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Trophozoites of Entamoeba histolytica in LPCB mount

Trophozoites of Entamoeba histolytica in LPCB mount of dysenteric fecal specimen at 40X objective microscope as shown above picture having following identification features-

• Size : 20 t0 20 µm
• Motility: Non-motile due to inactivation or sluggish in fresh stool preparation
• Cytoplasm: clearly defined into ectoplasm and endoplasm
• Cytoplasmic inclusions: RBCs, leukocytes, and tissue debris but lacking bacteria
• Nuclear characters: Central karyosome, fine chromatin granules line the delicate nuclear membrane

Introduction

LPCB is a combination of fixative, staining, and clearing agent. Its contents functions are as follows-

Lactic acid: It helps in preserving the morphology of the fungal elements.

Phenol: It acts as a disinfectant

Cotton blue: It stains the fungal elements as well as intestinal parasitic (cyst, ova, and oocyst) and non-parasitic structures(vegetable cells, mucus, muscle fibers, and other artifacts) and

Glycerol: It is a hygroscopic agent that prevents drying.

Note: In LPCB wet mount of stool phenol and lactic clear fecal debris.

Principle of LPCB mount

Cotton blue stains the chitin in the cell wall of fungi and identification of filamentous fungi is made by their characteristic microscopic morphology such as shape, size, arrangement of spores, and hyphae.

In the LPCB wet mount of stool, glycerol provides a semi-permanent preparation. Cyst of intestinal protozoa and ova takes blue color while ova of helminths are stained deep blue. An additional advantage of this stain is that it can also detect blue-colored Cyclospora and Isospora oocyst.

Requirements for test

• Compound light microscope
• LPCB stain
• Clean and grease-free microscopic slides
• Coverslip
• Dropper or bamboo sticks
• Fungal growth  in the medium
• Stool samples

Procedure of LPCB Preparation

Mycology

Scotch tape preparation

1. Take a clean and grease-free slide and place a drop of LPCB on it.
2. Touch the adhesive side of the tape of transparent scotch tape on the surface of the colony at a point intermediate between its center and periphery.
3. Fix the adhesive side of the tape over an area on the glass side containing the LPCB.
4. Focus the preparation at 10x and finally observe at 40x objective of a light microscope.

Tease mount preparation

1. Put a drop of LPCB on a clean grease-free glass slide.
2.  Take a small portion of the colony and the supporting agar at a point between the center and the periphery and place it in the drop.
3. With the help of a needle, tease the fungal culture first and spread in the LPCB.
4.  Wait for normally 30 minutes .i.e. sufficient time for the structures to take up the stain.
5. Focus on 10X objective and finally examine at 40X objective under a microscope.

Observation

Fungi appear as dark blue stained mycelium.

Results and interpretations

Different fungi under LPCB wet mount will show different types of morphological structures including hyphae and spores. We concern with Aspergillus as shown above picture.

Parasitology

1. Take a clean and grease-free slide.
2.  Using a glass dropper, put a drop of LPCB on the glass slide.
3. With the help of an applicator stick, take a small portion .i.e  match the sick head size of the stool sample and finally emulsify it in the drop of LPCB.
4. Put a coverslip over the  LPCB suspension of the stool and wait for at least 30 minutes.
5. Focus the preparation first under low power objective (10X) and then finally observe under high power objective (40X) of the microscope.

Observation

Observe larvae, ova, cysts, and oocyst of intestinal parasites and also non-parasitic structures that normally take blue color.

Result and interpretation

We concern with the cyst of Entamoeba histolytica/dispar as shown below.

Clues: A 10-15 µm sized, round, deep blue colored, structure seen with blue colored 1-4 nuclei and glycogen mass.

Cyst of Entamoeba histolytica or dispar on LPCB wet mount of stool

Keynotes

1. LPCB wet mount is always examined at least 30 minutes after preparation.
2. A wet mount preparation should neither be too thick or too thin.
3.  In this preparation, both bile-stained and non-bile-stained helminthic eggs are stained blue.
4. LPCB kills the trophozoites of Entamoeba and Trichomonas, hence, can not be demonstrated by this.

Further Readings

1. Medical Mycology. Editors:  Emmons and Binford, 2nd ed 1970, Publisher Lea and Febiger, Philadelphia.
2. Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
3. A Textbook of Medical Mycology. Editor: Jagdish Chander.  Publication Mehata, India.
4.  Practical Laboratory Mycology. Editors: Koneman E.W. and G.D. Roberts, 3rd ed 1985, Publisher Williams and Wilkins, Baltimore.
5. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
6. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.