled with a biohazard label. After autoclaving the biohazard label is removed.
• All biohazard materials must be autoclaved.
• Discard all non-sharp biologically contaminated items in the Petri plate container.
• Located on your work bench. Any ‘pointy’ item must be disposed in plastic lined bucket (not Petri Plate container), this includes paperman tips, disposable cuvettes, slides, Pasteur pipettes, micro titration plates, disposable 1 ml and 10 ml pipettes, broken glassware, brittle plastic objects, metal objects. This includes all items that are ‘pointy’ regardless of biological contact.
• Biological spills put on glove. Cordon off area to let aerosol settle for 10-30 min then wash container.
• Glassware (unbroken), remove tape and pen markings (use alcohol) from glassware before placing on discard trolley.
• Chemical hazardous material, organic solvents must be disposed of in organic solvent container.
• Biohazard sharps disposal: Dispose of all sharps (needles, syringe tops, razors, scalpel blades) in specified container (red or yellow).
• Wash bench area before and after with BDD (Back down Detergent Disinfectant containing nonyl-phenoxypolyethoxy ethanol, alkyl-aryl ammonium chloride and ethyl benzyl ammonium chlorides).
• First aid kit present in lab Location is safety station.
• Know location of exits, fire extinguisher, eye wash, full body shower.
• Know how to operate equipment before use.
• DO NOT use equipment unless you know exactly how to operate the equipment.
• Leave your bench area clean.
• All equipment and supplies should be returned to original place.
Reception of Pathology Laboratory:
It is a receiving point at which samples are received and reports are delivered to patients after performing the specific test in pathology laboratory.
Sample reception criteria
Following SOP, s should be followed at reception;
Checking the request form:
• Check the request form of the patient for tests.
• Check the sample according to the requirement of test that the sample is fulfilling the criteria or not.
Patient information:
• Check the patient name and forename.
• Check the age of patient.
• Check the phone number, ward name, category and fee paid slip.
Specific vials for specific tests:
• Check the vial, containing blood sample that, this vial is appropriate for the test which is prescribed by the doctor.
• For CBC, the vial should be purple headed containing EDTA.
• For coagulation tests, blue headed vial is used containing sodium citrate.
• For chemistry, green, red, yellow and syringe also used for sampling.
• For thyroid profile, yellow vial containing gel is used.
Amount of sample:
• For hematology tests, sample should be according to the requirements i.e. for CBC sample should not be more than 2ml and for PT, APPT and INR blood sample should be neither less nor more than 2.6 ml for 3ml vial and 1.8 ml for 2ml vial.
• In chemistry, for lipid profile, sugar level and HbsAg, HIV, anti Hcv screening sample should be 2ml for each.
• For thyroid profile 3ml blood sample is require.
• Elisa, Trop. I. and Trop. t. 2ml for each is require.
Form of sample
• For different tests, different form of sample is required i.e. for hematology, all blood gasses (ABG, s) troponin I and troponin T blood should be unclothed while for other chemistry tests like thyroid profile and lipid profile, blood should be in clotted form.
If sample is not fulfilling the following criteria, the sample is not acceptable for lab, so sample should be rejected and ask the patient to repeat the sample.
Computer section of pathology lab:
Computer section of pathology lab is an important section where report is generated in hard form. In big hospitals, online system is present to deliver the reports of admitted patients in wards. This is system based on a software like patient management system and hospital management system. After allotting the specific lab number to each sample, these lab numbers are entered in that software, where it connects with fee section of hospital. This provides the information about the patient, required test and payment of fee. When tests are done, their results will be saved in that software again. On the demand of patient, this report is generated in hard form and handed over to the patient.
OPD reception
Phlebotomy principles
Phlebotomy:
The process in which the blood is collected through venipuncture and sometimes by pricking the artery.
Purpose:
The purpose of phlebotomy is that, to perform the different test to diagnose any disease.
Required material and equipment:
Following are the material and equipment are required for phlebotomy;
• Subject
• Syringe or other pricking equipment
• Sample saving vials
• Disinfectant
• Tourniquet
• Cotton
• Sticking
• Yellow box
Procedure:
Following are the steps of phlebotomy;
• Ask the patient to sit in the chair and satisfy to the patient.
• Took the slip at which the tests are recommended by the doctor.
• Check the fee paid slip.
• Confirm that either venous blood is required or arterial.
• Identify the place of puncture site.
• Place the tourniquet 2cm above the picturing site.
• Disinfect the puncturing site with cotton swab.
• Open the syringe and tied up the needle.
• Syringe should be air free and easily moveable piston.
• Prick and collect the sample according to the requirement of test.
• Save the sample in the vials according the rules and regulations.
• Attach the sample with slip with the help of sticking.
• Dispose off the syringe into the yellow box.
• Send the sample in the lab.
• Use the syringe for single patient.
• Do not use same syringe for second prick.
Introduction:
Hematology is a branch of medicine which deals with the study of blood, blood formation, blood forming organs and blood diseases. Hematology test are used to diagnose the blood related disorders like anemia, blood cancer, viral infection, drug reaction etc. hematological tests like complete blood count, blood group, ESR, PT-INR etc.
Complete blood count (CBC)
Introduction:
CBC stands for complete blood count. In this test different types of blood cells are analyzed like red blood cells, white blood cells, platelets and some other factors like MCV, MCHC, RDW, PDW etc. this test is done to diagnose leukemia, anemia and infection. Beyond the normal limits of any type of blood cell, is the indication that you are underlying medical condition.
Principle:
By using cell pack method, count the blood cells on automated hematology analyzer. Different types of blood cells like red blood cells, white blood cells, platelets and other factors like MCV, MCHC etc. are analyzed in this test.
Requirements:
• Blood Sample
• EDTA vial
• Labeling marker
• Roller mixer
• Abascus-5 analyzer
Procedure:
• Sit the patient in the chair, sitting or lying on the bed.
• Identify the site for venipuncture.
• Draw the blood sample.
• Put the blood sample in to the EDTA vial and mix the sample.
• Properly label the sample test tube.
• Place the sample tube on the roller mixer to mix the sample properly.
• Place the sample tube into sample probe of the Abacus-5 hematology analyzer.
• Feed the sample “ID”.
• Press the button “DONE”.
• Press the button “ADD”.
• Press the button “start”.
• Automated hematology analyzer use cell pack for the differentiation of blood cells.
• Results are displayed on the display screen of the automated hematology analyzer.
Results & Evaluation:
Following are the normal range;
Different Blood Cell evaluation
Cell Name Normal Range Causes of Low Count Causes of High Count
White Blood Cell 4,500-11,000 cells/µL • Bone Marrow Damage
• Autoimmune disease
• Disease of Immune System e.g. HIV/AIDS • Viral Infection
• Inflammation
• Asthma
Neutrophils 1800-7800 cell/µL (56%) Do Do
Lymphocyte 1000-4800 cell/µL (34)% Do Do
Eosinophils 0-450 cell/µL (2.7%) Numbers are normally low in the blood. Medically insignificant • Drug Reaction
• Parasitic Infection
• Addison disease
Basophils 0-200 cell/µL (0.3%) Numbers are normally low in the blood. Medically insignificant • Allergic reactions
• Inflammation
• Uremia
Red Blood Cells 4.5-5.9x109cell/µL (For Male)
4.5-5.1 x109 cell/µL(For Female) Known as anemia
• Acute bleeding
• Nutritional deficiency
• Bone Marrow damage Known as polycythemia
• Dehydration
• Lung Disease
• Living high altitude
Hemoglobin 14-17.5g/dL (For Male)
12.3-15.3g/dL (For Female) Do Do
Hematocrit 41.5-50.4% (For Male)
36.9-44.6% (For Female) Do Do
Mean Corpuscular Volume 80-96 µm Indicates RBCs are smaller than normal (microcytic); caused by iron deficiency anemia Indicates RBCs are larger than normal (macrocytic),in anemia caused by vitamin B12 or folate deficiency, hypothyroidism
Mean Corpuscular Hemoglobin 27.5-33.2 pg Do Do
Mean Corpuscular Hemoglobin Conc. 33.4-35.5 g/Dl Decreased MCHC values are seen in conditions such as iron deficiency anemia and thalassemia. Increased MCHC values are seen in autoimmune hemolytic anemia
Platelets 150-450×103/µL Viral Infection, Cirrhosis Cancer, Arthritis
Precautions:
• Before analyzing the blood sample, be sure that the blood sample is well mixed and not clotted.
• For CBC, sample should be in EDTA tube.
• Sample should not old more than 24 hours.
Coagulation test
Introduction:
Coagulation tests i.e. PT –INR, APTT provides the basic information of hemostasis and thrombosis to prevent the blood loss from vascular damage sites.
Principle: A coagulation factor test is based on the determination of the relationship between the concentration of the factor and the resultant clotting time, other reagent concentrations being kept constant.
Requirement:
• Sample
• Vial containing Tri sodium citrate
• Centrifuge machine
• Human-clot pro hematology analyze
• Forceps
Procedure:
• Draw the sample of patient by using proper method.
• Put the sample into vial of blue color.
• Label the sample properly.
• Send the sample into hematology lab.
• Centrifuge the sample into centrifuge machine for 4 minutes.
• Feed the sample ID into the machine.
• Close the shield of machine.
• Press the button “load reagent”.
• Press the button “load sample”.
• Select the sample and test type i.e. PT, INR and APTT.
• Press the button “ok”.
• Press the button “start the run”.
Results:
Sr.no. Test name Normal value Causes of prolonged value Causes of shorting values
1 Prothrombin time (PT) 14 sec. • Factor vii deficiency
• Early oral anticoagulant therapy
• Vitamin K deficiency
• Liver deficiency • Thyroid disorders
• Cancer
• Estrogen containing medication
2 Activated partial thromboplastin time(aPTT) 30-45 sec. • Heparin use
• Inhibitor of vWF, factors VIII, IX, XI or XII
• Antiphospholipid antibodies
3 International normalized ratio (INR) 0.8-1.1 Same of PT
Erythrocyte sedimentation rate
Introduction:
An Erythrocyte sedimentation rate test also called as sed rate test or sedimentation rate test. This test is very helpful to diagnose an active disease like inflammatory diseases or autoimmune diseases i.e. rheumatoid arthritis and systemic lupus erythematosus, cancer and infections.
Methods of ESR MEASURMENTS:
Following are the two methods for the measurement of ESR.
• Wintergreen’s method
• Win Trobe’s method
Principle:
Erythrocyte sedimentation rate is a nonspecific test to detect the presence or absence of a disease. When blood is allowed to stand vertically and undisturbed position in ESR rage by SEDIPLAST WESTERGREN method, the red cells start settling down the bottom of the ESR vial.
Materials:
• Blood sample
• ESR vial
• ESR rage
• Stop watch
Procedure:
• Draw the sample by practicing a proper method.
• Put the sample in ESR vial.
• Properly label the sample with lab number.
• Place the sample vertically into the ESR rage.
• Note down the level of red cells (first level) on the ESR rage
• Note down the time or start the stop watch.
• After the one hour, note the erythrocyte sedimentation level second level).R
• Compare to the first level and second level.
• Calculate the sedimentation rate per hour.
Results:
Sr.no. Gender Age Normal value Causes of low ESR Causes of elevated ESR
I Female Less than 50 20mm/hour • Congestive heart failure.
• Hypofibrinogenemia.
• Leukocytosis.
• Low plasma protein.
• Polycythemia.
• Sickle cell anemia • inflammation
• anemia,
• infection,
• pregnancy
• Aging.
• severe infection
• increase in globulins
• Polymyalgia rheumatica
• Temporal arthritis
Ii Male Less than 50 15mm/hour
Iii Female More then50 30mm/hour
IV Male More than 50 20mm/hour
V Newborn 2mm/hour
Vi Children Before puberty 3-13mm/hour
Precautions:
• Only use the EDTA or Sodium Citrate containing tube for good results.
• Carefully note the start and stop time.
• Carefully note the first and second level of red cells.
• Always mix well to the sample before performing test.
• Do not use clotted sample for test.
• Sample should not old more than 24 hours.
Equipment for Hematology:
Abacus-5 Human clot pro
ESR Det
Definition:
The branch of biology that deals with the study of microorganism and their effects on other living things.
Introduction:
In microbiology different microorganism are studied like bacteria, viruses, fungi and protozoa. For clinical diagnosis different culture tests are used to identify the presence of type of microorganism.
Following are the cultures which are being used;
• Blood culture
• Puss culture
• Urine culture
• Body fluid culture
These culture tests are performed on specific culture media.
Following are the different types of culture media;
• MacConkey
• CLED for urine
• Mannitol salt
• Blood agar
Blood for culture and Sensitivity
Introduction:
It is a culture test through which blood culture is done. If microbe’s colony is found on culture, it means that an infection is present, then sensitivity can be applied according to the requirement. Through this culture, type of microbes can be identified.
Principle:
Several types of microbes may be present in blood which causes the hemolysis of RBC, s and hemoglobin by secreting enzymes like hemolysins. These bacteria may be Gram- positive or Gram-negative cocci. Through this culture, type of bacteria is identified.
Composition of Blood Agar:
• 0.5% NaCl
• 1.5% agar
• 5% Sheep Blood
• Buffer (pH 7.2-7.6)
• 0.3% yeast extract
• Distilled water
• 0.5% Peptones
Material:
• Petri dishes.
• Incubator.
• Autoclave.
• Blood sample.
• Glass slides.
• Microscope.
• Inoculating loop.
• Blood culture vials.
• Stains.
• Burner.
Procedure:
• Blood sample is collected into blood culture vials.
• Incubate the petri dishes at 37 C before 24 hours of use.
• Pour the already prepared blood agar into the petri dishes.
• Wait until this media become semi solidify.
• Take a drop of blood and pour it on that blood agar media.
• With the help of hot red inoculating loop, striking is done in zig zag manner.
• Again, incubate these petri dishes at 37 C for 24 hours.
• Observe the culture after 24 hours.
• If the growth appears, go for the further process like preparing slides and staining.
• If growth is not found, go for the subculture.
Results:
Following three types of hemolysis may be present;
• Alpha hemolysis: partial destruction of RBC, s and greenish color of agar is appeared.
• Beta hemolysis: complete destruction of hemoglobin and RBC, s and clear medium around colonies.
• Gemma hemolysis: it is non-hemolysis and simple growth appears.
Urine for culture and sensitivity
Introduction:
To diagnose the diseases of urinary tract, urine culture test is done to identify the microbes causing urinary tract infections (UTI, s). For this culture test cysteine-lactose-electrolyte-deficient (CLED) agar is used.
Composition of CLED agar:
• Agar 15g/L
• Bromothymol blue 20mg/L
• Tryptone 4g/L
• Peptone 4g/L
• Lactose 10g/L
• L-cysteine 128g/L
• Lab lemco powder 3g/L
Material:
• Urine sample.
• Petri dishes.
• CLED agar media.
• Microscope.
• Glass slides.
• Stains and dyes.
• Burner
• Inoculating loop.
• Autoclave.
• Incubator
Procedure:
• Collect urine sample.
• Incubate the petri dishes at 37 C for 24 hours before use.
• Take a drop of urine sample and spread it on the media by striking.
• Incubate that media plates for 24 hours.
• After 24 hours, observe the culture.
• If growth is present, go for further process like slide making and staining.
• If growth is not present, go for subculture.
Results:
Sr.no Name of Bacteria Appearance
1 Klebsiella Much mucoid, varying color from yellow to white blue.
2 Salmonella Blue in color and flat colonies.
3 Corynebacterial Gray and small colonies.
4 Staphylococcus aureus Deep yellow and uniform in color, 0.75 mm in diameter.
5 E. coli Yellow in color, 1.75 mm in diameter
6 Proteus species Smaller than E. coli in size, translucent in color.
7 Pseudomonas aeruginosa Green colony in color, rough periphery.
8 Enterococcus faecalis Yellow in color, 0.5 mm in size
Pus for culture and sensitivity
Introduction:
Pus is produced in wound when the wound is infected with bacteria. Pus culture test is done to identify the type of bacteria which causes the infection. For this test MacConkey agar is used to culture the pus.
Composition of agar:
Following is the composition;
No. Component name Quantity
1 Distilled water 1L
2 Agar 13.5g/L
3 Neutral red 0.03g/L
4 Crystal violet 0.001g/L
5 Proteose peptones 3g/L
6 Bile salt 1.5g/L
7 Sodium chloride 5g/L
8 Peptone 17g/L
9 Lactose 10g/L
Required materials:
Following materials are require;
• Pus sample
• Pus sample tubes
• MacConkey agar
• Petri dishes
• Burner
• Glass slides
• Microscope
• Stains and dyes
• Inoculating loop
• Autoclave machine
Procedure:
Following steps are involve;
• Draw pus sample in pus sample tube.
• Label it properly.
• Incubate to petri dishes at 37°C.
• Pour pus sample on petri dishes.
• Incubate the plate.
• Observe the culture after 24 hours.
• Record the observations.
Results:
Following results may be;
Sr.no. Organism Color Appearance of Colony
1 Aerobacter aerogens Pink Mucoid
2 Escherichia coli Red/pink Non-mucoid
3 Pseudomonas aeruginosa Green-Brown Fluorescent growth
4 Staphylococcus species Pale Pink Opaque
5
Enterococcus species Red Minute, Round
Fluid complete examination
Introduction:
In laboratory, many types of fluids can be tested other than blood because these fluids tests can give more precise information about any part of the body. These fluids may be paracardial, Extracellular fluid, cerebrospinal fluid, peritoneal fluid, and pleural fluid etc.
Principle:
Fluid examination may be of the three types;
• Physical Examination:
Volume, Color, turbidity, coagulum, deposits.
• Chemical Examination:
Glucose, total protein, albumin, LDH.
• Microscopic Examination:
RBCS, WBCs, total cells, lymphocytes, polycytes, AFBs, morphology of cells, gram positive or negative bacteria.
Microscopy is done with Neubauer chamber (hemocytometer) or stained slides.
Required materials:
Following materials are required;
• Cerebrospinal fluid sample.
• Neubauer chamber.
• Cover slip
• Microscope.
Procedure:
Following steps are involve;
• Make the dilution of blood sample.
• Place cover slip over the chamber.
• The tip of the pipette is placed near the cover slip.
• Capillary action will draw the fluid into the chamber
• It is important not to overload the chamber, as doing so will give an inaccurate count.
• Count the cells by applying formula;
Total cells = No. of cells x 2.5 x dilution facto
Staining:
It is a technique involve in artificial coloring of a microorganism, cell or tissue to facilitate in differentiation and classification.
Stain:
A penetrative dye which is used to coloring a microorganism, cell or tissue to facilitate in differentiation and classification.
Types of staining:
Following are the major types of staining;
• Gram staining.
• Giemsa staining.
• Ziehl-Neelsen staining.
Gram staining
Introduction:
Difference between gram- positive and gram-negative bacteria can be through this type of stain. Gram-negative bacteria stain red color while gram-positive bacteria stain purple brown.
Principle:
When the bacterial smear is stained with primary stain (Crystal Violet) and fixed by the iodine, some of the bacteria retain the primary stain and some are decolorized by alcohol. Gram positive bacteria have low peptidoglycan and lipid content due to which cell shrinkage occur and pores of the cell membrane also closed, and crystal violet stain is not removed. This causes the purple and blue appearance of gram positive bacteria. Gram negative bacteria have high content of peptidoglycan and lipids. So, gram negative bacteria stain red due to safranin.
Required material:
Following materials are required;
• Primary stain (crystal violet)
• Mordant (iodine)
• Decolorizer(methanol)
• Counterstain (safranin)
• Clean glass slide
• Microscope
• Inoculating loop
• Burner
• Bacterial culture
Procedure:
Following steps are involved;
• Take a grease free slide.
• Prepare a smear on the clean slide.
• Air dry.
• Heat fix
• Crystal Violet was poured.
• Wait for 30 seconds to 1 minutes.
• Rinse with tap water.
• Flood iodine for 1 minute.
• Wash with tap water.
• Wash with 95% alcohol for about 10-20 seconds.
• Rinse with tap water.
• pour safranin for about 1 minute.
• Wash with tap water.
• Air dry.
• Blot dry.
• Observe under Microscope.
Interpretation:
Gram positive bacteria like Clostridium, Actinomyces, Staphylococcus, Streptococcus etc. stained purple blue color while gram negative bacteria like Escherichia coli, Helicobacter, Salmonella, Pseudomonas etc. are stained red which is shown in figure.
Giemsa staining
Introduction:
Giemsa staining is commonly used for parasitic examination of blood. Nuclear differentiation and morphological characteristics can be studied under the microscope by Giemsa stain.
Principle:
Giemsa stain contains both the Acidic and Basic dyes like the affinity of blood cells. The acidic dye like Eosin and Azure stains the basic components of the cells like the cytoplasm etc. Basic dye e.g. Methylene blue stains the acidic components, especially to cell nucleus.
Required materials:
Following materials are required;
• Clean slide
• Distilled water
• Inoculating loop
• Burner
• Stock solution (Giemsa stain).
• Microscope
Procedure:
Following steps are involve;
• Take a grease free slide.
• Prepare a smear on the clean slide.
• Air dry.
• Heat fix
• Dissolve 1ml of Giemsa stain into 4ml of distilled water.
• Pour this dilution on smear which is prepared on the glass slide.
• Wait for 15 minutes.
• Wash with tap water.
• Observe under the microscope.
Interpretations:
Following are the interpretations;
Parts of Cell Color
• Nucleus • Blue
• Basophilic cytoplasm • Blue
• Neutrophilic granules • Lilac
• Eosinophilic granules • Orange
• Mast cell granules • Deep -violet
• Nucleolus • Blue
• Red cells • Pink
• Cytoplasm of mature monocytes • Grey blue
Ziehl-Neelsen staining
Introduction:
The Ziehl–Nieelsen stain, also called as the acid-fast stain. It is a special stain used to identify acid-fast organisms mainly Mycobacteria.
Principle:
This stain is used to identify mycobacterium tuberculosis and mycobacterium leprae (also called as acid-fast organisms). They are stained with red dye known as carbol fuschin. Their cell wall contains mycolic acid due to which they retain dye after the application of acid.
Required materials:
Following materials are required;
• Glass slide
• Inoculating loop
• Sample
• Burner
• Basic dye (carbol fuschin)
• Heat (mordant)
• Decolorizer
• Counter stain (methylene blue)
Procedure:
Following steps are involve;
• Make a smear of the specimen on the glass slide.
• Fix either by heating or methanol.
• Pour carbol fuschin on the smear.
• Wait for 5 minutes.
• Wash with tap water.
• Add 20% sulphuric acid.
• Wait for one minute.
• Wash off with tap water.
• add methylene blue.
• Wait for two minutes.
• Wash with tap water
• Air dry.
• Examine under microscope with oil immersion lens.
Results:
Acid fast bacilli stain pink. They may be straight or slightly curved rods. The background will appear blue due to methylene blue.
Interpretations:
If definite bacilli are seen, report as AFB positive.
S.r.no. No. of AFBs Results
1 ; 10 AFB/high power field –;+++
2 1-10 AFB/high power field –; ++
3 10-100 AFB/100 high power fields –; +
4 1-9 AFB/100 high power fields –; exact number
Figures of staining:
Giemsa stain Z.N. stain
Gram stain
Urinalysis:
Introduction:
Urinalysis is a chemical examination for identification of protein, glucose, blood cells, pH, urobilinogen, bilirubin, nitrites, ketone bodies and leukocyte esterase in urine sample.
Principle:
The Diagnostic Strips which are used to examine urine sample. These plastic strips contain several reagent areas separately. Urinalysis gives information regarding the status of kidney function, liver function, carbohydrate metabolism, urinary tract infection and acid-base balance.
Required materials:
Following materials are required;
• Urine sample
• Urine stripe
• Gloves
Procedure:
Following steps are involve;
• Place urine sample at balance surface.
• Wear gloves and face mask.
• Take a stripe.
• Dip the stripe for one minutes into urine sample until the all reagent areas dip into the sample.
• Wait for 2 minutes.
• Observe the color of reagent areas.
• Compare with chromatic scale.
• Note the results.
Normal Values:
• Color – Yellow (light/pale to dark/deep amber)
• Clarity/turbidity – Clear or cloudy ?
• pH – 4.5-8.
• Specific gravity – 1.005-1.025.
• Glucose – ?130 mg/d.
• Ketones – None.
• Nitrites – Negative.
• Leukocyte esterase – Negative.
Oxidase test
Introduction:
This test is used for identification of bacteria that produce cytochrome c oxidase enzyme. If cytochrome c oxidase is present in sample, it oxidizes tetramethyl-p-phenylenediamine into indophenols (product of purple color).
Principle:
When the sample is applied on the oxidase paper, it turned into purple color which is positive test. If cytochrome c oxidase is not present in sample, paper remains white which indicates to negative test.
Required materials:
• Moist filter paper with 1% tetramethyl-p-phenylenediamine dihydrochloride.
• sample.
• Pipet
Procedure:
Following steps are involve;
• Take a filter paper.
• moiest with tetramethyl-p-phenylenediamine dihydrochloride
• Socked the paper with sterile distilled water
• Pick the bacterial colony with inoculating loop.
• Spread on the filter paper.
• Wait for 10-30 second.
• Examine smeared area of paper.
Results:
If cytochrome c oxidase is present, then the color will be change into purple or deep blue, otherwise it remains white in color.
Catalase test
Introduction:
This test is used to identify catalase enzyme producing bacteria. Catalase is an enzyme which is secreted by bacteria to neutralize the bactericidal effect of H2O2. If catalase enzyme is present in sample, by applying H2O2 it produces bubbles which is the indication of positive test.
Principle:
Catalase is an enzyme which is present in some types of bacteria that converts to hydrogen peroxide into oxygen and water. The bacteria that contain catalase enzyme are either aerobic or facultative anaerobes.
Required materials:
Following materials are required;
• Glass slide.
• Sample.
• Inoculating loop.
• Hydrogen per oxide.
Procedure:
Following steps are involve;
• Take a clean and dry glass slide.
• Pour a small bacterial colony on the glass slide with inoculating loop.
• Add a drop of H2O2 on to the slide.
• Mix well.
• Wait for 5-10 seconds.
• Observe the slide.
Results:
• If bubbling appears, the test will be positive.
• If there is no bubble or a few scattered bubbles are present, it is the indication of negative test.
Coagulase test
Introduction:
This test is used for the differentiation between Staphylococcus aureus (positive) and Coagulase Negative Staphylococcus.
Principle:
The coagulase secreted by Staphylococcus Aureus reacts with factor (CRF) in plasma and makes a complex, known as thrombin. This converts fibrinogen to fibrin which causes clotting of plasma.
Required materials:
Following materials are required;
• Sample.
• Clean glass slide.
• Inoculating loop.
• Normal saline.
Procedure:
Following steps are involve;
• Take a clean and grease free slide.
• Label to both sides of glass slide as “test” and “control”.
• Pour a small colony on the both labelled sides of the glass slide.
• Mix a drop of plasma on this suspension.
• Wait for 5-10 seconds.
• Observe the slide.
Results:
If agglutination of cocci is occurred within 5-10 seconds, it is indication of positive test.
Oxidase test Coagulase test
Catalase test
Introduction:
In chemistry section of pathology lab, fluids are examined for diagnostic purpose. These fluids may be blood, CSF, plasma and serum. Plasma and Serum are the major fluids which are examined. There are two types of chemistry tests.
• Special chemistry tests.
• Routine chemistry tests.
Special chemistry:
In special chemistry, following tests are performed.
• Troponin I.
• Troponin T.
• Thyroid profile.
• Hepatitis B, C ELISA.
• All blood gases (ABGs)
Routine chemistry:
In routine chemistry, following tests are performed;
• Blood glucose.
• Renal function tests.
• Liver function tests.
• Lipid profile.
• Cardiac enzymes.
• Serum electrolytes.
Troponin I and T
Introduction:
A troponin test is performed to detect the levels troponin I or troponin T in the blood. When the heart muscles have been damaged due to heart attack, these both proteins are released in the blood. If heart muscles are more damaged, the greater amount of troponin I and T will be in the blood.
Principle:
In this test immunochromatographic method is used. When adding the specimen diluted in buffer to the sample well of the kit, it is absorbed, and it soaks a conjugate pad of kit. The immune complex is combined with the capture antibody which is fixed to the and it is colored as purple red by gold particle.
Required materials:
Following materials are required;
• Sample.
• Centrifuge machine.
• Forceps
• Advia Centaur immunoassay analyzer
Procedure:
Following steps are involve;
• Draw a sample.
• Label it properly.
• Centrifuge for 4 minutes.
• Put the sample in special rege.
• Enter the rage number into Advia Centaur immunoassay analyzer.
• Enter the lab number of sample into analyzer.
• Save information added into software of analyzer.
• Place the sample in sample place of analyzer.
• Click the button “start”.
• After some time usually after 30 minutes analyzer will display the results on the screen.
• Note the results.
Results:
Troponin I:
Normal values are;
• 0.30 ng/ml at any time high probability of Acute Myocardial Necrosis.
Troponin T:
Normal values are;
• Upto 14 pg/ml at 6-8 hours of symptoms excludes AMI.
• ;14 but 100 pg/ml at time high probability of AMI.
Thyroid profile
Introduction:
The thyroid is a small gland present in the lower-front of neck. It plays an important role to regulate many body’s processes, like metabolism, mood and energy production. It produces two major hormones i.e. Triiodothyronine (T3) and Thyroxine (T4).
Thyroid stimulating Harmons (TSH) is a hormone which is secreting from pituitary gland to control production of thyroid hormones. Three hormones are included in thyroid profile which are following;
• Triiodothyronine (T3).
• Thyroxine (T4).
• Thyroid Stimulating Hormone (TSH).
Principle:
The thyroid produces hormones called triiodothyronine, known as T3 and thyroxin, known as T4. Together, these hormones regulate your body’s process like temperature, metabolism, and heart rate. The levels of T3 and T4 helps determine the thyroid problems.
Required materials:
Following materials are required;
• Clotted blood sample.
• Centrifuge machine.
• Forceps
• RX Daytona immunoassay analyzer
Procedure:
Following steps are involve;
• Draw the sample through venipuncture.
• Give lab number.
• Centrifuge for 4 minutes.
• Enter rag number into the software part of analyzer.
• Enter lab number of sample.
• Select test name “thyroid profile”.
• Place the sample into sample place of analyzer.
• Close the shield of analyzer.
• Check the regent level.
• Check the distilled water level.
• Click the button “run” to start the test.
• Wait for results.
Normal values:
Following are the normal values;
Thyroid Test Reference Range
Total T3 80 -200 ng/dL
Total T4 4.5-12.5 µg/dL
TSH 0.5-4.70 µIU/mL
Enzyme Linked immunosorbent Assay (ELISA)
Introduction:
ELISA is used for the detection of antibodies of autoimmune diseases. A particular color is appeared when antigen reacts with antibody in the presence of linked enzyme. Therefor it is named as “enzyme-linked immunosorbent assay”, because this test is related to “enzyme” and “immune.
Principle:
When sample is added in microvalves which are already coated with specific antibodies or antigens (depends upon company of kit), if antibody or antigen is present in sample, it bounds and makes immunocomplex in the presence of linked enzyme and substrate. This conjugation produces a particular color which indicates either test is positive or negative.
Required material:
Following materials are required;
• Blood sample
• Centrifuge machine
• Forceps
• Serum cups
• ELISA kit
• LISYS Uno immunoassay analyzer
• Reagents for ELISA
Procedure:
Following steps are required;
• Draw a blood sample.
• Label it properly.
• Give a lab number to sample.
• Centrifuge to sample to get serum.
• Put 2ml of serum into serum cup.
• Label to serum cup just like sample and give same number to serum cup.
• Place the serum cup at sample place of analyzer.
• Select test type.
• Click the button “run”.
• Wait until the result showed on the screen.
Normal values:
Following are normal values;
• 1.00 mg/dl for hepatitis C virus.
• 1.00 mg/dl for hepatitis B virus.
Arterial Blood Gases (ABG, s)
Introduction:
An arterial blood gas (ABG) test detects the pH, the levels of oxygen(O2) and carbon dioxide (CO2) in the arterial blood. This test also tells that how well the lungs are able to exchange blood gases (remove carbon dioxide and move oxygen into the blood).
Principle:
pCO2, pH and pO2 are measured directly from blood sample by using electrodes. This measurement is done by using two types of electrodes. For detection of pH and pCO2, potentiometric electrodes are used. For these electrodes, semipermeable membrane is used where the voltage produced across this membrane. For the measurement of pO2, an amperometric electrode is used.
Required material:
Following materials are required;
• Arterial blood sample.
• Medica ABGs analyzer.
Procedure:
Following steps are involve;
• Draw a blood sample from artery.
• Label it properly.
• Give a lab number to sample.
• Mix well the sample.
• Put out the sample probe.
• Put the sample probe into the syringe and press the button yes for aspiration of blood.
• Remove sample when this comment showed on screen.
• Enter the data about sample.
• Wait for 2-3 minutes for results.
• Observe the results.
Normal values:
• PaO2 – 75 – 100 mmHg.
• PaCO2 – 38 – 42 mmHg.
• pH of 7.38 – 7.42.
• SaO2 – 94 – 100%
• HCO3 – 22 – 28 mEq/L.
Blood Glucose Level
Introduction:
Glucose testing is done to diagnose diabetes type 1, 2 and gestational diabetes. In diabetes, blood glucose level rises above normal value. Insulin is a hormone which controls the level of sugar in blood. In diabetes, either insulin is produced properly or not, it does not work. Which cusses the increased level of blood sugar. This increased level of blood sugar can cause the damage of organ if untreated. Blood sugar testing is of two types;
• Fasting blood glucose level.
• Random blood glucose level (RBS).
Principle:
Blood glucose level is detected by calorimetrically method. Glucose is converted into gluconate, when glucose is oxidized by glucose oxidase. After oxidation of glucose, hydrogen per oxide ions are produced which detected by chromogenic oxygen detector.
Required material:
Following materials are required;
• Pricking needle.
• Glucometer.
• Swab.
• Glucometer testing strip.
Procedure:
Following steps are involve;
• Change strip of glucometer.
• Select the prinking site on the finger.
• Disinfect the prinking site with swab.
• Press the finger tip 3 to 4 times to increase blood supply at pricking site.
• Prick the finger strip.
• Take a blood drop on testing strip.
• Wait for results.
Results:
• Fasting blood glucose level between 70 and 100 mg/dL is considered normal.
• Random blood glucose level below 125 mg/dl is considered normal.
Renal function test
Introduction:
Renal function test is performed to diagnose early kidney diseases. RFTs are included detection level of urea, creatinine and alkaline phosphate.
Creatinine:
This test examines whether creatinine is building up in blood. High levels of creatinine suggest a kidney problem.
Blood urea nitrogen:
It checks kidney functioning by measuring how much urea nitrogen in your blood. It reveals whether the levels are increasing than normal suggesting that kidneys or liver may not working properly.
Uric acid:
The blood uric acid test measures the amount of uric acid in a blood sample. Uric acid is produced from the natural breakdown of your body’s cells and from the foods. The uric acid blood test is used to detect high levels of this compound in the blood in order to help diagnose gout or Kidney stones.
Principle:
In RFTs different compounds are analyzed to diagnose any underlying condition about kidneys. Increased level of any compound suggests an underlying condition.
Required material:
Following materials are required;
• Clotted sample.
• Centrifuge machine.
• Forceps.
• Randox chemistry analyzer with essential components.
• Reagents for RFTs tests.
Procedure:
Following steps are involve;
• Draw a sample.
• Save into green, yellow or red vial.
• Give a lab number to sample.
• Wait for clotting the sample.
• Centrifuge the sample for 4 minutes.
• Enter the lab number into software part of Randox chemistry analyzer.
• Place the sample into rag of the analyzer.
• Select the test type.
• Close the shield of the analyzer.
• Check the regent level of the machine.
• Check the level of distilled water.
• Check the waste level.
• Press the button “run”.
• Wait for results.
Normal values:
Following are the normal values of RFTs;
Test Male Female
Creatinine 0.5 to 1.5 mg/dL 0.6 to 1.2mg/d
Blood urea nitrogen
4 to 20 mg/dL 4 to 20 mg/dL
Uric acid 3.4-7.0 mg/dL 2.4-6.0 mg/dL
Liver function test
Introduction:
It is also known as LFTs. This test is performed to diagnose early liver diseases. LFTs included Triglycerides, Cholesterol, LDH, LDL and Bilirubin.
HDL:
Helps remove fat from the body by binding with it in the blood stream and carrying it back to the liver.
LDL:
Carries mostly fat and only a small amount of protein from the liver to other parts of the body.
Cholesterol:
This test is also known as lipid profile. Doctor use this test to measure the amount of “good” and “bad” cholesterol and triglycerides, a type of fat in blood.
Bilirubin:
Bilirubin is produced by the breakdown of red blood cells. The liver is responsible for the detoxification excretion of bilirubin into bile. Increase in the total level, produces the jaundice. Yellow discoloration of skin and the sclera are symptoms of jaundice. It is increased in liver disease and also increase in other conditions that cause increased breakdown of red cells.
Principle:
Liver function tests are blood tests that detect the total quantity of fatty substances in the blood. These tests also help to tell how much the liver is damaged and help to monitor the response to drugs and treatments.
Required materials:
Following materials are required;
• Clotted blood sample.
• Centrifuge machine.
• Forceps.
• Randox chemistry analyzer.
• Waste bin.
• Jouster.
• Yellow and blue tips.
Procedure:
Following steps are involve;
• Draw a sample.
• Save into green, yellow or red vial.
• Give a lab number to sample.
• Wait for clotting the sample.
• Centrifuge the sample for 4 minutes.
• Enter the lab number into software part of Randox chemistry analyzer.
• Place the sample into rag of the analyzer.
• Select the test type.
• Close the shield of the analyzer.
• Check the regent level of the machine.
• Check the level of distilled water.
• Check the waste level.
• Press the button “run”.
• Wait for results.
Normal Values:
Following are the normal levels of LFTs;
Test Normal Value
HDL 40 to 49 mg/dL
LDL < 100mg/dL
Bilirubin < 20 umol/L
Cholesterol < 200 mg/dL
Cardiac enzymes
Introduction:
Cardiac enzymes tests can detect blood levels of the enzyme i.e. creatine phosphokinase (CPK) and CK-MB. Additionally, this test can be used to check blood levels of the myoglobin and troponin proteins. These tests are included CK-MB, CPK, SGOT.
Creatine kinase-MB (CK-MB):
This test is used to detect an elevated creatine kinase (CK) level due to heart muscle damage or skeletal muscle. The test is ordered if a person has chest pain, shortness of breath, dizziness, fatigue or nausea.
Creatine phosphokinase Test (CPK):
This test measures to creatine phosphokinase (CPK) in the blood. CPK is present in the heart, skeletal muscles and brain. CPK leaks in the blood after damaging the heart or skeletal muscles. Detection of different forms of CPK, helps to find out that which tissue is damaged.
SGOT/AST (serum glutamic oxaloacetic transaminase):
This enzyme is present in heart and liver muscles. when the heart or liver muscle is damaged, this enzyme is released in blood.
Principle:
This test is performed to analyzed that how much the heart is healthy. This test also gives the information about the damaging state of the heart. The more values of these enzymes indicate to more damage of heart.
Required material:
Following materials are required;
• Clotted blood sample.
• Centrifuge machine.
• Forceps.
• Randox chemistry analyzer.
• Waste bin.
• Jouster.
• Yellow and blue tips.
Procedure:
Following steps are involve;
• Draw a sample.
• Save into green, yellow or red vial.
• Give a lab number to sample.
• Wait for clotting the sample.
• Centrifuge the sample for 4 minutes.
• Enter the lab number into software part of Randox chemistry analyzer.
• Place the sample into rag of the analyzer.
• Select the test type.
• Close the shield of the analyzer.
• Check the regent level of the machine.
• Check the level of distilled water.
• Check the waste level.
• Press the button “run”.
• Wait for results.
Normal values:
Following are the normal levels of cardiac enzymes;
Test Normal values
SGOT 5 to 40 units per liter of serum
CPK 10 to 20 mcg/L
CKMB 3-5 % or 5 to 25 IU/L
Serum Electrolytes
Introduction:
This test helps to determine that either electrolytes are balance or not in the body. Electrolytes are minerals and salts i.e. sodium, chloride, potassium and bicarbonate. These are present in the blood. Their functions are to conduct electrical impulses and acid base balance in the body.
Principle:
The level of electrolytes is measured by a process called as potentiometry. Voltage is measured during potentiometry that develops across an ion selective electrode.
Required material:
Following materials are required;
• Clotted Blood sample.
• Centrifuge machine.
• Forceps.
• Electrolyte analyzer.
Procedure:
Following steps are involve;
• Draw a blood sample.
• Save it into green, yellow or red headed vials.
• Give a lab number.
• Centrifuge for 4 minutes.
• Prepare the electrolyte analyzer.
• Aspirate the serum of the sample through probe of the analyzer.
• Remove the sample after the aspiration.
• Enter the data about the sample.
• Press the button “start”.
• Wait for results.
Normal values:
Following are the normal values of serum electrolytes;
Electrolyte Normal value
Sodium 136 – 145mEq/L
Potassium 3.5 – 5.1 mEq/L
Chloride 98 – 107 mEq/L
Bicarbonate 23 – 29mEq/L
Equipment for Biochemistry:
Serum electrolyte analyzer
Learning outcomes
Following are the outcomes of internship;
• Internship has been a rewarding experience.
• I have learned time management skills and self-motivation.
• I have gained knowledge, skills and met new people.
• I have practiced all the methods and procedures.
• The internship was good to find out my strengths and weaknesses.
• Practical implementation of theory.
• Experience of professional field.
• Use my gained skills and knowledge.
• Build my self-confidence.
