I want to know how to stain malarial parasites with Wright-Giemsa Stain and how to make the stain at my lab ?
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Keep in mind various clinical algorithms have been suggested and the best of them predict only up to 50% of true malaria cases. This method has poor specificity and positive predictive value. It does not allow differentiation in between falciparum and vivax malaria. Moreover, many symptoms of presumptive malaria overlap with other diseases such as pneumonia, otitis, tonsillitis etc.
Methyllene blue, and eosin will make the Wright-Giemsa Stain you are seeking, but I dunno what amounts. It's easier and cheaper just to buy a kit.
Every clinic in the world has its own specifics in preparing malaria blood smear, yet there are general principles to follow. As a general rule, malaria blood film should be prepared as soon as possible after collecting venous blood. That will secure against the possible changes in parasite morphology and will also ensure the good staining characteristics. Particular attention should be given to the fixing stage of film preparation. Accidental fixing of the thick smear with methanol should be avoided, especially when both thick and thin blood smears are on the same slide. The examiner should let alcohol on patient's finger to dry before starting to prepare the thick smear. Enough care should be also taken to avoid fixing the thick smear while drying up the slide with heat.
So, the thin smear is fixed in methanol before staining; the thick smear is stained unfixed. Many hospitals have a Wright-Giemsa stain available, which is acceptable; however, Wright stain alone will not reliably stain Plasmodium parasites. For best results, the smear should be stained with a 3% Giemsa solution (pH of 7.2) for 30-45 minutes
The correct thickness of the thick film can be judged by the legibility of the printed text seen through the slide. The print should be just legible.
In resource limited settings, thin and thick films may be taken on the same slide. The important hometake message is that both thick and thin smears should be always prepared. Both of them provide important information for diagnostic purposes
The determination of the number of circulating parasites is exceedingly important for clinical purposes to monitor the evolution of the disease and the efficacy of therapy. Different methods have been proposed:
Number of parasites/µL of blood (thick film): This method requires observation of as many microscopic fields (100x oil immersion lenses) necessary to count 200 white blood cells (WBC). Number of asexual parasites and WBCs should be counted in each field until the number of WBCs reaches 200.
If number of WBCs is unknown, it can be assumed to be 8000//µL
Number of parasites/µL of blood (thin film): This method requires the preliminary determination of the number of erythrocytes (RBCs) present in the average microscopic field.
The number of asexual parasites is counted in at least 25 microscopic fields. The number of RBCs in the average microscopic field is about 200, so total RBCs counted in 25 fields is about 200*25 = 5000. If the hemogram is not available, RBCs/µL is assumed 5.000.000 for males and 4.500.000 for females.
Proportion of parasitized erythrocytes/total RBC count (thin film): This method will indicate the percentage of erythrocytes that are infected by malaria parasites.
The number of parasitized erythrocytes (asexual forms) present in 25 microscopic fields is counted divided by the total number of erythrocytes present in these fields (about 5000), and multiplied by 100.
Semi quantitative count (thick film): This alternative method of estimating parasite density is very quick but less accurate. It should be used only when it is not possible to perform more accurate methods. The following semi-quantitative scale is used. + 1-10 asexual parasites per 100 thick film fields
++ 11-100 asexual parasites per 100 thick film fields
+++ 1-10 asexual parasites per single thick film field
++++ > 10 asexual parasites per single thick film field
It is important to know that parasite blood density is correlated with the severity of clinical presentation
Source(s):
WHO
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