Apart from staining malarial parasites, giemsa stain has a variety. 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. Giemsa stain principle, procedure, results, interpretation. 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. 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. Dry vertically and examine under the 100x objective. Making and staining a blood smear university of vermont. 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.
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. 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. Most commonly blood smears are used for the differential leukocyte count dlc. 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. The thick blood film permits the examination of a large amount of blood for the presence of parasites. When using the regressive staining method, the nucleus is deliberately overstained with a nonacidified haematoxylin. A blood smear is a blood test used to look for abnormalities in blood cells. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. Dip the dried smear in giemsa plus fixative 5 times, 1 second per. The decolourising process is then stopped by immersing the slide in running tap water. During the performance of the simple staining procedure, you failed to heat fix your e.
Make blood smear on a clean glass slide and it is dried in the air. Allow the water to evaporate and the slide to air dry completely. 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. Staining procedure for thin blood film bp healthcare. A thick smear of proper density is one which, if placed wet over newsprint. Thick and thin blood films stained with giemsa hematological stains permit the detection of blood parasites including malarial parasites, trypanosomes, and microfilariae. In this lesson, readers will learn about the procedure, staining methods, and interpretation of the blood smear test.
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. For giemsa staining, the film was stood in 5% giemsa for 30 minutes, then washed gently in tap water and air dried. Thick blood smear and thin blood smear parasitology. 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 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. The excess stain is removed with dilute hydrochloric acid solution acid water. 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. Staining can be reduced by reducing the dilution of the stain.
The method for staining, concentration and timing of stain used varies according to the purpose, for example, thin blood smears use 1. Check out the giemsa staining protocol for staining blood smears. One needs to be careful about thick smears when taking the specimen from an agar medium. Good color differentiation of red and white cells is an indication of a good quality stain. Nov 09, 2017 thick and thin blood smear procedure 1. 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. Thick smears place a small drop of blood in the center of the precleaned, labeled slide. Without delay, the blood was spread with a glass spreader.
You can accelerate the drying by using a fan or hair dryer. Thick and thin blood smears for malaria cs mott children. Answers is the place to go to get the answers you need and to ask the questions you want. Thick and thin blood smear for malaria diagnosis learn. Giemsa staining technique principle, preparation, procedure.
Giemsa staining was used for the first two sporozoite challenge studies, and fields stain in coplin jars for the later two studies. 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. Terms in this set 43 why are thick or dense smears less likely to provide a good smear preparation for microscopic evaluation. Jun 24, 2018 the video describes the preparation and interpretation of a malarial thick smear for the detection of malarial parasites. Staining the thin and thick blood smear on the same slide with giemsa stain. A thick blood smear is a drop of blood on a glass slide. If the smear is opaque, it is too thick and should be emulsified with a drop or two of saline. Less sensitive than a thick film especially where there is a low parasitemia. 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. Carefully overlay the entire smear with demineralized water. 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. 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. Differences between thick blood smear and thin blood smear. Papanicolaou stain also papanicolaous stain and pap stain is a multichromatic staining histological technique developed by george papanikolaou, the father of cytopathology. Giemsa stain principle, results, procedure and more lab. This lesson also includes some general descriptions of. The bacterial smear will wash away during the staining procedure. Add a thick smear of blood and air dry for 1 hour on a staining rack. Check out the leishman staining protocol for staining blood smears. 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. 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. Introduction, principle, procedure and interpretation dr. Thick film staining add a thick smear of blood and air dry for 1 hour on a staining rack. Dec 30, 2019 i used giemsa staining for micronuclei test on blood smears of liza spp following this protocol.
Using the corner of another slide or an applicator stick, spread the drop in a circular pattern. Thick blood smears are most useful for detecting the presence of parasites, because they examine a larger sample of blood. Proceed with either the thin or thick smearfilm staining method. This is avoided by heat fixation, during which the bacterial proteins are coagulated and fixed to the glass surface.
A blood smear, thick andor thin, is run once each month or with each staining procedure if done less often than once each month. Two easy methods for blood film preparation and staining. Grampositive bacteria have cell walls that contain thick layers of. Thick smears are mainly used to detect infection and to estimate parasitemia. 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. Printerfriendly preparation and examination of skin smears pdf 225 kb. Thin blood smears helps to discover which species of parasite is causing the infection. A phlebotomist, a person specifically trained to draw blood, first cleans and sterilizes the injection site with an antiseptic.
I am a medical lab tech, a web developer and bibliophiliac. If fixed prior to staining, then the smear will be read as a thin blood film. 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. Gently flood the smear with grams iodine and let stand for 1 minute. Should you heat the slide lightly to lyse the erythrocytes and get those organisms out. Place the thick smear slide flat on a staining rack. 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. A thin blood smear is a drop of blood that is spread across a large area of the. Thick smears allow a more efficient detection of parasites increased sensitivity 11 times than thin smear. Thick smears consist of a thick layer of dehemoglobinized lysed red blood cells rbcs. The three main blood cells that the test focuses on are. Or the specimen being stained may be used as the organisms andor the white blood cells are a built in quality control.
The gram stain procedure is a differential staining procedure that involves multiple steps. Below, we discuss the procedures for preparing both thin and thick smear for giemsa staining technique, importance, and applications of blood smears, in. Large blood drops may extend the smear over too much of the base slide, while a small. 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. Prepare a thin and thick smear of the specimen on the same slide which can be done by dividing the slides into two parts and then making the thin smear on one side and thick on the other side. 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. The skin smear is a valuable, costeffective tool in the routine management of the patient with hansens disease.
Procedure for thick and thin blood film staining technique. Thick and thin blood smear procedure linkedin slideshare. You can use 2 slides, 1 for each bacterium, or you can divide one slide in. Fix in methanol for one minute or get spray easyfix. Tail is the last part of the smear with ragged margins. The dye will not penetrate well, and there will be far too many bacterial cells to see individual shapes and arrangements.
What if your smear was too thick in gram stain answers. Fix airdried film in absolute methanol by dipping the film briefly two dips. Thick and thin blood smears for malaria cs mott childrens. Learn about the different types of tests doctors use to test for malaria in your body and how the exam is performed.
The dried film should be of brownish yellow colour buff coloured. In order to stain the bacterial specimen for microscopy one must first prepare the smear on the slide. Through a series of staining and decolorization steps, organisms in the domain bacteria are differentiated according to cell wall composition. Remove thin smear slides and rinse by dipping 34 times in the giemsa buffer. Follows this procedure for preparation and staining of thin.
What is the disadvantage of having a really thick smear when. Do not use a wax pencil or marker or any pen as it dissolves and washed out during the staining process. The video describes the preparation and interpretation of a malarial thick smear for the detection of malarial parasites. What is the disadvantage of having a really thick smear when staining. After selecting a good smear with naked eyes focus it under low power of the microscope. Jan 12, 2020 16 responses to papanicolaou pap staining. The scratch method is an alternate method for making thick films that allows for improved adherence and faster turnaround times. Thick and thin blood films procedure laboratory stack. Pap staining is used to differentiate cells in smear preparations of various bodily secretions. The smear is greater than 25 mm long and the feathered edge stops approximately 10 mm from the end of the slide. Dip fixed smear to field stain b red stain for 5 to 6 seconds.
Gently flood smear with crystal violet and let stand for 1 minute. Take care to prevent overheating the slide because proteins in the specimen can coagulate causing cellular morphology to appear distorted. If there will be a delay in staining smears, dip the thick smear briefly in water to hemolyse the rbcs. The blood smear does not have to be positive for blood parasites. 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.
Decolorize the stained smears by immersion in distilled or deionized water and air dry 3. Place slide with heat fixed smear on staining tray. Giemsa stain is a type of romanowsky stain, named after gustav giemsa, a german chemist who created a dye solution. Conversely, the smear will be too thick if the spreader is moved slowly or if the angle is greater than 30.
There should not be marks or blank spaces in the smear. Do not heat or disturb the slide during this phase. Hi, im the founder and developer of paramedics world, a blog truly devoted to paramedics. Preparation and examination of skin smears official web.
Dip air dried blood film in undiluted stain for 15 to 30 seconds double the staining time for bone marrow smears. Staining of the thick thin smear with giemsa stain. What is the disadvantage of having a really thick smear. Thick blood film examination for plasmodium falciparum. 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. To heatfix a sample, a thin layer of the specimen is spread on the slide called a smear. 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. Thick blood smears are most useful for detecting the presence of parasites. The gram stain, the most widely used staining procedure in bacteriology, is a complex and differential staining procedure. Without delay, the blood was spread with a glass spreader held at a steep angle to achieve a thick smear.
Fix airdried film in absolute methanol by dipping the film briefly two dips in a coplin jar containing absolute methanol. Gram staining and acidfast staining and simple staining. For perfect malaria staining the ph of the buffer should be 7. Blood smears are needed for microscopic examination of the blood. 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. Microbiology lab test, micro lab 1 flashcards quizlet. Procedure of regressive papanicolaou staining method. Prepare within an accepted time frame, a wellmade blood smear or bone marrow smearfilm per your laboratories protocol, with a focus on uniform cell distribution. Examination of stained bacillus cereus slides revealed a great deal of color variability, ranging from an intense blue to shades of pink. If there will be a delay in staining smears, dip the thick smear briefly in water to hemolyse. 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. Dec, 2016 blood smear preparation and staining duration.
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