MP-TROPACON 2025.pptx WORKSHOP ON HAEMOPARASITE

WORKSHOP ON DIFFERET HAEMOPARASITE IDENTIFICATION ,ISOLATION

1. MP-TROPACON 2025 Workshop - Staining procedure and Haemo-Parasitology Co- ordinator & resource person – Dr Nonika Rajkumari , Additional Professor, Dept of Microbiology, AlIMS,Kalyani (Formerly in JIPMER, Puducherry ) & team Date-16/07/25 Presented by Dr Mohan Singh Dhakad PG 2 nd year Department of Microbiology Moderator- Dr Prafulla Songara Professor & head Department of Microbiology Dr Laxmi Narayan Pandey GMC Ratlam

2. S TAINNG METHODS OF BLOOD SPECIMENS Giemsa Stain Reagents: Stock Giemsa stain (100 ml) Giemsa stain powder (Azure B type) 0.6 gm Methanol 50 ml Glycerin 50 ml Stock Solution Preparation (if needed ) Dissolve Giemsa powder:  Combine 0.6 gm of Giemsa powder with 50 ml of Glycerin  Heat the mixture to approximately 60°C with continuous stirring until the powder is dissolved

3. Giemsa Stain Add methanol:  Slowly add 50ml of methanol to the solution. Mature the solution:  Store the solution in a dark bottle for a period (1-2 months is often recommended) to allow for maturation and improved staining quality. Filter:  Filter the solution before use.  2. Working Solution Preparation: Dilute the stock:  Dilute the Giemsa stock solution with buffered water (and methanol) to create a working solution. The dilution ratio is usually 1:10 (stock to buffer) or as specified by the staining protocol. Filter:  Filter the working solution before use

4. Staining Procedure for blood smears Prepare the smear Fix the smear: (thin smear) Air dry : . Stain:  Flood the slide with the diluted Giemsa working solution for a specified time (e.g., 20-30 minutes for a 5% solution). Wash :  T ap water or buffer Dry Examine  

5. Observations of Giemsa stain a . RBCs pale red; nuclei of WBCs purple with pale purple cytoplasm. b. If malaria parasites are present, the cytoplasm stains blue and nuclear material stains red to purple-red Schuffner's dots and other inclusions in the RBCs will stain. c . Nuclear and cytoplasmic staining characteristics of the other blood parasites such as Babesia spp., Trypanosoma and Leishmania are like those of the malarial parasites d. The sheath of microfilariae may not always stain with Giemsa and the nuclei within the microfilaria itself stain blue to purple .

6. Observations Giemsa stain

7. JSB Stain ( Jaswant -Singh-Bhattacharya-Stain) REAGENTS: Solution 1 Methylene -blue 0.5gm Potassium- di -chromate 0.5gm Sulphuric acid (1%) 3 ml Potassium hydroxide(1%) 10 ml Water 500 ml Solution 2 Eosin 1gm Water 500ml

8. Procedure of JSB Stain ( Thin flm ) Fix the smear with methanol and air dry Dip in solution 1 x 30 seconds Wash in jar containing acidic tap water Dip in solution 2 x1second Wash again Dip in solution 1 for 30 seconds or till the smear give a pink background Wash for 10 seconds Air dry and observe under oil immersion field THICK FILM: not fixed with methanol

9. JSB Stain (cont.) Rapid staining method primarily used for the detection of malaria parasites in blood smears, particularly in thick smears Limitations: The stain may fade over time, making it less suitable for permanent slide preparations.  Advantages: Inexpensive, easily available, and allows for clear visualization of malarial parasites in both thick and thin smears

10. REAL-TIME PCR FOR IDENTIFICATION OF MALARIA AIM: To rapidly , accurately detect and identify malaria parasites, including specific species, and sometimes quantify parasite load, especially in cases where other methods are less sensitive or inconclusive PRÌNCIPLE: 1.Denaturation: Separation of double-stranded DNA. 2.Annealing: Binding of primers and probes specific to the Plasmodium genome 3. Extension: DNA polymerase extends the primer, amplifying the DNA

11. REAL-TIME PCR FOR IDENTIFICATION OF MALARIA (cont.) Specimen: Blood Inclusion criteria Exclusion criteria

12. DNA Extraction Procedure 1. Lysis & Homogenization: Pipette 200 ul of whole blood into a l.5 ml micro centrifuge tube Add 20 ul of Proteinase Add 200 ul of Lysis Buffer (Buffer AL). Vortex for 15 sec and incubate at 56°C for 10 min. 2. Precipitation: Add 200 ul of absolute ethanol and mix by vortexing . Transfer the mixture to a spin column placed in a collection tube. Centrifuge at full speed (10,000 rpm) for 1 min.

13. DNA Extraction Procedure 3. Washing: Add 500 ul of Buffer A W1, centrifuge for 1 min.& discard the flowthrough and collection tube Add 500 ul of Buffer A W2, centrifuge for 3 min.& discard the flowthrough and collection tube 4.Elution: Transfer the spin column to a new microcentrifuge tube. Add 100 ul of Elute Buffer and incubate for l min. Centrifuge at full speed for 1 min. Store extracted DNA at -20°C until PCR analysis .

14. SECTION B - REAL-TIME PCR FOR MALARIA DETECTION Materials and Reagents Required: PCR Master Mix (e.g., Taq Man Universal PCR Master Mix) Forward & Reverse primers for: Plasmodium falciparum Plasmodium vivax Plasmodium malariae Plasmodium ovale Species-specific Taq Man probes labeled with FAM/VIC Nuclease-free water PCR tubes and strip caps PCR machine

15. PCR Cycling Conditions Stage Step Temp (°C) Time 1 Taq polymerase activation 95°c 10 min 2 Denaturation 95°c 15 sec 3 Annealing / Extension 60°c 60 sec (Repeat steps 2-3 for 40 cycles)

16. REAL-TIME PCR FOR MALARIA DETECTION

17. REAL TIME PCR Controls and Assay Setup Negative template control (NTC): Nuclease-free water instead of DNA. Positive control (PC): Known Plasmodium DNA. Internal control ( IC ): Ensures no PCR inhibition Result Interpretation: Positive sample: Amplification curve with Ct<35. Negative sample: No amplification or Ct >40. Invalid result: No amplification in positive control or Ct> 35 in internal control.

18. Documentation and Storage Retain negative samples at -80°C. Store positive samples at -80°C for further studies. Store results in an Excel file.