Dip the thick blood smear into diluted giemsa stain prepared by taking 1ml of the stock solution and adding to 49ml of phosphate buffer or. The excess stain is removed with dilute hydrochloric acid solution acid water. Allow the water to evaporate and the slide to air dry completely. Without delay, the blood was spread with a glass spreader held at a steep angle to achieve a thick smear.
Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. A thick smear of proper density is one which, if placed wet over newsprint. A wellprepared blood smear is important to produce good results on analysis after doing a giemsa stain, in identifying blood cells orand demonstrating the presence of parasites in a sample. If there will be a delay in staining smears, dip the thick smear briefly in water to hemolyse. Through a series of staining and decolorization steps, organisms in the domain bacteria are differentiated according to cell wall composition. Large blood drops may extend the smear over too much of the base slide, while a small. Jan 12, 2020 16 responses to papanicolaou pap staining. Dec, 2016 blood smear preparation and staining duration. Insufficiently dried smears andor smears that are too thick can detach from the slides during staining. Before staining and observing a microbe under a microscope, a smear must be prepared. Learn about the different types of tests doctors use to test for malaria in your body and how the exam is performed. Thick and thin blood smears will let doctors know the percentage of red blood cells that are infected parasite density and what type of parasites are present. If there will be a delay in staining smears, dip the thick smear briefly in water to hemolyse the rbcs.
Dip the thick blood smear into diluted giemsa stain prepared by taking 1ml of the stock solution and adding to 49ml of phosphate buffer or distilled water, but the results may vary differently. When using the regressive staining method, the nucleus is deliberately overstained with a nonacidified haematoxylin. Check out the giemsa staining protocol for staining blood smears. To heatfix a sample, a thin layer of the specimen is spread on the slide called a smear. The goal of smear preparation is to place an appropriate concentration of cells on a slide and then cement them there so that they do not wash off during the subsequent staining procedure. Introduction, principle, procedure and interpretation dr. Pap staining is used to differentiate cells in smear preparations of various bodily secretions. Preparation and examination of skin smears official web. One needs to be careful about thick smears when taking the specimen from an agar medium. Do not heat or disturb the slide during this phase. Remove thin smear slides and rinse by dipping 34 times in the giemsa buffer. Procedure for thick blood film staining technique a thick film was made by placing a large drop of blood about 15mm in size on the centre of greasefree microscopic slide.
For giemsa staining, the film was stood in 5% giemsa for 30 minutes, then washed gently in tap water and air dried. The thick blood film permits the examination of a large amount of blood for the presence of parasites. The bacterial smear will wash away during the staining procedure. The gram stain, the most widely used staining procedure in bacteriology, is a complex and differential staining procedure. The procedure of giemsa staining varies as per the purpose of staining that means whether the staining is done for the examination of blood cells or to find the parasites in the blood smear and accordingly the blood smears are prepared as thin blood films or thick blood films. Grampositive bacteria have cell walls that contain thick layers of. Fix airdried film in absolute methanol by dipping the film briefly two dips in a coplin jar containing absolute methanol. Thick and thin blood smear for malaria diagnosis learn. Giemsa staining technique principle, preparation, procedure. Take care to prevent overheating the slide because proteins in the specimen can coagulate causing cellular morphology to appear distorted. Check out the leishman staining protocol for staining blood smears. Proceed with either the thin or thick smearfilm staining method. The dye will not penetrate well, and there will be far too many bacterial cells to see individual shapes and arrangements.
Thick and thin blood smears for malaria cs mott childrens. Because of snowstorm, your regular laboratory session was canceled and the gram staining procedure was perform on cultures incubated for a longer period of time. The blood film occupies the central portion of the slide and has definite margins on all sides that are accessible to examination by oil immersion. Giemsa stain principle, procedure, results, interpretation. Nov 09, 2017 thick and thin blood smear procedure 1. Thick smears consist of a thick layer of dehemoglobinized lysed red blood cells rbcs.
The smear is greater than 25 mm long and the feathered edge stops approximately 10 mm from the end of the slide. Thick and thin blood films stained with giemsa hematological stains permit the detection of blood parasites including malarial parasites, trypanosomes, and microfilariae. A thick blood smear is a drop of blood on a glass slide. Most commonly blood smears are used for the differential leukocyte count dlc. Gram staining and acidfast staining and simple staining. Papanicolaou stain also papanicolaous stain and pap stain is a multichromatic staining histological technique developed by george papanikolaou, the father of cytopathology. Principles of the procedure thick and thin blood films are a routine practice in the hematological department to diagnose various pathogens that cause ailments in the.
Decolorize the stained smears by immersion in distilled or deionized water and air dry 3. Printerfriendly preparation and examination of skin smears pdf 225 kb. Add a thick smear of blood and air dry for 1 hour on a staining rack. Thick film staining add a thick smear of blood and air dry for 1 hour on a staining rack. The skin smear is a valuable, costeffective tool in the routine management of the patient with hansens disease. Answers is the place to go to get the answers you need and to ask the questions you want.
The three main blood cells that the test focuses on are. Place the thick smear slide flat on a staining rack. You can use 2 slides, 1 for each bacterium, or you can divide one slide in. Staining the thin and thick blood smear on the same slide with giemsa stain. The video describes the preparation and interpretation of a malarial thick smear for the detection of malarial parasites. Dip the dried smear in giemsa plus fixative 5 times, 1 second per. This method produces a gradual decrease in thickness of the blood from thick to thin ends with the smear terminating in a feathered edge approximately 2 mm long.
If the smear is opaque, it is too thick and should be emulsified with a drop or two of saline. Thick smears allow a more efficient detection of parasites increased sensitivity 11 times than thin smear. Gently flood smear with crystal violet and let stand for 1 minute. Or the specimen being stained may be used as the organisms andor the white blood cells are a built in quality control. There should not be marks or blank spaces in the smear. What if your smear was too thick in gram stain answers.
Making and staining a blood smear university of vermont. This is avoided by heat fixation, during which the bacterial proteins are coagulated and fixed to the glass surface. Do not use a wax pencil or marker or any pen as it dissolves and washed out during the staining process. In order to stain the bacterial specimen for microscopy one must first prepare the smear on the slide. A blood smear, thick andor thin, is run once each month or with each staining procedure if done less often than once each month. Staining of the thick thin smear with giemsa stain. Thin blood smears helps to discover which species of parasite is causing the infection.
If you are using venous blood, blood smears should be prepared as soon as possible after collection delay can result in changes in parasite morphology and staining characteristics. It can be stained with any of the romanowsky stains listed above except that before staining, the smear is dehaemoglobinised by putting it in distilled water for 10 minute autostainers currently, automatic staining machines are available which enable a large batch of slides to be stained with a uniform quality. You can accelerate the drying by using a fan or hair dryer. Fix airdried film in absolute methanol by dipping the film briefly two dips. The gram stain procedure is a differential staining procedure that involves multiple steps. What is the disadvantage of having a really thick smear when. After the smear has airdried, hold the slide at one end and pass the entire slide through the flame of a bunsen burner two to three times with the smearside up. Fix in methanol for one minute or get spray easyfix. It was primarily designed for the demonstration of malarial parasites in blood smears, but it is also employed in histology for routine examination of blood smear. I am a medical lab tech, a web developer and bibliophiliac.
Staining procedure for thin blood film bp healthcare. Less sensitive than a thick film especially where there is a low parasitemia. Giemsa staining was used for the first two sporozoite challenge studies, and fields stain in coplin jars for the later two studies. Thick and thin blood smear procedure linkedin slideshare. A peripheral blood smear pbs or blood film is required to be made from capillary blood or from a drop of blood from an edta anticoagulated blood sample. Staining can be reduced by reducing the dilution of the stain.
The extra time and care taken during the field season will be rewarded later when the smears must be scanned, and parasites identified and counted. A thin blood smear is a drop of blood that is spread across a large area of the. Using the corner of another slide or an applicator stick, spread the drop in a circular pattern. Place slide with heat fixed smear on staining tray. Terms in this set 43 why are thick or dense smears less likely to provide a good smear preparation for microscopic evaluation. Dry vertically and examine under the 100x objective. Hi, im the founder and developer of paramedics world, a blog truly devoted to paramedics. What is the disadvantage of having a really thick smear when staining. Label the smear and circle the material to be stained with a diamond point pen for easier identification and location of the material after staining. Dec 30, 2019 i used giemsa staining for micronuclei test on blood smears of liza spp following this protocol. Without delay, the blood was spread with a glass spreader held at a steep angle to achieve a thick smear covering an area of 15 x 15mm. The method for staining, concentration and timing of stain used varies according to the purpose, for example, thin blood smears use 1.
A blood smear is a blood test used to look for abnormalities in blood cells. A phlebotomist, a person specifically trained to draw blood, first cleans and sterilizes the injection site with an antiseptic. Procedure for thick and thin blood film staining technique. Below, we discuss the procedures for preparing both thin and thick smear for giemsa staining technique, importance, and applications of blood smears, in.
Good color differentiation of red and white cells is an indication of a good quality stain. The blood smear does not have to be positive for blood parasites. During the performance of the simple staining procedure, you failed to heat fix your e. The dried film should be of brownish yellow colour buff coloured. The combination thick thin blood film provides both options on one glass slide and the slide can be stained as either a thick or thin blood film. Giemsa stain is a type of romanowsky stain, named after gustav giemsa, a german chemist who created a dye solution. The decolourising process is then stopped by immersing the slide in running tap water. Conversely, the smear will be too thick if the spreader is moved slowly or if the angle is greater than 30. Jun 24, 2018 the video describes the preparation and interpretation of a malarial thick smear for the detection of malarial parasites. Without delay, the blood was spread with a glass spreader. Heat fixing kills the bacteria in the smear, firmly adheres the smear to the slide, and allows the sample to more readily take up stains.
In this lesson, readers will learn about the procedure, staining methods, and interpretation of the blood smear test. Make blood smear on a clean glass slide and it is dried in the air. Quality control to ensure that proper staining results have been achieved, if a positive smear malaria is available it may be included with each new batch of working giemsa stain. Carefully overlay the entire smear with demineralized water. The scratch method is an alternate method for making thick films that allows for improved adherence and faster turnaround times. Procedure of regressive papanicolaou staining method. It was developed by danish microbiologist hans christian gram in 1884 as an effective method to distinguish between bacteria with different types of cell walls, and even today it remains one of the most frequently used staining techniques. Apart from staining malarial parasites, giemsa stain has a variety. Giemsa stain principle, results, procedure and more lab.
The combination thickthin blood film provides both options on one glass slide and the slide can be stained as either a thick or thin blood film. Making and staining a blood smear a wellmade blood smear is a beauty to behold, and likely to yield interesting and significant information for a research project. Dip fixed smear to field stain b red stain for 5 to 6 seconds. If fixed prior to staining, then the smear will be read as a thin blood film. For perfect malaria staining the ph of the buffer should be 7. Microbiology lab test, micro lab 1 flashcards quizlet. Examination of stained bacillus cereus slides revealed a great deal of color variability, ranging from an intense blue to shades of pink. The thin blood film should be thick enough at the starting point 1, becoming less thick in the middle 2, and have a flamelike shape at the end 3. Thick smears are mainly used to detect infection and to estimate parasitemia. Dip air dried blood film in undiluted stain for 15 to 30 seconds double the staining time for bone marrow smears. Differences between thick blood smear and thin blood smear.
Thick smears place a small drop of blood in the center of the precleaned, labeled slide. Thick and thin blood films procedure laboratory stack. Thick blood smear and thin blood smear parasitology. What is the disadvantage of having a really thick smear. Should you heat the slide lightly to lyse the erythrocytes and get those organisms out. Tail is the last part of the smear with ragged margins. Follows this procedure for preparation and staining of thin. Blood smears are needed for microscopic examination of the blood.
Thick and thin blood smears for malaria cs mott children. Thick blood film examination for plasmodium falciparum. This lesson also includes some general descriptions of. Two easy methods for blood film preparation and staining.
Thick blood smears are most useful for detecting the presence of parasites, because they examine a larger sample of blood. Thick blood smears are most useful for detecting the presence of parasites. After selecting a good smear with naked eyes focus it under low power of the microscope. This basically involves three stepstransferring a liquid suspension of the bacterium on the slide, drying the smear, and then heating slightly to firmly attach the smear to the slide. February 21, 2017 by dramjad leave a comment the purpose of preparation of blood smear blood film is to study the morphology of rbcs, differential leukocyte count and reticulocyte count. Gently flood the smear with grams iodine and let stand for 1 minute.
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